Human Reproduction, Vol. 18, No. 10, 2151-2156,
October 2003
© 2003 European Society of Human Reproduction and Embryology
Vitrification of zona-free rabbit expanded or hatching blastocysts: a possible model for human blastocysts
Laboratory of Animal Reproduction and Biotechnology (LARB-UPV), Politechnical University of Valencia, Camino de Vera, 14, 46071 Valencia, Spain
1 To whom correspondence should be addressed. e-mail: ritcer{at}dca.upv.es
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Abstract |
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BACKGROUND: The purpose of this study was to test the effectiveness of one two-step (A) and two one-step (B1 and B2) vitrification procedures on denuded expanded or hatching rabbit blastocysts held in standard sealed plastic straws as a possible model for human blastocysts. The effect of blastocyst size was also studied on the basis of three size categories (I: diameter <200 µm; II: diameter 200–299 µm; III: diameter
300 µm). METHODS: Rabbit expanded or hatching blastocysts were vitrified at day 4 or 5. Before vitrification, the zona pellucida was removed using acidic phosphate buffered saline. For the two-step procedure, prior to vitrification, blastocysts were pre- equilibrated in a solution containing 10% dimethyl sulphoxide (DMSO) and 10% ethylene glycol (EG) for 1 min. Different final vitrification solutions were compared: 20% DMSO and 20% EG with (A and B1) or without (B2) 0.5 mol/l sucrose. RESULTS: Of 198 vitrified blastocysts, 181 (91%) survived, regardless of the vitrification procedure applied. Vitrification procedure A showed significantly higher re-expansion (88%), attachment (86%) and trophectoderm outgrowth (80%) rates than the two one-step vitrification procedures, B1 and B2 (46 and 21%, 20 and 33%, and 18 and 23%, respectively). After warming, blastocysts of greater size (II and III) showed significantly higher attachment (54 and 64%) and trophectoderm outgrowth (44 and 58%) rates than smaller blastocysts (I, attachment: 29%; trophectoderm outgrowth: 25%). CONCLUSIONS: These result demonstrate that denuded expanded or hatching rabbit blastocysts of greater size can be satisfactorily vitrified by use of a two-step procedure. The similarity of vitrification solutions used in humans could make it feasible to test such a procedure on human denuded blastocysts of different sizes.
Key words: blastocyst size/in-vitro implantation/rabbit/standard sealed straws/vitrification
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Introduction |
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The high incidence of multiple pregnancy is a major side effect of human-assisted reproductive technology (Dhont, 2001
). The development of sequential media has led to the ability to routinely culture the human embryo to the viable blastocyst stage. Transfer of such self-selected blastocysts has resulted in a significant increase in implantation rates (Gardner et al., 2000). This fact has led to a reduction in the number of transferred embryos to only one or two, thus considerably reducing the number of multiple pregnancies (Gardner et al., 1998; Gorrill et al., 1999), but requires the cryopreservation of the spare embryos at the blastocyst stage (Fong et al., 1998).
It is possible that in zona-intact blastocysts, the in-vitro changes to the zona pellucida (ZP) could interfere with hatching in vivo, or if enzymes are released by the endometrium at the time of implantation their concentrations may not be sufficient to dissolve an in-vitro altered ZP (Fong et al., 1998). In consequence, if zona-free human blastocysts were transferred, better cell-to-cell interactions and anchoring of the embryo to the endometrium might be expected, with improved implantation rates and reduced embryonic losses (Fong et al., 1997; 1998; Urman et al., 2002).
There are few published studies on vitrification of zona-free blastocysts. Only Landa et al. (1991), working in mouse, have vitrified denuded expanding blastocysts in microvolumes. However, the cryopreservation strategy used by these authors does not avoid direct contact of the blastocysts with the liquid nitrogen, with the subsequent risk of the free access of the viruses present in some vitrified samples, such as hepatitis B virus, hepatitis C virus and HIV to the rest of samples (Steyaert et al., 2000), as well as the microorganisms that could be present in the liquid nitrogen (Bielanski et al., 2000). This drawback is avoided if vitrification is carried out in sealed straws, but we were only able to find one report in which hatched blastocysts were vitrified in standard sealed straws (in mouse: Zhu et al., 1996). In this sense, it must be pointed out that blastocyst vitrification procedures currently assayed in different species, such as electron microscopy grids, cryoloop and open pulled straws, imply the direct contact of the embryos with liquid nitrogen.
One-step vitrification procedures reduce the toxicity of cryoprotectants, but because the ZP may cause interchanging difficulties of such cryoprotectants during the vitrification process in young and expanded blastocysts, vitrification carried out in two or more steps (Valdez et al., 1992; Mahmoudzadeh et al., 1995; Vatja et al., 1996; Ohboshi et al., 1998; Lane et al., 1999; Yeoman et al., 2001; Zhu et al., 2001) confers both higher survival rates (ovine: two-step procedure 68–93% versus one-step procedure 50–78%; Zhu et al., 2001) and hatchability (ovine: two-step procedure 32–49% versus one-step procedure 21–28%; Zhu et al., 2001) than one-step vitrification procedures. Unfortunately, one-step vitrification procedures have only been studied in early or expanded blastocysts (Valdez et al., 1992; Zhu et al., 1993; 2001; Mahmoudzadeh et al., 1995; Saha and Suzuki, 1997; Sommerfeld and Niemann, 1999), but not at more advanced developmental stages.
As an expanded or hatching blastocyst is composed of a simple sheet of cells of epithelioid characteristics [trophectoderm (TE)] and inner cell mass (ICM), the cryoprotectant agents permeate easily to both TE and ICM. The absence of ZP could enhance this phenomenon, and so the use of a one-step vitrification procedure may be suitable. Indeed, effects on osmotic equilibration of ZP during and after the addition process of the vitrification solution are avoided if the ZP is first removed, with no negative effects on embryo survival (Vatja et al., 1996).
At the blastocyst stage, a larger size implies a more advanced degree of embryonic development, which could affect the efficiency of the vitrification procedure (Kaidi et al., 2000; Vanderzwalmen et al., 2002). On the other hand, at the same age, different blastocoelic cavity sizes could indicate a different embryo quality, which in turn could also affect vitrification results either by size effects and/or by embryo quality. Only one reference has been found in this respect (Hochi et al., 1995).
Because of the similarity of vitrification procedures and solutions used in both human (Mukaida et al., 2003; Son et al., 2003) and rabbit (Kasai et al., 1992; Silvestre et al., 2003) embryos, the establishment of suitable conditions for the vitrification of rabbit denuded blastocysts would be useful as a model for vitrification of human embryos at this stage.
In the present work, one- (B1 and B2) and two-step (A) vitrification procedures were tested on denuded expanded or hatching rabbit blastocysts, taking into account the effect of the blastocyst size on post-warming in-vitro survival and TE outgrowth.
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Rabbit embryo recovery
Adult mixed-breed females were mated with fertile bucks immediately before the GnRH injection (20 µg i.m.; GnRH Veterín-Receptal, Hoechst Russel Vet GmbH, Barcelona, Spain). At 14–15 h post-coitum rabbit zygotes were recovered from oviducts in HEPES-buffered Ham’s F-10 (Invitrogen, Barcelona, Spain) supplemented with bovine serum albumin (BSA; Invitrogen; hereafter BSA-H-Ham’s). Rabbit embryos were cultured in bicarbonate buffered-Ham’s F-10 (Invitrogen) supplemented with 20% (v/v) fetal bovine serum (FBS; Invitrogen) (hereafter B-Ham’s) in 7% CO2 in air at 39°C until they reached almost the expanded blastocyst stage. At days 4 and 5 post-coitum, in-vitro obtained expanded and hatching blastocysts were denuded. Briefly, blastocysts were put in acidified phosphate-buffered saline (PBS; pH 2.5) until ZP was dissolved, after which they were transferred to 0.25% trypsin 1:250 dilution (Cat. No. 25050; Invitrogen) until the mucin coat (where it was present) disappeared. The denuded blastocysts were washed with BSA-H-Ham’s to block the enzyme action. Denuded blastocysts were held in B-Ham’s in 7% CO2 in air at 39°C for 5–6 h before their vitrification. At this time, the maximum diameter of blastocysts was measured individually. Blastocysts were classified into three categories according to their diameter (category I: diameter <200 µm; category II: diameter 200–299 µm; category III: diameter
300 µm).
Cryopreservation procedures
Taking into account that an expanded or hatching blastocyst shows a simple sheet of cells of epithelioid characteristics around the blastocoele, this special cell organization can resemble a monolayer sheet of cultured cells, although in this case no cavity exists.
In our laboratory, two vitrification procedures, A and B, have been defined previously. Taking into account the special characteristics of hatching blastocysts, both procedures may be used for their cryopreservation. In fact, procedure A has been proved efficient in intact rabbit advanced embryos (Vicente and García-Ximénez, 1994; Silvestre et al., 2003). It is noteworthy that this procedure is similar to that applied for human blastocyst (Mukaida et al., 2003; Son et al., 2003) and embryonic stem cell (Reubinoff et al., 2001) vitrification. Procedure B, with two variations (B1 and B2), is used to vitrify sheets of cultured cells (Saeed et al., 2000). The procedures are described as follows.
In procedure A cryoprotectants are added in two-steps. Step 1: denuded blastocysts were placed in vitrification solution I (VSI), which contained 10% DMSO (Panreac Química SA, Barcelona, Spain) and 10% ethylene glycol (EG; Panreac Química SA) in Dulbecco’s PBS (Invitrogen) supplemented with 20% (v/v) FBS (hereafter: S-PBS) for 1 min. Step 2: blastocysts were transferred to vitrification solution II (VSII), which contained 20% DMSO, 20% EG and 0.5 mol/l sucrose (Panreac Química SA), and held in plastic straws for a maximum of 30 s before immersion in liquid nitrogen. It should be noted that in the present work, the time of exposure to cryoprotectants has been reduced compared with the original procedure, taking into account the small cell size of blastocysts.
Procedure B1: denuded blastocysts were directly equilibrated in the VSII and held in the straws for a maximum time of 30 s before immersion in liquid nitrogen.
Procedure B2: denuded blastocysts were placed in a modified VSII (mVSII), which contained only 20% EG and 20% DMSO in S-PBS without sucrose, for a maximum of 30 s in a one-step procedure before immersion of straws in liquid nitrogen.
In all cases, cryoprotectant solutions were prepared on the day of use and stored in darkness at room temperature (22–24°C) until use.
For vitrification, two to six denuded blastocysts of a specific category were transferred to 0.25 ml plastic straws (IMV Technologies, L’aigle, France) as follows: S-PBS (final column length 1.5 cm), air bubble, vitrification solution containing blastocysts (2.0 cm), air bubble and S-PBS (1.5 cm). Straws were identified and sealed with modelling clay and plunged vertically into liquid nitrogen.
Straws were stored in liquid nitrogen for periods of 3–6 weeks.
Regardless of the cryopreservation procedure applied and the blastocyst category, the same warming and cryoprotectant dilution procedures were performed, at room temperature. Briefly, each straw was warmed horizontally in air for 5 s and then for 10 s in a water bath at room temperature. The straw fraction containing the blastocysts in vitrification solution was then expelled into a watch glass containing 1 ml of 0.25 mol/l sucrose in S-PBS and gently mixed. After 5 min, blastocysts were transferred in the least amount of media possible to another watch glass containing 0.125 mol/l sucrose in S-PBS in which they were held for 5 min. Finally, blastocysts were washed in S-PBS for 5 min and cultured in B-Ham’s in 35 mm plates under equilibrated mineral oil (7% CO2 in air, 95% relative humidity, 39°C).
Evaluation criteria and statistical analysis
The efficiency of three vitrification procedures (A, B1 and B2) in cryopreserving blastocysts of categories I, II and III was tested according to post-warming recovery rate, blastocyst re-expansion and further in-vitro attachment to the substratum. Blastocyst re-expansion was examined after 4 h of in-vitro culture. At this time, apparent morphological damage was also evaluated by clarity of blastocyst cells and the presence of detached cells.
On re-expanded and non-re-expanded blastocysts, both attachment to the substratum and TE growth were assessed after 24 h in culture. After an additional 24 h of culture, TE growth was reassessed. Presence and growth of TE was given following a score between 0 and 3 based on the criterion established by Spindle and Pederson (1973), in which grade 0 was no growth and grade 3 was extensive outgrowth.
The percentage of blastocysts that reached extensive outgrowth from these initially vitrified were used as global efficiency evaluation parameter.
At least three replicates were performed in all vitrification procedures assayed. The results were analysed by the 
2-test. When a single degree of freedom was involved, the Yates’ correction for continuity was carried out.
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One hundred and eighty-one denuded rabbit blastocysts were recovered from 198 vitrified blastocysts (91%).
From the 198 vitrified blastocysts 46% were of category I, 31% of category II and 22% were of category III (data not shown). Blastocysts of category III appeared preferentially on day 5 [day 4: 2/65 (3%) versus day 5: 42/133 (32%); P < 0.05; data not shown].
Within vitrification procedure A, category I blastocysts showed a significantly lower global efficiency (Table I) than the rest of blastocyst categories [A-I: 50% (10/20) versus A-II: 96% (22/23) and A-III: 100% (11/11); P < 0.05]. Such differences were mainly due to the lower attachment ability of blastocysts of category I [A-I: 59% (10/17) versus A-II: 100% (22/22) and A-III: 100% (11/11); P < 0.05; Table I].
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Blastocysts of different categories vitrified by procedure B1 did not show differences in global efficiency [B1-I: 16% (5/32) versus B1-II: 16% (3/19) versus B1-III: 27% (4/15); P > 0.05; Table I]. In contrast, in procedure B2, category III blastocysts showed a significantly higher global efficiency than categories I and II [B2-I: 19% (6/32) and B2-II: 0% (0/15) versus B2-III: 57% (8/14); P < 0.05; Table I], mainly due to their greater ability to attach to the substratum [B2-I: 25% (6/24) and B2-II: 0% (0/9) versus B2-III: 80% (8/10); P < 0.05; Table I].
When the results were analysed regardless of the category of the blastocysts, the global efficiency of procedure A was significantly higher than for B1 and B2 procedures [A: 80% (43/54) versus B1: 18% (12/66) and B2: 23% (14/61); P < 0.05; Table II]. This difference was due to both the significantly higher re-expansion rate [A: 88% (44/50) versus B1: 46% (28/61) versus B2: 21% (9/43); P < 0.05; Table II] and attachment rate [A: 86% (43/50) versus B1: 20% (12/61) and B2: 33% (13/43); P < 0.05; Table II].
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In vitrification procedures A and B1, re-expansion did not determine further attachment ability of the blastocysts. However, in procedure B2 the percentage of re-expanded blastocysts that were able to subsequently attach to the substratum was significantly higher than for non-re-expanded blastocysts (67 versus 24%; P < 0.05; Table II).
Significant differences in TE outgrowth were only detected between procedures A and B1 until grades 1 and 2 [grade 1: A: 88% (38/43) versus B1: 42% (5/12); grade 2: A: 58% (25/43) versus B1: 8% (1/12); P < 0.05; Table III], but not until grade 3.
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When the results were analysed regardless of the vitrification procedure, TE outgrowth ability of vitrified blastocysts of categories II and III was significantly higher than in blastocysts of category I [I: 25% (21/84) versus II: 44% (25/57) and III: 58% (23/40); P < 0.05; Table IV]. This fact could be explained by the lower ability of blastocysts of category I put into culture, to attach to the substratum [I: 29% (21/72) versus II: 54% (25/46) and III: 64% (23/36); P < 0.05; Table IV]. When attachment rate was evaluated only in re-expanded blastocysts the same tendency was observed [I: 34% (13/38) versus II: 75% (21/28) and III: 100% (15/15); P < 0.05; Table IV]. Moreover, TE outgrowth ability of category I blastocysts was clearly lower until grade 2 than for categories II and III [I: 14% (3/21) versus II: 56% (14/25) and III: 57% (13/23); P < 0.05; Table V], but until grade 3, such differences were only maintained between categories I and III [I: 0% (0/21) versus III: 26% (6/23); P < 0.05; Table V].
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Discussion |
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Denuded rabbit blastocysts were satisfactorily vitrified by two-step vitrification procedure A. The one-step vitrification procedures, B1 and B2, which had previously been applied satisfactorily in our laboratory for the vitrification of monolayer sheets of cultured cells (Saeed et al., 2000
), conferred poor results on denuded rabbit blastocysts. In these procedures, the short exposure time of the cells to the final high concentration of cryoprotectants, together with the need to establish an osmotic equilibrium between the outer medium and the blastocoelic fluid, may cause the cryoprotectants to permeate too rapidly firstly into the TE cells and through them into the blastocoelic cavity. This fact could enhance the osmotic effects and/or cryoprotectant toxicity, as has been proposed by Zhu et al. (1996). Moreover, the exposure time to cryoprotectants could be insufficient to complete their permeation into the blastocoelic cavity (Zhu et al., 1993), which might lead to ice crystal formation. The post-warming detachment of TE cells observed after the application of these procedures suggested the existence of some kind of mechanical damage, possibly due to cryoinjury. This phenomenon was not observed when in-vitro cultured cells were vitrified by these procedures, probably due to the absence of the blastocoelic cavity and to the different water permeability coefficient specific of each cellular type (Leibo and Mazur, 1978). In fact, in human blastocysts results were improved when the blastocoelic cavity was artificially shrunk in order to reduce the ice crystal formation (Vanderzwalmen et al., 2002; Son et al., 2003). In our case, the reduction of blastocoelic cavity (our personal observation) observed during the two-step cryoprotectant addition (procedure A), although incomplete, could be the reason why no such cryoinjury damage was observed.
The majority of authors working in ART have transferred either fresh or cryopreserved human blastocysts at the expanded stage (day 5 or 6), obtaining an in-vivo implantation rate of approximately 30–40% in the case of fresh blastocysts (51%: Gardner et al., 1998; 25%: Jones et al., 1998; 30%: Rijnders and Jansen, 1998; 44%: Gorrill et al., 1999; 26%: Huisman et al., 2000; 33–69%: Khorram et al., 2000; 19–25%: Urman et al., 2002; 100%: Sagoskin et al., 2002) and around 10–20% in cryopreserved blastocysts (5%: Pantos et al., 2001; 16%: Behr et al., 2002; 15%: Reed et al., 2002; 13%: Vanderzwalmen et al., 2002; 20%: Mukaida et al., 2003; 100%: Sills et al., 2003; 29%: Son et al., 2003). However, as was studied by Khorram et al. (2000), hatching of human blastocysts by day 6 is a favourable prognostic factor for IVF outcome. In fact, embryos that fail to hatch by day 6 may have a lower implantation potential. Furthermore, hatching and hatched blastocysts can be frozen, resulting in pregnancies after transfer (Khorram et al., 2000). In this same sense, our results showed that categories of hatching rabbit denuded blastocysts (II and III) have a higher ability to survive, attach and proliferate (91, 100 and 97%, respectively; data not shown) than those of category I (expanded blastocysts: 82, 59 and 50%, respectively; data not shown), when vitrified at the same age, by procedure A. This fact would reflect that, at a given age, the initial size of the embryos expresses the embryo quality (Goto et al., 1992; Iwasaki, 1992).
Whatever the category, it was interesting to note that the denudation treatment applied was a safe and a non-invasive procedure because the majority of denuded blastocysts re-expanded 1–2 h later.
As has been proposed by Fong et al. (1998), a high in-vitro implantation rate on feeders of non-vitrified human blastocysts appeared to correlate to some extent with high in-vivo implantation rates. In consequence, the ability of vitrified rabbit blastocysts to attach to the substratum and to proliferate could be a possible in-vitro indicator of their in-vivo implantation ability. Unfortunately, due to rabbit embryos requiring ZP and mucin coat to implant in vivo, this correlation could not be made. Our results showed an effect of the vitrification procedure applied on in-vitro attachment and TE outgrowth rates. On the other hand, the initial ability to attach (as was reflected by TE outgrowth until grade 1) was not affected by the initial blastocyst size, although a marked effect of initial blastocyst size was observed on further TE outgrowth. Therefore, the initial blastocyst diameter could be used prospectively to select blastocysts that will ‘in-vitro implant’ after vitrification.
In human species, survival rates around 80% (50–83%: Cho et al., 2002; 100%: Reed et al., 2002; 80%: Mukaida et al., 2003; 90%: Son et al., 2003) were obtained when expanded ZP-intact blastocysts (not hatched and not denuded) were vitrified. In our case, similar survival rates (evaluated as re-expanded blastocysts) were obtained (91%; data not shown) when rabbit blastocysts, both denuded and in more advanced developmental stages (hatching and hatched blastocysts), were vitrified by procedure A. However, the special relevance of the embryo quality (expressed by their size at the same age) in these results must be emphasized.
As a result of the present work, testing of the two-step vitrification procedure on human denuded hatching/hatched blastocysts of different sizes, but at the same age, can be proposed. It should be emphasized that this vitrification procedure permits the use of standard sealed straws that avoid both microbial and viral contamination during storage.
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Acknowledgements |
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The authors thank Mr Neil Macowan for revising the English version of the manuscript. This work was supported by CICYT (AGL 2000-0595-C03-01).
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References |
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Submitted on April 17, 2003; accepted on July 4, 2003.
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