Department of Reproductive Biology, Univ. MacDonald Women's Hospital, Case Western Reserve University, 11100 Euclid Av., Cleveland, OH 44106, USA
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Abstract |
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Key words: blastocyst/co-culture/extracellular matrix/growth factor/Matrigel
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Introduction |
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The potential negatives of co-culture during human IVF are exposure of embryos to animal cells/proteins and the possibility of disease transmission. In addition, the co-culture technique is fairly labour intensive and requires tissue culture expertise, and the use of dividing cells can potentially introduce considerable variability into the embryo culture system (Desai and Goldfarb, 1998).
For the above reasons, an alternative to co-culture is highly desirable. Data from several investigations (Carnegie et al., 1995; Piekos et al., 1995
) indicate that cultivation of embryos on basement membrane protein coated substrates can enhance aspects of early embryonic development. Culture supplementation with specific growth factors/cytokines may be another approach to further mimic the co-culture environment and influence in-vitro development (Dunglison et al., 1996
).
In this study, the potential use of human extracellular matrix (HECM) proteins isolated from human placenta was investigated as an alternative culture system for embryo development. The effect of exogenous interleukin-6 (IL-6) supplementation on embryo growth was also studied. This cytokine has been identified in embryo culture supernatants (Murray et al., 1990; Austgulen et al., 1995
) and is also secreted at high concentrations by cell lines with embryotrophic properties (Desai and Goldfarb, 1996
, 1998
). Yet little is known about this cytokine and its interaction with the preimplantation embryo. Subsequent to the completion of the initial testing on mouse IVF embryos, a pilot study was conducted to determine if this culture system could be applied to human embryos.
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Materials and methods |
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Minimum essential medium alpha (-MEM; Gibco, Grand Island, NY, USA) supplemented with a plasma protein preparation (5%; Cutter Biological, Elkhart, IN, USA) was utilized for these experiments. Culture medium was supplemented with IL-6 (Biosource; Camarillo, CA, USA) and tested at two concentrations, 500 and 1000 pg/ml. Human extracellular matrix (HECM; Collaborative; Bedford, MA, USA) was coated onto dishes at a concentration of 5.0 or 10.0 µg/ml. Matrix was thawed on ice and pipetted into organ culture dishes. Dishes were allowed to set for 2 h at room temperature before rinsing with culture medium. Control dishes contained protein-supplemented medium alone.
Six to eight week old female CB6F1 mice purchased from Charles River (Wilmington, MA, USA) were superovulated to obtain oocytes. Animals were injected with 7.5 IU (i.p.) of pregnant mare serum gonadotrophin (Sigma, St. Louis, MO, USA). A second injection, 10 IU of human chorionic gonadotrophin (HCG) (Sigma), was given 48 h later. Mice were killed 1618 h post-HCG injection by cervical dislocation and their oviducts were excised. Cumuli containing unfertilized oocytes were isolated and placed in organ culture dishes containing pre-equilibrated medium. Dishes were incubated at 37°C in a humidified incubator with 5% CO2. Spermatozoa isolated from the epididymis of a male CB6F1 mouse (810 weeks) were used for oocyte insemination.
Fertilization was determined the following morning by assessing cleavage to the 2-cell stage. The 2-cell embryos were pooled and randomly distributed amongst control and treatment dishes, and incubated at 37°C with 5% CO2. At 96 h post-insemination, the percentage of 2-cell embryos undergoing blastocyst transformation and hatching was calculated for each treatment group.
In the last experiment, HECM and IL-6 treatment were combined. Each treatment was tested at its maximal concentration (10 µg/ml and 1000 pg/ml, respectively). At termination of the experiment, blastocysts from each treatment group were stained using the Hoechst nuclear staining protocol (Handyside and Hunter, 1984). Cell nuclei were counted with the aid of a computer based image analysis system. The total cell number per blastocyst was recorded.
Human embryo development
A pilot study of this culture system with human embryos was conducted using embryos arising from abnormally fertilized tripronucleate eggs from our IVF programme. The use of these triploid embryos for research was approved by our institutional review board. Dishes were coated with 1000 µg/ml of HECM, as described earlier. The basal culture medium was -MEM with 5% plasma protein preparation. This was further supplemented with IL-6 at a concentration of 1000 pg/ml. Triploid embryos were placed in coated dishes containing the test medium on the day after fertilization. Dishes were incubated at 37°C with 5% CO2. Development and morphology of embryos was monitored daily over a 5 day interval.
Statistical analysis
Differences in the rates of blastulation and hatching between treatment groups were analysed by the 2 test with the Yates correction to determine statistical significance. The Student's t-test was utilized to compare the mean cell numbers per blastocyst between treatment groups. Values of P < 0.05 were reported as being statistically significant.
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Results |
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Discussion |
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Embryo development on fibronectin coated dishes has been compared to that in an oviduct co-culture system (Piekos et al., 1995). Blastocyst development was similar in both treatments and significantly greater than in the control group. Yet hatching and the total cell number per embryo was still greater in the co-cultured blastocysts, suggesting that fibronectin coating alone was not an adequate substitute for co-culture. Matrigel, a commercially available extracellular matrix product, containing collagen IV, laminin, entactin and proteoglycans isolated from mouse tumour cells has also been tested (Carnegie et al., 1995
). Development of preimplantation embryos cultivated in conventional culture was contrasted to that of embryos grown on either Matrigel or on Vero cell monolayers. With both matrix and co-culture, in-vitro blastocyst formation and hatching were significantly enhanced over that observed in the medium-alone control. These investigators concluded that Matrigel might be an acceptable alternative to co-culture. Contradictory findings have been reported (Dawson et al., 1997
). Their data in fact suggest that the extracellular matrix product might be inhibitory. The variable nature of commercial ECM products may in part account for these findings.
In the present study, an extracellular matrix product isolated from human placenta was chosen. To our knowledge, embryo growth on placenta-derived matrix proteins has never before been tested. It was felt that an extracellular matrix similar in composition to Matrigel but of non-tumour origin might be more desirable should this product later be considered for introduction into the clinical laboratory for human embryo culture. Extracellular matrix was not found to be inhibitory to either mouse or human embryos. The observed positive effect of HECM on mouse blastocyst formation may be related in part to the low concentrations of growth factors associated with the matrix components. While the specific growth factor profile of the placenta-derived HECM has not yet been determined, Matrigel has been shown to contain variable amounts of epidermal growth factor (EGF) (0.51.3 ng/ml), platelet-derived growth factor (PDGF) (548 pg/ml), insulin-like growth factor-I (IGF-I) (1124 ng/ml) and transforming growth factor-ß (TGFß) (1.74.7 ng/ml) (Mannuzza, 1994). All of these identified factors can influence specific aspects of embryonic development. Cell metabolism and cell proliferation, especially in the inner cell mass of the developing blastocyst, have been shown to be stimulated by IGF-I (Smith et al., 1993
). Addition of EGF or TGFß to mouse embryo culture media enhances in-vitro blastulation (Paria and Dey, 1990
).
The most pronounced effect of the HECM was on the hatching rate. One explanation is that the zona hardening phenomenon often associated with in-vitro fertilized embryos is attenuated by culture on a more physiological substratum. Co-culture systems likely provide a similar advantage. Studies with both in-vivo and in-vitro fertilized mouse zygotes have shown that cultivation of embryos on cell monolayers enhances blastocyst hatching (Lai et al., 1992; Desai et al., 1994
; Piekos et al., 1995
). Human embryos cultivated on bovine oviductal cell monolayers exhibit more zona thickness variation than embryos grown directly on conventional tissue culture dishes (Morgan et al., 1995
). This non-uniformity in zona thickness could potentially facilitate embryonic hatching.
Protease activity in the early embryo can also be affected by culture substrata and extracellular matrix components. Embryonic secretion of basement membrane degrading proteases, such as urokinase plasminogen activator and type IV collagenase, is believed to be crucial in the implantation process, allowing penetration of the embryo through the decidua (Turpeenniemi-Hujanen et al., 1995). The effect of extracellular matrix components, fibronectin, collagen type IV and laminin on trophectodermal and inner cell mass cells isolated from sheep blastocysts has been examined (Bartlett and Menino, 1995
). The type of ECM was shown directly to influence total cellular outgrowth and secretion of urokinase plasminogen activator. Higher collagenase type IV activity when trophoblasts were cultured with laminin versus on plastic substrata was measured (Emonard et al., 1990
). In human embryo cultures, type IV collagenase activity was shown to peak, just prior to implantation and to decrease thereafter (Turpeenniemi-Hujanen et al., 1992
). Elevated type IV collagenase activity could be sustained by addition of laminin to the culture system. Laminin also promoted in-vitro hatching of cultured human embryos (Turpeenniemi-Hujanen et al., 1995
). All of these data clearly suggest that incorporation of ECM components into an embryo culture system may serve to augment the implantation process, allowing the embryo to continue with its `differentiated functions' (e.g. protease secretion) in the in-vitro environment and later upon transfer.
We initially became interested in understanding the role of IL-6 during embryo development because of work in our laboratory characterizing growth factor/cytokine secretion by the Vero cell line used for human embryo co-culture (Desai and Goldfarb et al., 1998). Of the factors secreted, IL-6 was measured in patient co-culture supernatants at relatively high concentrations (3001000 pg/ml). It was felt important to clarify further the interaction of this factor with the developing embryo. Studies demonstrating the presence of this cytokine in human and mouse embryo culture fluids, along with the detection of mRNA transcripts for IL-6 in mouse blastocysts, all point to an autocrine role for this factor (Murray et al., 1990
; Austgulen et al., 1995
). In addition, IL-6 shares a common signal transducer molecule with leukaemia inhibitory factor (LIF), a molecule identified to play an important role in the implantation process in mouse (Gearing et al., 1992
). Both factors are present in the uterus at elevated concentrations just prior to implantation (Charnock-Jones et al., 1994
; Tabibzadeh et al., 1995
). IL-6 secretion by endometrial cells is apparently stimulated by interferon-
(IFN-
), another immunomodulator found to be critical for pregnancy (Nasu et al., 1998
).
Results from this study indicate that the development of in-vitro fertilized oocytes to the hatched blastocyst stage can be modulated by addition of IL-6 to the culture medium. This finding is the first direct evidence of IL-6 influence on preimplantation embryonic development and will require further study. The combination of IL-6 supplementation with extracellular matrix coating of dishes resulted in a culture system that distinctly enhanced at least three parameters of embryonic quality, namely blastocyst formation, hatching, and total cell number per blastocyst. The latter parameter is of especial importance since it is often compromised in embryos developing to blastocysts under suboptimal conditions. Deficiencies in overall blastomere number may hinder proper allocation of cells to the inner cell mass, thereby impairing subsequent development.
Compared to the murine model, human embryo cultivation to the blastocyst stage and production of healthy offspring has been much more of a challenge. Until recently, the best method for obtaining high quality human blastocysts was through the utilization of co-culture techniques. The availability of newer media formulations that better support human blastocyst development may, however, completely eliminate the necessity of co-culture (Forsdahl et al., 1994; Gardner et al., 1998
). While initial trials in these new serum-free blastocyst culture media look promising, it still remains to be seen if these media alone can sufficiently support the embryos of all categories of patients. The combination of blastocyst culture media with growth factor and ECM components may prove to be a new avenue for formulating more physiological culture systems.
In summary, the data presented in this study suggest that the design of a culture system for in-vitro embryo development needs not only to examine the basal medium and nutrient requirement of embryos at various cell stages but should also consider the choice of culture substrata and the effect of growth promoting factors. As IVF clinics consider shifting to day 5 transfers with blastocysts, the in-vitro environment will be likely to impact even more on successful pregnancy outcomes. Implantation failure after human IVF may be related to zona hardening and the subsequent inability of transferred embryos to hatch appropriately. This study has identified two culture components that have a striking effect on embryonic hatching, namely IL-6 and human extracellular matrix proteins. In combination, these factors also promoted cell proliferation in the developing embryo.
Despite the positive results presented in this study, it is recognized that contaminating factors associated with the commercial ECM products may ultimately limit their clinical application. Growth factors associated with matrix products can act as either activators or inhibitors, thereby having contradictory effects on differentiation of the blastocyst inner cell mass and trophectodermal compartments. Additional testing of individual matrix components in combination with a variety of growth factors and cytokines should therefore be conducted with the goal of optimizing culture strategies and media formulations. Future studies should include analysis of cell distribution between the trophectoderm and inner cell mass with each treatment. This may ultimately aid in understanding the nature of the interaction between the embryo, the extracellular matrix and associated growth factors.
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Notes |
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References |
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Submitted on December 7, 1998; accepted on March 4, 1999.