Assessment of growth factor effects on post-thaw development of cryopreserved mouse morulae to the blastocyst stage

Nina Desai1, Julia Lawson and James Goldfarb

Department of Reproductive Biology, University MacDonald Women's Hospital, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The objective of this study was to assess the influence of specific factors on post-thaw development of mouse cryopreserved morulae. Thawed morulae (n = 206) were randomly distributed between 10 treatment groups: medium alone control (CT), Vero (VR) cells, leukaemia inhibitory factor (1 ng/ml), interleukin-6 (1 ng/ml), transforming growth factor (TGF) {alpha} (2 ng/ml), epidermal growth factor (EGF) (4 ng/ml), platelet-derived growth factor (1 ng/ml), insulin-like growth factor (IGF)-I (30 ng/ml), IGF-II (1 ng/ml) and TGFß (2 ng/ml). At 4, 8, 20, 30 and 48 h, a digitized image of each thawed embryo was captured and stored for later analysis. The following parameters were examined: blastocoel formation, blastocyst expansion, zona thickness and hatching. At termination of the experiment, cell number per embryo was determined by bisbenzimide staining. When contrasted to the medium alone control, co-culture consistently accelerated the development of frozen–thawed morulae to the hatched blastocyst stage, allowing embryos to recover rapidly from any damage sustained during the cryopreservation process. While no single growth factor/cytokine was able to completely mimic the results achieved with co-culture, all of the growth factors impacted positively on at least one of the morphological parameters studied. Cell proliferation was significantly stimulated by just 48 h exposure to growth factors, either through co-culture or by direct media supplementation. Co-culture again yielded the best results with a mean cell count of 217 ± 76 cells per blastocyst as compared with 131 ± 36 in control medium alone. Amongst the factors tested, IGF-I, IGF-II and EGF had the greatest impact, with mean cell counts of 172 ± 50, 168 ± 50 and 179 ± 55 respectively. Whereas only 5% of CT embryos developed to blastocysts with > 200 cells, 51% of thawed embryos placed on co-culture monolayers and 25–32% of embryos cultured with IGF-I, IGF-II or EGF had > 200 cells. This study for the first time systematically describes the effect of culture regimen and growth factor additives on the post-thaw development of cryopreserved embryos.

Key words: blastocyst/co-culture/cryopreservation/EGF/growth factors/IGF


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Interest in the culture and transfer of human embryos at the blastocyst stage has been spurred by availability of newer culture media formulations that better support the nutritional requirements of the developing embryo. To maximize the opportunity for pregnancy in any given in-vitro fertilization (IVF) cycle, `spare' untransferred embryos will need to be cryopreserved and thawed as needed in subsequent cycles. To date, culture factors influencing the post-thaw development of embryos frozen at late cleavage stages, such as morula and blastocyst, have not been extensively characterized.

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., 1990Go), Vero monkey kidney epithelial cells (Ménézo et al., 1990Go), bovine oviductal cells (Wiemer et al., 1993Go) and human uterine endometrial cells (Birkenfield and Navot, 1991Go; Desai et al., 1994Go) 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., 1994Go; Kauma and Matt, 1995Go; Desai and Goldfarb, 1996Go). In the human, LIF is expressed and secreted by cells of the oviduct (Keltz et al., 1996Go) and the endometrium (Charnock-Jones et al., 1994Go; Arici et al., 1995Go; Cullinan et al., 1996Go). Additional potential modulators of cell development identified in co-culture cell types include insulin-like growth factor (IGF)-I and -II (Boehm et al., 1990Go; Giudice et al., 1993Go; Pfeifer and Chegini, 1994Go), epidermal growth factor (EGF) and transforming growth factor (TGF) {alpha} (Haining et al., 1991Go; El Dansouri et al., 1993Go; Morishege et al., 1993), platelet-derived growth factor (PDGF) (Boehm et al., 1990Go; Chegini et al., 1992Go; Desai and Goldfarb, 1996Go, 1998Go) and interleukin (IL)-6 (Desai and Goldfarb, 1996Go, 1998Go; Desai et al., 1999Go). Trials with growth factor supplementation of human embryo culture medium have been very limited but appear quite promising. IGF-I (Lighten et al., 1998Go), heparin binding EGF (Martin et al., 1998Go) and LIF (Dunglison et al., 1996Go) 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., 1997Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Zygote isolation and cultivation
Female B6C3F1 mice 6–8 weeks old purchased from Charles River (Wilmington, MA, USA) were superovulated to obtain oocytes. Animals were injected with 10 IU (intraperitoneal) of pregnant mare serum gonadotrophin (Sigma, St Louis, MO, USA). A second injection of 10 IU of human chorionic gonadotrophin (HCG) (Sigma) was given 48 h later and the animals were mated with B6D2F1 males. Mice were killed 16 h post-HCG injection by cervical dislocation and their oviducts were excised. Cumuli containing zygotes were isolated and placed in organ culture dishes containing pre-equilibrated {alpha} modified Minimum Essential Medium ({alpha}-MEM; Gibco, Grand Island, NY, USA) supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific, Irvine, CA, USA). Dishes were incubated at 37°C in a humidified incubator with 5% CO2. All zygotes were cultured for 72 h to the morula stage and frozen.

Morula cryopreservation
A two-step glycerol freeze protocol (Ménézo et al., 1992Go) 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 {alpha}-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., 1992Go). 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{alpha} (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 {alpha}-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 5Go).



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Figure 5. Variation in zona thickness. The percentage zona variation for individual embryos in each treatment group was calculated at 4, 8 and 20 h using the following formula:

The calculated zona variation for individual embryos within a treatment was then averaged and is plotted on this graph. A high percentage zona variation is indicative of non-uniform zonae. At 20 h, zona variation in all treatment groups was higher than that observed in the medium alone control (CT). Statistically significant increase in zona variation was observed with Vero cells (VR), TGF{alpha}, TGFß (P < 0.005), and with growth factors IGF-I, IGF-II, LIF, PDGF (P < 0.05).

 
At termination of the experiment, embryos from each treatment group were stained using the Hoechst nuclear staining protocol (Handyside and Hunter, 1984Go) for determination of cell number. Cell number per embryo was tabulated by computer-assisted counting of fluorescing blastomeres. Embryos with <60 cells were categorized as non-blastocysts and excluded from calculations of the mean cell number per blastocyst.

Statistical analysis
The {chi}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 {chi}2 test. The mean cell number per blastocyst and the percentage zona thickness variation for each treatment group were contrasted using the t-test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The morphology of thawed morulae placed in different post-thaw culture environments was carefully monitored. Morula post-thaw survival rate was 84%. A total of 1278 thawed mouse morulae were studied. Eight hours after thawing, 70–80% of all embryos had blastulated regardless of the treatment. The post-thaw regimen did, however, significantly influence the rate of blastocyst maturation. Figure 1Go illustrates the percentage of initial morulae reaching each stage of maturity. Stage A and B blastocysts showed no signs of expansion and are depicted on the graph as early cavitating blastocysts. Although 45% of thawed morulae cultured in the medium alone control had developed into early cavitating blastocysts, only 3% reached the final stage of maturation (stage D), the fully expanded blastocyst. In contrast, thawed morulae placed on Vero cell monolayers developed rapidly, with 22% reaching the fully expanded stage within 8 h of culture. A similar pattern was observed with the growth factors TGF{alpha}, EGF, PDGF, IGF-I and IGF-II. Each of these treatments was found to be significantly different from the control (P < 0.005).



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Figure 1. Pattern of blastocyst development and maturity amongst the different treatment groups 8 h after morula thaw. Blastocysts were categorized as early cavitating (cav) (stages A and B), expanding (stage C) or fully expanded blastocysts (stage D) based on cavity size and level of expansion. The percentage of thawed morulae reaching each level of maturity was calculated and compared to the control. *Maturation pattern significantly different from the control (P < 0.005). CT = control; VR = Vero, LIF = leukaemia inhibitory factor, IL = interleukin, TGF = transforming growth factor, EGF = epidermal growth factor, PDGF = platelet-derived growth factor, IGF = insulin-like growth factor.

 
These data suggested that thawed morulae progressed much more rapidly to fully expanded blastocysts either in the presence of specific growth factors or in the co-culture environment. Even at 30 h post-thaw (Figure 2Go), control morulae were lagging behind the co-culture and growth factor-treated embryos. Only 73% of thawed morulae in the control group were able to complete development to the fully expanded blastocyst stage. In contrast, 99% of co-cultured morulae and 90–96% of morulae from growth factor-treated groups were fully differentiated after 30 h of culture. At the 30 h observation point, we also noted a significant difference in embryonic hatching amongst treatment groups. With the exception of PDGF, all growth factors significantly enhanced embryonic hatching. The hatching rate was 61% for control embryos as compared to 82–89% upon supplementation of post-thaw culture medium with individual growth factors (LIF 84%; IL6 85%; TGF{alpha} 84%; EGF 87%; IGF-I 82%; IGF-II 89% and TGF ß 83%; P < 0.005). Co-culture yielded the highest percentage of hatching, 96%.



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Figure 2. A comparison of the percentage of thawed morulae reaching the fully expanded blastocyst stage at 8 h versus 30 h under different treatment regimens. For abbreviations, see Figure 1Go.

 
In Figure 3Go, the time course of progression from morula to fully expanded blastocyst stage was plotted under the various treatment regimens. Our data indicated that control embryos required additional time in culture to catch up to their counterparts cultivated either on cell monolayers or alternatively with growth factor additives. It was not until the 48 h time point that control embryos were observed to finally complete their differentiation to fully expanded blastocysts. The final rate of stage D blastocyst formation was comparable between the treatments and light microscopic evaluation of blastocysts failed to provide any further evidence of differences between the treatments.



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Figure 3. Summary of the time course of progression from morula to fully expanded blastocyst. Blastocyst maturity evaluated at 4, 8, 20, 30 and 48 h. For abbreviations, see Figure 1Go.

 
To evaluate further the quality of the blastocysts being produced under each of the test conditions, we determined the total cell number per blastocyst at the final 48 h observation point. The mean cell count per blastocyst was distinctly affected by the post thaw culture regimen (Table IGo). Thawed control embryos cultured in medium alone had a mean cell count of 131 ± 36. With the exception of LIF and TGF{alpha}, all of the growth factor additives significantly increased total blastomere number in developing embryos when contrasted to the control (P < 0.005). Amongst the factors tested, IGF-I, IGF-II and EGF had the greatest impact, with mean cell counts of 172 ± 50, 168 ± 50 and 179 ± 55 respectively. Blastomere number in the IL-6, PDGF and TGF ß treatment groups ranged from 153–162 cells per embryo but was still significantly greater than the control group. Co-culture once again yielded the best results with a mean cell count of 217 ± 76 cells per blastocyst.


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Table I. Effect of culture regimen on blastocyst cell number
 
Figure 4Go further accentuates the differences between treatments and resultant cell number per blastocyst. The percentage of thawed morulae developing to blastocysts with 100–150 cells is shown in the bottom panel, 151–200 cells in the middle and >200 cells in the top panel, for each of the test groups and the control. What was particularly striking was that only 5% of control thawed embryos cultivated in medium alone developed to blastocysts with cell counts of >200 cells. This was in stark contrast to the co-culture group, where >51% of thawed embryos developed to blastocysts with >200 cells. Of the tested growth factors, EGF and both of the IGFs influenced total cell count the most. With EGF, IGF-I or IGF-II supplementation, 25–32% of thawed embryos reached >200 cells within 48 h of culture. Despite a shared receptor protein (reviewed by Wiley et al., 1995), TGF{alpha} treatment did not enhance cell counts to the same extent as EGF. We did however, find an upward shift in the percentage of blastocysts with >150 cells (Figure 4Go). In the untreated control embryos, only 21% of blastocysts had >150 cells as compared with 42% of TGF{alpha} derived blastocysts.



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Figure 4. Effect of treatment regimen on cell number. Blastocysts were categorized according to cell number. The percentage of total blastocyst population with 100–150 cells (lower panel), 150–200 cells (middle panel) and >200 cells (upper panel) is shown for each test group. For abbreviations, see Figure 1Go.

 
The last parameter that we examined was the variation in zona thickness around individual embryos and whether or not this feature was influenced by culture regimen. The percentage zona variation is a calculation that reflects zona thickness at several points around an embryo and how `different' these measurements were from the mean zona thickness of that embryo (Wiemer et al., 1995Go). Thus, the greater the percentage difference in zona measurements, the less `uniform' the zona. The mean percentage zona variation for embryos in each treatment group was calculated at 4, 8 and 20 h and is shown in Figure 5Go. Zona of control embryos underwent only a moderate change between the first observation at 4 h and the final observation at 20 h (11–20%). In contrast, by 20 h post-thaw, the mean zona variation of co-cultured embryos was quite high, ~40% (P < 0.005). A mean percentage zona thickness variation of >=25% was also seen with all of the growth factor additives except IL-6. Of the factors tested, TGF{alpha} and ß showed the most significant influence on zona variation (44.8 and 36.7% respectively; P < 0.005). To look at this further, we took the data from the 20 h endpoint and tabulated the number of embryos in each test regimen exhibiting >25% zona thickness variation (Table IIGo). The proportion of embryos in each test regimen with high zona variation is displayed. Zona variation appeared to be significantly influenced by co-culture, LIF, TGF {alpha} and ß, PDGF, IGF-I and II (P <= 0.005).


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Table II. Proportion of embryos exhibiting increased zona variation
 
The effects of each culture regimen on the various parameters examined in this study are summarized in Table IIIGo.


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Table III. Summary of co-culture and growth factor effects on in-vitro development of thawed morulae
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of the present study clearly demonstrate that the post-thaw development of embryos can be modulated by addition of growth factors. We were able to show that culture factors can accelerate the progression of thawed morulae to the hatched blastocyst stage, increase total cell number per embryo and affect zona characteristics. In addition, our findings offer further evidence supporting the embryotrophic effect of co-culture. While growth factor additives were clearly beneficial to thawed embryos, no single growth factor was able completely to mimic the results attained with co-culture. An enhancement in post-thaw development with co-culture has been observed with both human zygotes (Wiker et al., 1992Go) and cleavage stage embryos (Tucker et al., 1995Go). To our knowledge, this is the first study to systematically explore and contrast the effect of growth factors and co-culture on frozen–thawed embryos.

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{alpha} (Paria and Dey, 1990Go; Dardik et al., 1992Go; Chia et al., 1995Go; Wiley et al., 1995Go; Brison and Schultz, 1996Go). EGF receptor protein has been identified on cells of both the trophectoderm and inner cell mass (ICM) in the mouse (Brison and Schultz, 1996Go) and human (Chia et al., 1995Go). 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., 1995Go). Receptors for PDGF (Palmieri et al., 1992Go) and IGF-I and -II (Harvey and Kaye, 1991Go; Rappolee et al., 1992Go; Smith et al., 1993Go) 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., 1996Go). 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, 1992Go; Yoshida et al., 1994Go; Kane et al., 1997Go; Desai et al., 1999Go).

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, 1997Go) 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 10–100 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, 1997Go), TGF{alpha} (Brison and Schultz, 1997Go) and TGFß plus EGF (Paria and Dey, 1990Go).

Embryonic culture in vitro may potentially compromise autocrine secretion of necessary growth factors and timely expression of specific receptors (O'Neill, 1998Go). 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, 1997Go) studying embryonic apoptosis, have reported a role for TGF{alpha} 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{alpha} may be acting as a `survival factor' in embryos. Other putative `survival factors' for in-vitro cultured embryos include IGF-I (Herrler et al., 1998Go) and PAF (O'Neill, 1997Go). LIF has also been proposed to play a role in maintaining the proliferative ability of ICM cells (Stewart et al., 1992Go; Nichols et al., 1996Go).

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 frozen–thawed 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{alpha} 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 4Go). We must also consider the possibility that TGF {alpha} and LIF preferentially target the ICM cells (Threadgill et al., 1995Go; Nichols et al., 1996Go; Brison and Schultz, 1997Go). 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 frozen–thawed 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., 1988Go). 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., 1995Go). 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 frozen–thawed blastocysts have come from laboratories practising co-culture (Ménézo et al., 1993Go; Kaufman et al., 1995Go). 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 freeze–thaw process. Blastomere numbers in co-cultured human blastocysts have ranged from 120–240 cells on day 6 (Ménézo et al., 1992Go; Vlad et al., 1996Go) as compared to 40–82 cells (Hardy et al., 1989Go; Ray et al., 1995Go) 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.


    Notes
 
1 To whom correspondence should be addressed

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    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 14, 1999; accepted on October 28, 1999.