Department of Reproductive Biology, University MacDonald Women's Hospital, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106, USA
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Abstract |
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Key words: blastocyst/co-culture/cryopreservation/EGF/growth factors/IGF
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Introduction |
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Data supporting a vital role for growth factors during early embryonic development have been steadily accumulating (reviewed by Adamson, 1993; Kane et al., 1997; O'Neill, 1998). Indirect evidence for the beneficial effect of growth factors comes from co-culture studies. A wide variety of cell types such as human oviduct (Bongso et al., 1990), Vero monkey kidney epithelial cells (Ménézo et al., 1990
), bovine oviductal cells (Wiemer et al., 1993
) and human uterine endometrial cells (Birkenfield and Navot, 1991
; Desai et al., 1994
) have been demonstrated to improve in-vitro embryo development. Release of low amounts of growth promoting factors and cytokines by somatic cell monolayers may be one mechanism by which co-culture cells might exert an influence on embryonic development. One such factor, leukaemia inhibitory factor (LIF), has been demonstrated in cell lines exhibiting embryotrophic properties (Papaxanthos-Roche et al., 1994
; Kauma and Matt, 1995
; Desai and Goldfarb, 1996
). In the human, LIF is expressed and secreted by cells of the oviduct (Keltz et al., 1996
) and the endometrium (Charnock-Jones et al., 1994
; Arici et al., 1995
; Cullinan et al., 1996
). Additional potential modulators of cell development identified in co-culture cell types include insulin-like growth factor (IGF)-I and -II (Boehm et al., 1990
; Giudice et al., 1993
; Pfeifer and Chegini, 1994
), epidermal growth factor (EGF) and transforming growth factor (TGF)
(Haining et al., 1991
; El Dansouri et al., 1993
; Morishege et al., 1993), platelet-derived growth factor (PDGF) (Boehm et al., 1990
; Chegini et al., 1992
; Desai and Goldfarb, 1996
, 1998
) and interleukin (IL)-6 (Desai and Goldfarb, 1996
, 1998
; Desai et al., 1999
). Trials with growth factor supplementation of human embryo culture medium have been very limited but appear quite promising. IGF-I (Lighten et al., 1998
), heparin binding EGF (Martin et al., 1998
) and LIF (Dunglison et al., 1996
) have each been shown significantly to improve in-vitro blastulation of human embryos cultured in serum-free media.
In our IVF laboratory, we have noted that when cryopreserved human blastocysts are placed on Vero cell monolayers immediately after thaw, re-expansion proceeds much more quickly and we have had better overall pregnancy outcomes. To look at this in a more controlled fashion, we conducted a study comparing the post-thaw development of day 4 mouse embryos in medium alone versus on co-culture cells (Desai et al., 1997). These preliminary data suggested that the co-culture cells and/or their secretions accelerated the in-vitro development of thawed embryos.
The primary objective of the current study was to determine if direct supplementation with specific growth factors and/or cytokines could mimic the co-culture environment and produce similar improvements in post-thaw development of mouse embryos. We also hoped to gain a better understanding of the effect of individual factors on embryonic progression from morula to the blastocyst stage.
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Materials and methods |
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Morula cryopreservation
A two-step glycerol freeze protocol (Ménézo et al., 1992) was utilized to cryopreserve the morula stage embryos. In the first step, mouse morulae were incubated in 5% glycerol for 10 min. This was followed by a second 10 min incubation in a solution containing 9% glycerol and 0.2 mol/l sucrose, prior to loading in Nunc cryovials. All freeze solutions were prepared using
-MEM + 10% SSS. A controlled rate Planer freezer was used for embryo cryopreservation.
Morula thaw and culture
Morulae were thawed and rehydrated using a seven-step protocol (Ménézo et al., 1992). Thawed morulae were pooled and randomly distributed between the 10 treatment groups. The treatment groups were as follows: control (CT), Vero (VR) cells, LIF (1 ng/ml), IL-6 (1 ng/ml), TGF
(2 ng/ml), EGF (4 ng/ml), PDGF (1 ng/ml), IGF-I (30 ng/ml), IGF-II (1 ng/ml) and TGFß (2 ng/ml). Growth factors were added to the basal
-MEM supplemented with 10% SSS at the concentrations indicated. Thawed embryos were cultured in 100 µl media drops under oil, except for the co-culture treatment group (VR). Co-culture was performed in Nunc four-well dishes seeded 48 h before the thaw with Vero cells (100 000 per well). Aliquots of 1 ml of fresh equilibrated medium were added to each well prior to co-incubation with thawed morulae.
Data collection and embryonic assessment
Thawed embryos were observed at 4, 8, 20, 30 and 48 h on an Olympus IX 70 microscope fitted with Hoffman modulation contrast optics. Images were captured and stored for later analysis using the MetaMorph Imaging System (Universal Imaging Corporation; West Chester, PA, USA). Morphometric analysis of digitized images was performed at each successive time point. The parameters evaluated were blastocoel formation, expansion, blastocyst maturity, zona uniformity and hatching. Blastocysts were evaluated as to maturity and inner cell mass (ICM) development. Maturity was graded as follows: A, cavity just starting; B, cavity less than half volume; C, expanding, distinctly increased embryo diameter with cavity greater than half embryo volume; D, fully expanded. The percentage of thawed embryos progressing from morula through the different stages of blastocyst development was calculated for each time point. A single focal plane that allowed sharp definition of inner and outer zona edges was selected for zona measurements. Images were captured and zona thickness measurements were made on each embryo, at the 3, 6, 9 and 12 o'clock positions. The mean zona thickness was calculated for each embryo. Within-embryo percentage zona variation was determined by subtracting the zona measurement most deviant from the mean and expressing this as a percentage of the mean zona thickness (see Figure 5).
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Statistical analysis
The 2 test and Student's t-test with the Bonferroni correction were utilized to determine statistically significant differences in treatment regimens. Values of P < 0.005 were reported as being statistically significant. Rates of hatching and blastulation were compared between culture regimens using the
2 test. The mean cell number per blastocyst and the percentage zona thickness variation for each treatment group were contrasted using the t-test.
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Results |
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Discussion |
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The selection of frozen morula versus frozen blastocyst stage embryos for this study offered the advantage of additional time in culture to delineate more clearly the differences amongst treatments. Initial experiments were in fact performed with blastocyst stage embryos (unpublished data). Analysis of data was complicated by the fact that we often were freezing blastocysts of maturity levels varying from early cavitating to fully expanded blastocysts. It was extremely difficult to synchronize the stage of blastocyst maturity and moreover, we frequently ended up with a large proportion of hatching blastocysts by the time the freeze was initiated. Use of early morula resolved these difficulties. Blastocyst freezing methodology can be quite successfully applied to embryos at the morula stage.
Autocrine secretion of growth factors by embryos and expression of specific receptors at particular cell stages strongly implicate growth factors as mediators in early embryonic events (reviewed by Adamson, 1993; Kane et al., 1997; O'Neill, 1998). With the exception of EGF and IGF-I, all of the growth factor ligands selected for testing in the present study are also expressed by the preimplantation mouse embryo. Growth factor and receptor interaction during the regulation of early embryo development has been most clearly documented with the EGF receptor and its ligands, EGF and TGF (Paria and Dey, 1990
; Dardik et al., 1992
; Chia et al., 1995
; Wiley et al., 1995
; Brison and Schultz, 1996
). EGF receptor protein has been identified on cells of both the trophectoderm and inner cell mass (ICM) in the mouse (Brison and Schultz, 1996
) and human (Chia et al., 1995
). Selective ablation of the gene for this receptor was found to be lethal, resulting in blastocysts that failed to develop an ICM (Threadgill et al., 1995
). Receptors for PDGF (Palmieri et al., 1992
) and IGF-I and -II (Harvey and Kaye, 1991
; Rappolee et al., 1992
; Smith et al., 1993
) have also been identified in mouse embryos. In the mouse model, LIF receptor mRNA transcripts have been detected in ICM cells but not in trophectodermal cells (Nichols et al., 1996
). Although little information is currently available on IL-6 and TGFß receptor expression and distribution in mouse blastocysts, there is certainly evidence to support a role for these factors during embryo development (reviewed by Lee, 1992
; Yoshida et al., 1994
; Kane et al., 1997
; Desai et al., 1999
).
In designing this study, we were careful to keep the number of embryos cultured in individual 100 µl drops to between one and three to reduce any potential autocrine effect of embryo-derived growth factors. It has recently been reported (O'Neill, 1997) that there is an enhancement in blastulation and cell number per embryo when embryos were cultured at concentrations of one embryo per µl of culture medium. Reducing embryo concentration to one embryo per 10100 µl of medium resulted in a loss of this autocrine embryotrophic effect. Similar observations have been reported by other investigators. Partial compensation for the adverse effects of culture at low embryo concentration could be achieved by addition of specific factors such as IGF-I, IGF-II, platelet activating factor (PAF) (O'Neill, 1997
), TGF
(Brison and Schultz, 1997
) and TGFß plus EGF (Paria and Dey, 1990
).
Embryonic culture in vitro may potentially compromise autocrine secretion of necessary growth factors and timely expression of specific receptors (O'Neill, 1998). This, combined with the absence of paracrine influence from oviductal and uterine derived growth factors, may make the in-vitro cultured embryo particularly vulnerable. The deprivation of necessary growth factors has been shown to trigger apoptosis or programmed cell death in a wide variety of in-vitro cultured cells (reviewed by Wyllie et al., 1980; Collins et al., 1994). Brison and Schultz (Brison and Schultz, 1997
) studying embryonic apoptosis, have reported a role for TGF
in regulation of cell death in mouse blastocysts. Addition of this growth factor to singly cultured embryos resulted in decreased cell death in the ICM of developing blastocysts and also immunosurgically isolated ICMs. These data, taken together with information from gene knockout experiments with the EGF receptor, suggest that TGF
may be acting as a `survival factor' in embryos. Other putative `survival factors' for in-vitro cultured embryos include IGF-I (Herrler et al., 1998
) and PAF (O'Neill, 1997
). LIF has also been proposed to play a role in maintaining the proliferative ability of ICM cells (Stewart et al., 1992
; Nichols et al., 1996
).
Cryothaw procedures often result in cell loss or damage. It is likely that such damage may also result in alterations in autocrine secretion of growth factors and therefore heighten the impact of the post-thaw culture environment. In this study, all embryos had been cultured identically until cryopreservation at the morula stage, yet after as little as 8 h of post-thaw exposure to the different treatment regimens we could visualize morphological differences between groups. Cell proliferation in frozenthawed embryos was significantly stimulated by just 48 h of exposure to growth factors, either through co-culture or by direct media supplementation with specific factors. Of the tested growth factors, only LIF failed to enhance overall cell number per blastocyst. TGF treatment resulted in a marginal increase in the calculated mean cell number per embryo, most evident when the blastocyst population was further stratified according to cell number (Figure 4
). We must also consider the possibility that TGF
and LIF preferentially target the ICM cells (Threadgill et al., 1995
; Nichols et al., 1996
; Brison and Schultz, 1997
). Since only total cell counts were performed, any stimulatory effect might have been masked by a simultaneous decrease in the trophectoderm cell number. Differential staining of treated blastocysts and analysis of cell distribution in both trophectodermal and ICM compartments upon growth factor treatment will be needed to interpret these data further. We know very little about the action of growth factors as `survival factors' in frozenthawed embryos. It is possible that their inclusion in post-thaw culture media may serve to minimize any further cell death, especially of the ICM cells, which are fewer in number.
Besides cell number and accelerated development, the increased zona thickness variation observed in growth factor and co-cultured embryos may be another prognostic indicator of a beneficial culture regimen. Increased zona thickness variation may aid in identifying embryos more likely to implant (Cohen et al., 1988). Wiemer et al. 1995 observed that human embryos cultivated on bovine oviductal cells exhibited more zona thickness variation than those grown directly on conventional tissue culture dishes (Wiemer et al., 1995
). The transfer of at least one embryo with >20% zona pellucida variation was associated with positive pregnancy outcomes. The earlier timing of hatching for co-culture and growth factor-exposed embryos observed in the current study could in part be related to the manner in which these culture components alter zona properties. Irregularities in zona thickness around embryos could potentially facilitate the process of hatching. In human IVF, incomplete hatching of in-vitro derived blastocysts has been linked to monozygotic twinning. As we study different culture regimens for human in-vitro blastocyst formation, it may be of value also to study their effects on zona uniformity.
In summary, these results for the first time describe the effect of culture regimen on development after cryopreservation and thaw. As programmes move away from co-culture to the new generation of sequential media for human blastocyst culture, one area of concern should be the establishment of a successful blastocyst cryopreservation programme. To date, the most extensive studies and the best pregnancy outcomes with frozenthawed blastocysts have come from laboratories practising co-culture (Ménézo et al., 1993; Kaufman et al., 1995
). A major determinant of success with frozen blastocysts may be the presence of sufficient cell numbers in trophectodermal and ICM compartments to overcome cell damage during the freezethaw process. Blastomere numbers in co-cultured human blastocysts have ranged from 120240 cells on day 6 (Ménézo et al., 1992
; Vlad et al., 1996
) as compared to 4082 cells (Hardy et al., 1989
; Ray et al., 1995
) reported for traditional culture media. Cell number in the newer sequential culture regimens such as Gardner's G1/G2 (Scandinavian IVF Science, Gothenberg, Sweden), Medicult's EBSS/M3 (Medicult, Copenhagen, Denmark), Quinn's Enhance-B XI/Enhance-D3 (Conception Technology, San Diego, CA, USA) and Irvine's P1/Blastocyst medium combination (Irvine Scientific, Santa Ana, CA, USA) still need to be studied in detail. It remains to be seen if blastocysts derived from these newer culture regimens do as well after cryopreservation as co-cultured blastocysts. Exogenous stimulation of post-thaw blastocyst expansion and cell division though growth factor additives may be another avenue to explore as a potential means to `jumpstart' cryopreserved human embryos, especially those arising from non-co-culture systems.
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Notes |
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References |
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Submitted on July 14, 1999; accepted on October 28, 1999.