- Email: [email protected]
In vitro maturation (IVM) of murine and human germinal vesicle (GV)–stage oocytes by coculture with immortalized human fallopian tube epithelial cells
In vitro maturation (IVM) of murine and human germinal vesicle (GV)–stage oocytes by coculture with immortalized human fallopian tube epithelial cells Ho-Joon Lee, Ph.D.,a Alexander M. Quaas, M.D., Ph.D.,a Diane L. Wright, Ph.D.,b Thomas L. Toth, M.D.,b and Jose M. Teixeira, Ph.D.a a Vincent Center for Reproductive Biology and b Division of Reproductive Medicine and IVF, Department of Obstetrics and Gynecology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Objective: To improve the maturation rate of murine and human germinal vesicle (GV) oocytes using human tubal epithelial cells (hTECs). Design: Murine and human GV oocytes were randomized to human tubal fluid (HTF) media alone or cocultured with mouse embryonic fibroblasts (MEFs) or primary hTECs or immortalized hTECS (ihTECs) for various times. Rates of maturation to meiosis II (MII) were compared between groups. Intervention(s): TECs were isolated from discarded salpingectomy specimens. One batch was immortalized with TERT and SV40 large T-antigen. GVoocytes (n ¼ 710) were isolated from 8-week-old-mice at 40 hours after pregnant mare’s serum gonadotropin stimulation. Discarded human GV oocytes (n ¼ 62) were obtained from intracytoplasmic sperm injection cycle IVF center patients. Oocytes were cultured in HTF media alone or with MEFs, hTECs, or ihTECs. Main Outcome Measure(s): Maturation rates were assessed by standard morphological criteria and compared. Result(s): The maturation rate of murine GVoocytes to MII at 12 and 24 hours increased significantly in coculture with hTECS and ihTECS compared with MEF and HTF media alone. In addition, the development rate after IVF was significantly higher with hTECS and ihTECS than in MEF and HTF media alone. Maturation of human GV oocytes to MII at 24 and 48 hours was significantly higher in hTECS and ihTECS compared with HTF media alone. Conclusion(s): Coculture with either primary or immortalized TECs might improve oocyte quality and significantly raise in vitro maturation rates for GV oocytes. (Fertil Steril 2011;95:1344–8. 2011 by American Society for Reproductive Medicine.) Key Words: Oocytes, in vitro maturation, tubal feeder cells
In vitro maturation (IVM) of human and murine oocytes is an evolving technique that holds promise for use in both clinical and the basic laboratory settings. In vivo, human oocytes are arrested at prophase in meiosis I (MI) for 12–50 years before ovulation, which involves an intricate process of dominant follicle selection followed by nuclear maturation, germinal vesicle (GV) breakdown, chromosomal arrangement, and completion of MI by extrusion of the first polar body, all of which occur concurrently with a not as well characterized process of cytoplasmic maturation (1). Accordingly, during IVM, these reciprocally choreographed cytoplasmic and nuclear processes must be properly managed by intrinsic and extrinsic environmental factors such as hormones, growth factors, and nutrients, to ensure faithful maturation. The advantages of IVM for clinical use include the decreased need or elimination of gonadotroReceived April 12, 2010; revised July 29, 2010; accepted August 13, 2010; published online September 23, 2010. H-J.L. has nothing to disclose. A.M.Q. has nothing to disclose. D.L.W. has nothing to disclose. T.L.T. has nothing to disclose. J.M.T. has nothing to disclose. Supported by a grant from the Harvard Stem Cell Institute and by the Vincent Memorial Research Fund (to JMT). Presented as an oral presentation at the 65th Annual Meeting of the American Society for Reproductive Medicine, which was held in Atlanta, GA, on October 17–21, 2009. Reprint requests: Jose M. Teixeira, Ph.D., Vincent Center of Reproductive Biology/Thier 931, Massachusetts General Hospital and Harvard Medical School, 55 Fruit Street, Boston, Massachusetts 02114 (E-mail: [email protected]).
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pin and GnRH agonist use, leading to decreased risks associated with controlled ovarian hyperstimulation (2). For patients with polycystic ovary syndrome who are anovulatory (3, 4) and for patients with a high risk of future infertility, such as young females undergoing aggressive gonadotoxic chemotherapy (5), IVM has been proposed as a possible component for preserving their fertility options (6). Recent reports have described the use of IVM for oocytes retrieved from antral follicles with subsequent successful fertilization and pregnancy (7, 8). However, the efficiency of human oocyte IVM is suboptimal when comparing outcomes with standard IVF approaches, which indicates that better methods for IVM are needed. One possible mechanism for improving IVM outcomes would be to use a coculture system. For example, mouse embryonic fibroblasts (MEFs) or mouse fibroblast cell lines are routinely used as feeder cells to derive and maintain embryonic stem cells. However, MEFs are not optimal for use with human embryonic stem cell (hESC) if they are ever to be used clinically owing to the risk of contamination with zoonotic pathogens (9). To avoid this risk, hESCs have been successfully derived and maintained on various feeder cells isolated from human fetal skin, muscle cells, fallopian tubal epithelial cells (10), foreskin fibroblasts (11, 12), placentaderived cells (13), and uterine endometrial cells (14). In addition, feeder layer-free culture systems have been shown to maintain hECSs, providing a safer alternative for clinical application of hECSs in the future (15–17). Coculture systems using various cell types have also been used in assisted reproduction. Coculturing early embryos with human
Fertility and Sterility Vol. 95, No. 4, March 15, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.08.034
granulosa cells or tubal or endometrial epithelial cells has been shown to improve the development of embryos, blastocyst formation, and pregnancy rates in both mice and humans (18–23), and human tubal epithelial cells (hTECs) have been shown to promote mouse embryo hatching in vitro (18, 24). Coculture and IVM have not been widely studied, but improved GV to MII transition by IVM with coculture of oocytes and cumulus granulosa cells has been reported, although it is not clear whether the coculture alone is sufficient to develop ‘‘usable’’ oocytes (25, 26). Despite the source, most cells used in coculture are derived from primary tissues and have a limited life span in culture. Oocytes and fertilized embryos spend many days in the fallopian tube, suggesting that the surrounding tubal cells provide a beneficial environment for - oocytes and for embryo development. An ideal method for IVM would be one that combines the benefits of the fallopian tube environment with the sustained, consistent culture conditions of immortalized cells. Here we show the generation and establishment of immortalized hTECs and compare maturation of GV oocytes in coculture with primary and immortalized hTECs.
MATERIALS AND METHODS Isolation and Immortalization of hTECs Human fallopian tube ampullary tissue was obtained in a Massachusetts General Hospital (MGH) Institutional Review Board (IRB)-approved study from consented premenopausal patients undergoing salpingectomy for gynecological conditions other than tubal disease. The hTECs were obtained from the ampullae by enzymatic digestion as described elsewhere (27). Briefly, the lumen of the fallopian tube was exposed and placed in center well organ culture dishes with culture media (Dulbecco’s minimum essential medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS), 1% insulin, transferrin, and selenium solution, 0.25% collagenase type IA (Invitrogen, Carlsbad, CA), and 10 U/mL DNase I (Sigma, St. Louis, MO) for 30–60 minutes in 5% CO2 at 37 C. After enzymatic digestion, the epithelial lining of the lumen was gently scraped off with a scalpel blade and the remaining tissue was discarded. The tubal cells suspension was diluted with an equal volume of culture medium and centrifuged twice at 300 g for 5 minutes at 4 C. The supernatant was then removed, and the pellet of tubal cells was resuspended in culture media and seeded in 25 cm2 flasks. Cultured media were changed every 3 days, and the cells were normally passaged every 5 days. For longterm storage, tubal cells were resuspended in DMEM/F12 supplemented with 10% FBS and 15% dimethyl sulfoxide, placed at 80 C overnight, and then stored in liquid nitrogen. Immortalized hTECs (ihTECs) were prepared by transduction with lentiviruses expressing human TERT and SV40 large T-antigen (28) and placed in selective media for a minimum of 3 days before culture as above for primary cells. MEFs (Invitrogen) were used as controls for coculture.
Collection of GV Oocytes GVoocytes were collected from mice in an MGH Institutional Animal Care and Use Committee-approved study. Female 6-week-old B6D2F1 mice (Charles River Laboratories, Wilmington, MA) were injected with 7.5 IU of pregnant mare’s serum gonadotropin (PMSG; Sigma) and were euthanized 40 hours later. Ovaries were removed and placed in human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS. Cumulus-oocyte complexes were obtained by puncturing the antral follicles, denuded by repeat pipetting using a stripper (Mid Atlantic Diagnostics, Marlton, NJ), and classified by GV stage. Unfertilized denuded human oocytes at the GV stage that would normally be discarded were collected from consented patients on standard IVF stimulation protocols whose MII oocytes were undergoing intracytoplasmic sperm injection in the MGH Fertility Center IVF laboratory using an MGH IRB-approved protocol. The oocytes were transferred to laboratory immediately and classified under the microscope. Oocytes with GV breakdown and atretic oocytes were discarded, and oocytes with intact GV were used in this study.
Fertility and Sterility
Coculture The day before oocyte collection, hTECs and ihTECs were thawed, washed, and resuspended in culture media and seeded at 8 104 cells/well in 4-well Nunc dishes (ThermoFisher Scientific, Waltham, MA). On the day of oocyte collection, the medium was changed to HTF with 10% FBS for murine oocytes or to HTF with 10% Synthetic Serum Substrate (Irvine Scientific) for human oocytes. GV-stage oocytes were cultured with culture media as control, primary hTECs, or ihTECs. Mouse oocytes were observed at 12 and 24 hours, and human oocytes were observed at 24 and 48 hours for oocyte maturation. Grading of oocytes was performed by microscopic examination in the standard fashion. Progression to the MII stage was identified by extrusion of the first polar body into the perivitelline space.
IVF and Development with Coculture IVF with mouse IVM oocytes was performed to assess their quality essentially as described elsewhere (29). Briefly, mouse sperm was obtained from caudal epididymides of 3-month-old males (B6D2F1), released into HTF media, and dispersed by incubating them at 37 C for 30 minutes. An appropriate volume of sperm suspension (1 106 sperm/mL final concentration) was transferred to each insemination drop and allowed to capacitate at 37 C for 1 hour before fertilization. IVM oocytes were transferred into the insemination drops containing capacitated sperm and incubated for 6 hours. Fertilized oocytes were washed several times through drops of HTF with 10% FBS and then transferred to plates seeded with MEFs, hTECs, or ihTECs for further coculture. Both IVF and subsequent culture of embryos were done at 37 C under a humidified atmosphere of 5% CO2. Fertilization was assessed by the presence of male and female pronuclei as well as cleavage to the two-cell stage at 24 hours after sperm insemination. Evaluation of embryonic development was then followed through to the blastocyst stage for 96 hours.
Statistical Analyses Data were analyzed by analysis of variance with the Tukey post hoc method for determining significant differences using Prism software (GraphPad Software, La Jolla, CA). P<.05 was considered statistically significant.
RESULTS IVM of Murine Oocytes The maturation rates of murine GV oocytes to the MII stage in control HTF medium or cocultured with MEFs, primary tubal cells (hTECs), and immortalized tubal cells (ihTECs) was compared (Fig. 1A). After 12 hours in culture, whereas approximately 17% of murine oocytes remained in the GV stage when incubated in control medium, only 9%, 4%, and 9% of the oocytes remained in the GV stage when cocultured with MEFs, hTECs, or ihTECs, respectively. By 24 hours, the majority of these 12-hour GV oocytes remained in GV. The number of oocytes undergoing GV breakdown was not significantly different between the control incubations and those incubated with coculture at 12 hours but was significantly lower at 24 hours in coculture with tubal cells compared with MEFs. Half the oocytes in HTF medium alone matured to MII within 12 hours, which was little improved with MEF coculture (54%). In contrast, a significantly higher percentage of the oocytes cocultured with hTECS and ihTECS, 70% and 63%, respectively, were MII by 12 hours compared with media alone. By 24 hours, the number of oocytes in MII increased in HTF to 64% but was still significantly lower than that of oocytes incubated with the hTECs and ihTECs, which also increased to 81% and 76%, respectively. Coculture with hTECs also significantly improved the MII maturation rate compared with MEFs at both 12 and 24 hours. In all cases, no statistically significant difference was ever observed between oocytes cocultured with hTECS and those incubated with ihTECs.
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FIGURE 1 IVM of GV-stage oocytes by coculture. (A) Immature GV oocytes were collected from PMSG-primed mice or (B) from human IVF patients and incubated in control HTF medium or cocultured with either MEFs, hTECs, or ihTECs as indicated. Murine oocytes (n ¼ 20 in each group, repeated 4–9 times; total ¼ 80–180 oocytes in each group) were examined after 12 and 24 hours in culture and scored for degree of maturation. Human oocytes (n ¼ 1–6 in each group, repeated 6–8 times; total ¼ 20–22 oocytes in each group) were cultured for 24 and 48 hours. The mean percent of oocytes observed in each stage is shown plotted with error bars that represent SEM. An ‘‘a’’ and/or ‘‘b’’ above bars indicates a significant difference (P< .05) when compared with control medium and/or MEFs, respectively.
observed that maturation to the MII stage was nearly four-fold higher when human oocytes were incubated in coculture with either hTECs (69%) or ihTECs (70%) compared with control medium (19%) at 24 hours and approximately 50% higher at 48 hours. There was no statistically significant difference in human oocyte maturation rates between oocyte coculture with hTECS or ihTECs. Additionally, two atretic oocytes were observed in the culture with control medium and one atretic oocyte was observed with ihTECs (data not shown). Samples of human oocytes from different stages of maturation are shown in Figure 2.
Embryonic Development of IVM Mouse Oocytes The fertilization rates and blastocyst development rates of the IVM oocytes after coculture were compared with HTF alone (Table 1). The fertilization and development rates of oocytes cocultured with MEFs were not significantly different than with HTF. Fertilization was significantly higher with coculture using hTECs but not ihTECs compared with either HTF or MEFs. The rate of development to blastocyst was significantly improved if IVM was performed with either hTECs or ihTECs compared with MEFs and HTF. These results indicate that the quality of oocytes that have undergone IVM in coculture with human tubal cells is significantly better than the quality of those that were matured with media alone or with MEFs. Additionally, both IVM and blastocyst development rates are significantly improved with human tubal cell coculture.
DISCUSSION
Lee. IVM with tubal epithelial cell coculture. Fertil Steril 2011.
IVM of Human Oocytes As we observed with murine oocytes, the number of human oocytes remaining in GVafter 24 hours of culture of GVoocytes was lower if the oocytes were cocultured with either hTECs or ihTECs (Fig. 1B). Although fewer GV oocytes were observed after 48 hours culture in all three culture conditions compared with after 24 hours, the number was still lower with coculture. Unlike murine oocytes and although not statistically significant, we consistently observed fewer human oocytes in GV breakdown with either coculture, particularly at the 24-hour time point. At 24 hours, 19% of the human GV oocytes had matured to the MII stage in HTF control medium. We
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IVM with tubal epithelial cell coculture
One obvious approach for oocyte maturation with improved oocyte quality would be to mimic its prefertilization in vivo environment with primary human tubal cells during IVM. However, important obstacles for using primary cells limit their implementation in coculture systems, primarily their limited life span and loss of cellular characteristics with each subsequent passage. Coculturing methods are used in assisted reproduction to improve embryo development and to increase clinical pregnancy rates for infertility patients because of the embryotrophic factors secreted by the various cell types used (30, 31). IVM medium has been supplemented with several factors, including growth factor and cytokines, FSH, LH, PMSG, and hCG. However, coculture systems for oocyte maturation have not been widely adopted. We have established immortalized hTECs with characteristics of primary tubal cells. These cells had the typical morphology of primary cells (data not shown) and can functionally substitute for primary hTECs in a coculture system to improve oocyte maturation rates and embryonic development rates after IVF over MEF or over medium alone. The length of incubation time during IVM with human immature oocytes varies between 24 and 52 hours (32), but oocytes that mature within 30 hours after retrieval have been shown to be developmentally more effective than those that require more time to mature (33). Also, the reported maturation rates of unstimulated oocytes with the shortest incubation times of 24–28 hours were generally in the 40%–60% range (32). Our data show that 70% of human GV stage oocyte cocultured with hTECs or ihTECs reached the MII stage within 24 hours, suggesting that fertility outcomes could be improved if IVM were performed with these coculture systems. However, important caveats remain, tempering any expectation of widespread, routine IVM. For example, retrieval from unstimulated patients is technically challenging and might be a probable limitation to any IVM protocol. Additionally, autologous tubal cells would not normally be available for IVM, which would require that any primary cells or cell lines be certified pathogen-free before use. Vol. 95, No. 4, March 15, 2011
FIGURE 2 Stages of human oocyte maturation in coculture. Collected oocytes are shown with GV (arrowheads) shortly after plating in (A) HTF control medium, (C) hTECs, and (E) ihTECS. MII oocytes with polar bodies (arrowheads) are observed after 24 hours of culture in (B) HTF, (D) hTECs, and (F) ihTECS.
Lee. IVM with tubal epithelial cell coculture. Fertil Steril 2011.
The advantages of oocyte IVM in assisted reproduction as an alternative to hormone stimulation for patients with polycystic ovary syndrome are clear, as is its use for those wishing to avoid ovarian hyperstimulation syndrome. There is, however, another population of women emerging who would also benefit from IVM: cancer patients about to undergo aggressive life-saving therapy who wish to preserve their fertility options (34). Over 1.3 million persons are diagnosed with cancer every year in the United States. Of these, 4%, or approximately 55,000, are under the age of 35, and the option of preserving their fertility should be considered when discussing their cancer treatment choices, particularly if permanent infertility or compromised fertility is a well-known side effect of the treatment. Additionally, standard IVF stimulation protocols might be contraindicated in patients with hormonally sensitive tumors. Developing Fertility and Sterility
TABLE 1 Developmental rate of IVM oocytes after IVF. Group (n [ 80/group) HTF MEF HTECs ihTECs
MII (% of total)
2-Cell (% of MII)
Blastocyst (% of MII)
54 (67.5) 55 (68.8) 68 (85.0)a 66 (82.5)a
26 (48.2) 28 (50.1) 41 (60.3)b 37 (56.1)
19 (35.2) 20 (36.4) 33 (48.5)c 30 (45.5)c
Note: Experiments were replicated 4 times. In each column, numbers without matching superscripts are statistically significant. P< .01. Lee. IVM with tubal epithelial cell coculture. Fertil Steril 2011.
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a widespread, safe, and effective method to extract oocytes from patients followed by IVM would allow these patients to produce embryos with their partners that can be frozen for later use. Alternatively, they might choose to store their IVM MII oocytes for future use as oocyte vitrification methods improve. It is unlikely that success rates of assisted reproductive technology (ART) with IVM will ever approach that of standard ART; however, for these patients an improvement in IVM methods should help mitigate their possible loss of fertility. Here we have described a method that improves oocyte IVM precisely with that intent.
In conclusion, this study shows that coculture with either primary or immortalized tubal epithelial cells improved IVM rates for murine and human GV oocytes, suggesting a promising new technique with future applications in clinical reproductive medicine or in basic science research. Further studies will be needed to elucidate the embryotrophic factors secreted by the hTECs and ihTECs and the mechanisms and pathways activated that support oocyte maturation. Acknowledgments: The authors thank Dr. Darrell R. Borger for his assistance with the immortalization of the hTECs.
REFERENCES 1. Picton HM, Harris SE, Muruvi W, Chambers EL. The in vitro growth and maturation of follicles. Reproduction (Cambridge, England) 2008;136:703–15. 2. Aboulghar M. Symposium: update on prediction and management of OHSS. Prevention of OHSS. Reprod Biomed Online 2009;19:33–42. 3. Cha KY, Lee DR, Cho JH, Yoon TK. In vitro maturation of immature oocytes and IVF/ICSI in PCOS patients. J Indian Med Assoc 2006;104:446. 8, 73. 4. Chian RC. In-vitro maturation of immature oocytes for infertile women with PCOS. Reprod Biomed Online 2004;8:547–52. 5. Maltaris T, Beckmann MW, Dittrich R. Review. Fertility preservation for young female cancer patients. In Vivo 2009;23:123–30. 6. Cao YX, Chian RC. Fertility preservation with immature and in vitro matured oocytes. Semin Reprod Med 2009;27:456–64. 7. Le Du A, Kadoch IJ, Bourcigaux N, Doumerc S, Bourrier MC, Chevalier N, et al. In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome: the French experience. Hum Reprod 2005;20:420–4. 8. Soderstrom-Anttila V, Makinen S, Tuuri T, Suikkari AM. Favourable pregnancy results with insemination of in vitro matured oocytes from unstimulated patients. Hum Reprod 2005;20:1534–40. 9. Chen HF, Chuang CY, Shieh YK, Chang HW, Ho HN, Kuo HC. Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions. Hum Reprod 2009;24:1114–25. 10. Richards M, Fong CY, Chan WK, Wong PC, Bongso A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotech 2002;20:933–6. 11. Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–9. 12. Unger C, Gao S, Cohen M, Jaconi M, Bergstrom R, Holm F, et al. Immortalized human skin fibroblast feeder cells support growth and maintenance of both human embryonic and induced pluripotent stem cells. Hum Reprod 2009;24:2567–81.
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13. Miyamoto K, Hayashi K, Suzuki T, Ichihara S, Yamada T, Kano Y, et al. Human placenta feeder layers support undifferentiated growth of primate embryonic stem cells. Stem cells (Dayton, Ohio) 2004;22:433–40. 14. Lee JB, Lee JE, Park JH, Kim SJ, Kim MK, Roh SI, et al. Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition. Biol Reprod 2005;72:42–9. 15. Amit M, Shariki C, Margulets V, Itskovitz-Eldor J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837–45. 16. Navarro-Alvarez N, Soto-Gutierrez A, Yuasa T, Yamatsuji T, Shirakawa Y, Nagasaka T, et al. Longterm culture of Japanese human embryonic stem cells in feeder-free conditions. Cell Trans 2008;17:27–33. 17. Wagner KE, Vemuri MC. Serum-free and feeder-free culture expansion of human embryonic stem cells. Meth Mol Biol 2010;584:109–19. 18. Bongso A, Ng SC, Fong CY, Anandakumar C, Marshall B, Edirisinghe R, et al. Improved pregnancy rate after transfer of embryos grown in human fallopian tubal cell coculture. Fertil Steril 1992;58:569–74. 19. Bongso A, Ng SC, Fong CY, Ratnam S. Cocultures: a new lead in embryo quality improvement for assisted reproduction. Fertil Steril 1991;56: 179–91. 20. Goldberg JM, Khalifa EA, Friedman CI, Kim MH. Improvement of in vitro fertilization and early embryo development in mice by coculture with human fallopian tube epithelium. Am J Obstet Gynecol 1991;165:1802–5. 21. Thibodeaux JK, Godke RA. In vitro enhancement of early-stage embryos with co-culture. Arch Pathol Lab Med 1992;116:364–72. 22. Barmat LI, Liu HC, Spandorfer SD, Xu K, Veeck L, Damario MA, et al. Human preembryo development on autologous endometrial coculture versus conventional medium. Fertil Steril 1998;70: 1109–13. 23. Spandorfer SD, Pascal P, Parks J, Clark R, Veeck L, Davis OK, et al. Autologous endometrial coculture in patients with IVF failure: outcome of the first 1,030 cases. J Reprod Med 2004;49:463–7.
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24. Frasor J, Sherbahn R, Soltes B, Molo MW, Binor Z, Radwanska E, et al. Optimizing tubal epithelial cell growth promotes mouse embryo hatching in coculture. J Assist Reprod Genet 1996;13:423–30. 25. Johnson JE, Higdon 3rd HL, Boone WR. Effect of human granulosa cell co-culture using standard culture media on the maturation and fertilization potential of immature human oocytes. Fertil Steril 2008;90:1674–9. 26. Eppig JJ. A comparison between oocyte growth in coculture with granulosa cells and oocytes with granulosa cell-oocyte junctional contact maintained in vitro. J Exp Zool 1979;209:345–53. 27. Levanon K, Ng V, Piao HY, Zhang Y, Chang MC, Roh MH, et al. Primary ex vivo cultures of human fallopian tube epithelium as a model for serous ovarian carcinogenesis. Oncogene 2010;29:1103–13. 28. Hahn WC, Dessain SK, Brooks MW, King JE, Elenbaas B, Sabatini DM, et al. Enumeration of the simian virus 40 early region elements necessary for human cell transformation. Mol Cell Biol 2002;22:2111–23. 29. Lee HJ, Elmoazzen H, Wright D, Biggers J, Rueda BR, Heo YS, et al. Ultra-rapid vitrification of mouse oocytes in low cryoprotectant concentrations. Reprod Biomed Online 2010;20:201–8. 30. Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update 1995;1: 91–148. 31. Simon C, Mercader A, Garcia-Velasco J, Nikas G, Moreno C, Remohi J, et al. Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure. J Clin Endocrinol Metab 1999;84:2638–46. 32. Jurema MW, Nogueira D. In vitro maturation of human oocytes for assisted reproduction. Fertil Steril 2006;86:1277–91. 33. Son WY, Lee SY, Lim JH. Fertilization, cleavage and blastocyst development according to the maturation timing of oocytes in in vitro maturation cycles. Hum Reprod 2005;20:3204–7. 34. Lee SJ, Schover LR, Partridge AH, Patrizio P, Wallace WH, Hagerty K, et al. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. J Clin Oncol 2006;24:2917–31.
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Objective: To improve the maturation rate of murine and human germinal vesicle (GV) oocytes using human tubal epithelial cells (hTECs). Design: Murine and human GV oocytes were randomized to human tubal fluid (HTF) media alone or cocultured with mouse embryonic fibroblasts (MEFs) or primary hTECs or immortalized hTECS (ihTECs) for various times. Rates of maturation to meiosis II (MII) were compared between groups. Intervention(s): TECs were isolated from discarded salpingectomy specimens. One batch was immortalized with TERT and SV40 large T-antigen. GVoocytes (n ¼ 710) were isolated from 8-week-old-mice at 40 hours after pregnant mare’s serum gonadotropin stimulation. Discarded human GV oocytes (n ¼ 62) were obtained from intracytoplasmic sperm injection cycle IVF center patients. Oocytes were cultured in HTF media alone or with MEFs, hTECs, or ihTECs. Main Outcome Measure(s): Maturation rates were assessed by standard morphological criteria and compared. Result(s): The maturation rate of murine GVoocytes to MII at 12 and 24 hours increased significantly in coculture with hTECS and ihTECS compared with MEF and HTF media alone. In addition, the development rate after IVF was significantly higher with hTECS and ihTECS than in MEF and HTF media alone. Maturation of human GV oocytes to MII at 24 and 48 hours was significantly higher in hTECS and ihTECS compared with HTF media alone. Conclusion(s): Coculture with either primary or immortalized TECs might improve oocyte quality and significantly raise in vitro maturation rates for GV oocytes. (Fertil Steril 2011;95:1344–8. 2011 by American Society for Reproductive Medicine.) Key Words: Oocytes, in vitro maturation, tubal feeder cells
In vitro maturation (IVM) of human and murine oocytes is an evolving technique that holds promise for use in both clinical and the basic laboratory settings. In vivo, human oocytes are arrested at prophase in meiosis I (MI) for 12–50 years before ovulation, which involves an intricate process of dominant follicle selection followed by nuclear maturation, germinal vesicle (GV) breakdown, chromosomal arrangement, and completion of MI by extrusion of the first polar body, all of which occur concurrently with a not as well characterized process of cytoplasmic maturation (1). Accordingly, during IVM, these reciprocally choreographed cytoplasmic and nuclear processes must be properly managed by intrinsic and extrinsic environmental factors such as hormones, growth factors, and nutrients, to ensure faithful maturation. The advantages of IVM for clinical use include the decreased need or elimination of gonadotroReceived April 12, 2010; revised July 29, 2010; accepted August 13, 2010; published online September 23, 2010. H-J.L. has nothing to disclose. A.M.Q. has nothing to disclose. D.L.W. has nothing to disclose. T.L.T. has nothing to disclose. J.M.T. has nothing to disclose. Supported by a grant from the Harvard Stem Cell Institute and by the Vincent Memorial Research Fund (to JMT). Presented as an oral presentation at the 65th Annual Meeting of the American Society for Reproductive Medicine, which was held in Atlanta, GA, on October 17–21, 2009. Reprint requests: Jose M. Teixeira, Ph.D., Vincent Center of Reproductive Biology/Thier 931, Massachusetts General Hospital and Harvard Medical School, 55 Fruit Street, Boston, Massachusetts 02114 (E-mail: [email protected]).
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pin and GnRH agonist use, leading to decreased risks associated with controlled ovarian hyperstimulation (2). For patients with polycystic ovary syndrome who are anovulatory (3, 4) and for patients with a high risk of future infertility, such as young females undergoing aggressive gonadotoxic chemotherapy (5), IVM has been proposed as a possible component for preserving their fertility options (6). Recent reports have described the use of IVM for oocytes retrieved from antral follicles with subsequent successful fertilization and pregnancy (7, 8). However, the efficiency of human oocyte IVM is suboptimal when comparing outcomes with standard IVF approaches, which indicates that better methods for IVM are needed. One possible mechanism for improving IVM outcomes would be to use a coculture system. For example, mouse embryonic fibroblasts (MEFs) or mouse fibroblast cell lines are routinely used as feeder cells to derive and maintain embryonic stem cells. However, MEFs are not optimal for use with human embryonic stem cell (hESC) if they are ever to be used clinically owing to the risk of contamination with zoonotic pathogens (9). To avoid this risk, hESCs have been successfully derived and maintained on various feeder cells isolated from human fetal skin, muscle cells, fallopian tubal epithelial cells (10), foreskin fibroblasts (11, 12), placentaderived cells (13), and uterine endometrial cells (14). In addition, feeder layer-free culture systems have been shown to maintain hECSs, providing a safer alternative for clinical application of hECSs in the future (15–17). Coculture systems using various cell types have also been used in assisted reproduction. Coculturing early embryos with human
Fertility and Sterility Vol. 95, No. 4, March 15, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.08.034
granulosa cells or tubal or endometrial epithelial cells has been shown to improve the development of embryos, blastocyst formation, and pregnancy rates in both mice and humans (18–23), and human tubal epithelial cells (hTECs) have been shown to promote mouse embryo hatching in vitro (18, 24). Coculture and IVM have not been widely studied, but improved GV to MII transition by IVM with coculture of oocytes and cumulus granulosa cells has been reported, although it is not clear whether the coculture alone is sufficient to develop ‘‘usable’’ oocytes (25, 26). Despite the source, most cells used in coculture are derived from primary tissues and have a limited life span in culture. Oocytes and fertilized embryos spend many days in the fallopian tube, suggesting that the surrounding tubal cells provide a beneficial environment for - oocytes and for embryo development. An ideal method for IVM would be one that combines the benefits of the fallopian tube environment with the sustained, consistent culture conditions of immortalized cells. Here we show the generation and establishment of immortalized hTECs and compare maturation of GV oocytes in coculture with primary and immortalized hTECs.
MATERIALS AND METHODS Isolation and Immortalization of hTECs Human fallopian tube ampullary tissue was obtained in a Massachusetts General Hospital (MGH) Institutional Review Board (IRB)-approved study from consented premenopausal patients undergoing salpingectomy for gynecological conditions other than tubal disease. The hTECs were obtained from the ampullae by enzymatic digestion as described elsewhere (27). Briefly, the lumen of the fallopian tube was exposed and placed in center well organ culture dishes with culture media (Dulbecco’s minimum essential medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS), 1% insulin, transferrin, and selenium solution, 0.25% collagenase type IA (Invitrogen, Carlsbad, CA), and 10 U/mL DNase I (Sigma, St. Louis, MO) for 30–60 minutes in 5% CO2 at 37 C. After enzymatic digestion, the epithelial lining of the lumen was gently scraped off with a scalpel blade and the remaining tissue was discarded. The tubal cells suspension was diluted with an equal volume of culture medium and centrifuged twice at 300 g for 5 minutes at 4 C. The supernatant was then removed, and the pellet of tubal cells was resuspended in culture media and seeded in 25 cm2 flasks. Cultured media were changed every 3 days, and the cells were normally passaged every 5 days. For longterm storage, tubal cells were resuspended in DMEM/F12 supplemented with 10% FBS and 15% dimethyl sulfoxide, placed at 80 C overnight, and then stored in liquid nitrogen. Immortalized hTECs (ihTECs) were prepared by transduction with lentiviruses expressing human TERT and SV40 large T-antigen (28) and placed in selective media for a minimum of 3 days before culture as above for primary cells. MEFs (Invitrogen) were used as controls for coculture.
Collection of GV Oocytes GVoocytes were collected from mice in an MGH Institutional Animal Care and Use Committee-approved study. Female 6-week-old B6D2F1 mice (Charles River Laboratories, Wilmington, MA) were injected with 7.5 IU of pregnant mare’s serum gonadotropin (PMSG; Sigma) and were euthanized 40 hours later. Ovaries were removed and placed in human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS. Cumulus-oocyte complexes were obtained by puncturing the antral follicles, denuded by repeat pipetting using a stripper (Mid Atlantic Diagnostics, Marlton, NJ), and classified by GV stage. Unfertilized denuded human oocytes at the GV stage that would normally be discarded were collected from consented patients on standard IVF stimulation protocols whose MII oocytes were undergoing intracytoplasmic sperm injection in the MGH Fertility Center IVF laboratory using an MGH IRB-approved protocol. The oocytes were transferred to laboratory immediately and classified under the microscope. Oocytes with GV breakdown and atretic oocytes were discarded, and oocytes with intact GV were used in this study.
Fertility and Sterility
Coculture The day before oocyte collection, hTECs and ihTECs were thawed, washed, and resuspended in culture media and seeded at 8 104 cells/well in 4-well Nunc dishes (ThermoFisher Scientific, Waltham, MA). On the day of oocyte collection, the medium was changed to HTF with 10% FBS for murine oocytes or to HTF with 10% Synthetic Serum Substrate (Irvine Scientific) for human oocytes. GV-stage oocytes were cultured with culture media as control, primary hTECs, or ihTECs. Mouse oocytes were observed at 12 and 24 hours, and human oocytes were observed at 24 and 48 hours for oocyte maturation. Grading of oocytes was performed by microscopic examination in the standard fashion. Progression to the MII stage was identified by extrusion of the first polar body into the perivitelline space.
IVF and Development with Coculture IVF with mouse IVM oocytes was performed to assess their quality essentially as described elsewhere (29). Briefly, mouse sperm was obtained from caudal epididymides of 3-month-old males (B6D2F1), released into HTF media, and dispersed by incubating them at 37 C for 30 minutes. An appropriate volume of sperm suspension (1 106 sperm/mL final concentration) was transferred to each insemination drop and allowed to capacitate at 37 C for 1 hour before fertilization. IVM oocytes were transferred into the insemination drops containing capacitated sperm and incubated for 6 hours. Fertilized oocytes were washed several times through drops of HTF with 10% FBS and then transferred to plates seeded with MEFs, hTECs, or ihTECs for further coculture. Both IVF and subsequent culture of embryos were done at 37 C under a humidified atmosphere of 5% CO2. Fertilization was assessed by the presence of male and female pronuclei as well as cleavage to the two-cell stage at 24 hours after sperm insemination. Evaluation of embryonic development was then followed through to the blastocyst stage for 96 hours.
Statistical Analyses Data were analyzed by analysis of variance with the Tukey post hoc method for determining significant differences using Prism software (GraphPad Software, La Jolla, CA). P<.05 was considered statistically significant.
RESULTS IVM of Murine Oocytes The maturation rates of murine GV oocytes to the MII stage in control HTF medium or cocultured with MEFs, primary tubal cells (hTECs), and immortalized tubal cells (ihTECs) was compared (Fig. 1A). After 12 hours in culture, whereas approximately 17% of murine oocytes remained in the GV stage when incubated in control medium, only 9%, 4%, and 9% of the oocytes remained in the GV stage when cocultured with MEFs, hTECs, or ihTECs, respectively. By 24 hours, the majority of these 12-hour GV oocytes remained in GV. The number of oocytes undergoing GV breakdown was not significantly different between the control incubations and those incubated with coculture at 12 hours but was significantly lower at 24 hours in coculture with tubal cells compared with MEFs. Half the oocytes in HTF medium alone matured to MII within 12 hours, which was little improved with MEF coculture (54%). In contrast, a significantly higher percentage of the oocytes cocultured with hTECS and ihTECS, 70% and 63%, respectively, were MII by 12 hours compared with media alone. By 24 hours, the number of oocytes in MII increased in HTF to 64% but was still significantly lower than that of oocytes incubated with the hTECs and ihTECs, which also increased to 81% and 76%, respectively. Coculture with hTECs also significantly improved the MII maturation rate compared with MEFs at both 12 and 24 hours. In all cases, no statistically significant difference was ever observed between oocytes cocultured with hTECS and those incubated with ihTECs.
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FIGURE 1 IVM of GV-stage oocytes by coculture. (A) Immature GV oocytes were collected from PMSG-primed mice or (B) from human IVF patients and incubated in control HTF medium or cocultured with either MEFs, hTECs, or ihTECs as indicated. Murine oocytes (n ¼ 20 in each group, repeated 4–9 times; total ¼ 80–180 oocytes in each group) were examined after 12 and 24 hours in culture and scored for degree of maturation. Human oocytes (n ¼ 1–6 in each group, repeated 6–8 times; total ¼ 20–22 oocytes in each group) were cultured for 24 and 48 hours. The mean percent of oocytes observed in each stage is shown plotted with error bars that represent SEM. An ‘‘a’’ and/or ‘‘b’’ above bars indicates a significant difference (P< .05) when compared with control medium and/or MEFs, respectively.
observed that maturation to the MII stage was nearly four-fold higher when human oocytes were incubated in coculture with either hTECs (69%) or ihTECs (70%) compared with control medium (19%) at 24 hours and approximately 50% higher at 48 hours. There was no statistically significant difference in human oocyte maturation rates between oocyte coculture with hTECS or ihTECs. Additionally, two atretic oocytes were observed in the culture with control medium and one atretic oocyte was observed with ihTECs (data not shown). Samples of human oocytes from different stages of maturation are shown in Figure 2.
Embryonic Development of IVM Mouse Oocytes The fertilization rates and blastocyst development rates of the IVM oocytes after coculture were compared with HTF alone (Table 1). The fertilization and development rates of oocytes cocultured with MEFs were not significantly different than with HTF. Fertilization was significantly higher with coculture using hTECs but not ihTECs compared with either HTF or MEFs. The rate of development to blastocyst was significantly improved if IVM was performed with either hTECs or ihTECs compared with MEFs and HTF. These results indicate that the quality of oocytes that have undergone IVM in coculture with human tubal cells is significantly better than the quality of those that were matured with media alone or with MEFs. Additionally, both IVM and blastocyst development rates are significantly improved with human tubal cell coculture.
DISCUSSION
Lee. IVM with tubal epithelial cell coculture. Fertil Steril 2011.
IVM of Human Oocytes As we observed with murine oocytes, the number of human oocytes remaining in GVafter 24 hours of culture of GVoocytes was lower if the oocytes were cocultured with either hTECs or ihTECs (Fig. 1B). Although fewer GV oocytes were observed after 48 hours culture in all three culture conditions compared with after 24 hours, the number was still lower with coculture. Unlike murine oocytes and although not statistically significant, we consistently observed fewer human oocytes in GV breakdown with either coculture, particularly at the 24-hour time point. At 24 hours, 19% of the human GV oocytes had matured to the MII stage in HTF control medium. We
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IVM with tubal epithelial cell coculture
One obvious approach for oocyte maturation with improved oocyte quality would be to mimic its prefertilization in vivo environment with primary human tubal cells during IVM. However, important obstacles for using primary cells limit their implementation in coculture systems, primarily their limited life span and loss of cellular characteristics with each subsequent passage. Coculturing methods are used in assisted reproduction to improve embryo development and to increase clinical pregnancy rates for infertility patients because of the embryotrophic factors secreted by the various cell types used (30, 31). IVM medium has been supplemented with several factors, including growth factor and cytokines, FSH, LH, PMSG, and hCG. However, coculture systems for oocyte maturation have not been widely adopted. We have established immortalized hTECs with characteristics of primary tubal cells. These cells had the typical morphology of primary cells (data not shown) and can functionally substitute for primary hTECs in a coculture system to improve oocyte maturation rates and embryonic development rates after IVF over MEF or over medium alone. The length of incubation time during IVM with human immature oocytes varies between 24 and 52 hours (32), but oocytes that mature within 30 hours after retrieval have been shown to be developmentally more effective than those that require more time to mature (33). Also, the reported maturation rates of unstimulated oocytes with the shortest incubation times of 24–28 hours were generally in the 40%–60% range (32). Our data show that 70% of human GV stage oocyte cocultured with hTECs or ihTECs reached the MII stage within 24 hours, suggesting that fertility outcomes could be improved if IVM were performed with these coculture systems. However, important caveats remain, tempering any expectation of widespread, routine IVM. For example, retrieval from unstimulated patients is technically challenging and might be a probable limitation to any IVM protocol. Additionally, autologous tubal cells would not normally be available for IVM, which would require that any primary cells or cell lines be certified pathogen-free before use. Vol. 95, No. 4, March 15, 2011
FIGURE 2 Stages of human oocyte maturation in coculture. Collected oocytes are shown with GV (arrowheads) shortly after plating in (A) HTF control medium, (C) hTECs, and (E) ihTECS. MII oocytes with polar bodies (arrowheads) are observed after 24 hours of culture in (B) HTF, (D) hTECs, and (F) ihTECS.
Lee. IVM with tubal epithelial cell coculture. Fertil Steril 2011.
The advantages of oocyte IVM in assisted reproduction as an alternative to hormone stimulation for patients with polycystic ovary syndrome are clear, as is its use for those wishing to avoid ovarian hyperstimulation syndrome. There is, however, another population of women emerging who would also benefit from IVM: cancer patients about to undergo aggressive life-saving therapy who wish to preserve their fertility options (34). Over 1.3 million persons are diagnosed with cancer every year in the United States. Of these, 4%, or approximately 55,000, are under the age of 35, and the option of preserving their fertility should be considered when discussing their cancer treatment choices, particularly if permanent infertility or compromised fertility is a well-known side effect of the treatment. Additionally, standard IVF stimulation protocols might be contraindicated in patients with hormonally sensitive tumors. Developing Fertility and Sterility
TABLE 1 Developmental rate of IVM oocytes after IVF. Group (n [ 80/group) HTF MEF HTECs ihTECs
MII (% of total)
2-Cell (% of MII)
Blastocyst (% of MII)
54 (67.5) 55 (68.8) 68 (85.0)a 66 (82.5)a
26 (48.2) 28 (50.1) 41 (60.3)b 37 (56.1)
19 (35.2) 20 (36.4) 33 (48.5)c 30 (45.5)c
Note: Experiments were replicated 4 times. In each column, numbers without matching superscripts are statistically significant. P< .01. Lee. IVM with tubal epithelial cell coculture. Fertil Steril 2011.
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a widespread, safe, and effective method to extract oocytes from patients followed by IVM would allow these patients to produce embryos with their partners that can be frozen for later use. Alternatively, they might choose to store their IVM MII oocytes for future use as oocyte vitrification methods improve. It is unlikely that success rates of assisted reproductive technology (ART) with IVM will ever approach that of standard ART; however, for these patients an improvement in IVM methods should help mitigate their possible loss of fertility. Here we have described a method that improves oocyte IVM precisely with that intent.
In conclusion, this study shows that coculture with either primary or immortalized tubal epithelial cells improved IVM rates for murine and human GV oocytes, suggesting a promising new technique with future applications in clinical reproductive medicine or in basic science research. Further studies will be needed to elucidate the embryotrophic factors secreted by the hTECs and ihTECs and the mechanisms and pathways activated that support oocyte maturation. Acknowledgments: The authors thank Dr. Darrell R. Borger for his assistance with the immortalization of the hTECs.
REFERENCES 1. Picton HM, Harris SE, Muruvi W, Chambers EL. The in vitro growth and maturation of follicles. Reproduction (Cambridge, England) 2008;136:703–15. 2. Aboulghar M. Symposium: update on prediction and management of OHSS. Prevention of OHSS. Reprod Biomed Online 2009;19:33–42. 3. Cha KY, Lee DR, Cho JH, Yoon TK. In vitro maturation of immature oocytes and IVF/ICSI in PCOS patients. J Indian Med Assoc 2006;104:446. 8, 73. 4. Chian RC. In-vitro maturation of immature oocytes for infertile women with PCOS. Reprod Biomed Online 2004;8:547–52. 5. Maltaris T, Beckmann MW, Dittrich R. Review. Fertility preservation for young female cancer patients. In Vivo 2009;23:123–30. 6. Cao YX, Chian RC. Fertility preservation with immature and in vitro matured oocytes. Semin Reprod Med 2009;27:456–64. 7. Le Du A, Kadoch IJ, Bourcigaux N, Doumerc S, Bourrier MC, Chevalier N, et al. In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome: the French experience. Hum Reprod 2005;20:420–4. 8. Soderstrom-Anttila V, Makinen S, Tuuri T, Suikkari AM. Favourable pregnancy results with insemination of in vitro matured oocytes from unstimulated patients. Hum Reprod 2005;20:1534–40. 9. Chen HF, Chuang CY, Shieh YK, Chang HW, Ho HN, Kuo HC. Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions. Hum Reprod 2009;24:1114–25. 10. Richards M, Fong CY, Chan WK, Wong PC, Bongso A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotech 2002;20:933–6. 11. Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–9. 12. Unger C, Gao S, Cohen M, Jaconi M, Bergstrom R, Holm F, et al. Immortalized human skin fibroblast feeder cells support growth and maintenance of both human embryonic and induced pluripotent stem cells. Hum Reprod 2009;24:2567–81.
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Lee et al.
13. Miyamoto K, Hayashi K, Suzuki T, Ichihara S, Yamada T, Kano Y, et al. Human placenta feeder layers support undifferentiated growth of primate embryonic stem cells. Stem cells (Dayton, Ohio) 2004;22:433–40. 14. Lee JB, Lee JE, Park JH, Kim SJ, Kim MK, Roh SI, et al. Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition. Biol Reprod 2005;72:42–9. 15. Amit M, Shariki C, Margulets V, Itskovitz-Eldor J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837–45. 16. Navarro-Alvarez N, Soto-Gutierrez A, Yuasa T, Yamatsuji T, Shirakawa Y, Nagasaka T, et al. Longterm culture of Japanese human embryonic stem cells in feeder-free conditions. Cell Trans 2008;17:27–33. 17. Wagner KE, Vemuri MC. Serum-free and feeder-free culture expansion of human embryonic stem cells. Meth Mol Biol 2010;584:109–19. 18. Bongso A, Ng SC, Fong CY, Anandakumar C, Marshall B, Edirisinghe R, et al. Improved pregnancy rate after transfer of embryos grown in human fallopian tubal cell coculture. Fertil Steril 1992;58:569–74. 19. Bongso A, Ng SC, Fong CY, Ratnam S. Cocultures: a new lead in embryo quality improvement for assisted reproduction. Fertil Steril 1991;56: 179–91. 20. Goldberg JM, Khalifa EA, Friedman CI, Kim MH. Improvement of in vitro fertilization and early embryo development in mice by coculture with human fallopian tube epithelium. Am J Obstet Gynecol 1991;165:1802–5. 21. Thibodeaux JK, Godke RA. In vitro enhancement of early-stage embryos with co-culture. Arch Pathol Lab Med 1992;116:364–72. 22. Barmat LI, Liu HC, Spandorfer SD, Xu K, Veeck L, Damario MA, et al. Human preembryo development on autologous endometrial coculture versus conventional medium. Fertil Steril 1998;70: 1109–13. 23. Spandorfer SD, Pascal P, Parks J, Clark R, Veeck L, Davis OK, et al. Autologous endometrial coculture in patients with IVF failure: outcome of the first 1,030 cases. J Reprod Med 2004;49:463–7.
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24. Frasor J, Sherbahn R, Soltes B, Molo MW, Binor Z, Radwanska E, et al. Optimizing tubal epithelial cell growth promotes mouse embryo hatching in coculture. J Assist Reprod Genet 1996;13:423–30. 25. Johnson JE, Higdon 3rd HL, Boone WR. Effect of human granulosa cell co-culture using standard culture media on the maturation and fertilization potential of immature human oocytes. Fertil Steril 2008;90:1674–9. 26. Eppig JJ. A comparison between oocyte growth in coculture with granulosa cells and oocytes with granulosa cell-oocyte junctional contact maintained in vitro. J Exp Zool 1979;209:345–53. 27. Levanon K, Ng V, Piao HY, Zhang Y, Chang MC, Roh MH, et al. Primary ex vivo cultures of human fallopian tube epithelium as a model for serous ovarian carcinogenesis. Oncogene 2010;29:1103–13. 28. Hahn WC, Dessain SK, Brooks MW, King JE, Elenbaas B, Sabatini DM, et al. Enumeration of the simian virus 40 early region elements necessary for human cell transformation. Mol Cell Biol 2002;22:2111–23. 29. Lee HJ, Elmoazzen H, Wright D, Biggers J, Rueda BR, Heo YS, et al. Ultra-rapid vitrification of mouse oocytes in low cryoprotectant concentrations. Reprod Biomed Online 2010;20:201–8. 30. Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update 1995;1: 91–148. 31. Simon C, Mercader A, Garcia-Velasco J, Nikas G, Moreno C, Remohi J, et al. Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure. J Clin Endocrinol Metab 1999;84:2638–46. 32. Jurema MW, Nogueira D. In vitro maturation of human oocytes for assisted reproduction. Fertil Steril 2006;86:1277–91. 33. Son WY, Lee SY, Lim JH. Fertilization, cleavage and blastocyst development according to the maturation timing of oocytes in in vitro maturation cycles. Hum Reprod 2005;20:3204–7. 34. Lee SJ, Schover LR, Partridge AH, Patrizio P, Wallace WH, Hagerty K, et al. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. J Clin Oncol 2006;24:2917–31.
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