Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification

P. Vanderzwalmen1,4, G. Bertin1, Ch. Debauche1, V. Standaert1, E. van Roosendaal1, M. Vandervorst1, N. Bollen1, H. Zech2, T. Mukaida3, K. Takahashi3 and R. Schoysman1

1 Schoysman Infertility Management Foundation, Vaartstraat 42, 1800 Vilvoorde, Belgium, 2 Institut fur In vitro Fertilisierung und Embryo Transfer, Bregenz, Austria and 3 Hiroshima HART Clinic, Hiroshima, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Discussion
 References
 
BACKGROUND: In 1996, with the introduction of sequential media, we set up a programme of cryopreservation of supernumerary morulae (day 4) and blastocysts (day 5) using a vitrification procedure. Our results showed that the efficiency of the vitrification method was dependent on the stage of embryo development and was negatively correlated with the expansion of the blastocoele. We postulated that a large blastocoele might disturb cryopreservative potential due to ice crystal formation during the cooling step. We analysed therefore the effectiveness of reducing before vitrification the volume of the blastocoelic cavity. METHOD: Day 4 and day 5 embryos were vitrified in 40% ethylene glycol–18% Ficoll and 0.3 mol/l sucrose before plunging the straws directly into liquid nitrogen. Artificial shrinkage of the blastocyst was achieved after pushing a needle into the blastocoele cavity until it contracted. RESULTS: The survival rate post-thawing of day 4 and intact day 5 embryos was correlated with the volume of the blastocoele. In the control group only 20.3% blastocysts or expanded blastocysts survived as compared with 54.5 and 58.5% with morulae and early blastocyst respectively. After puncturing the blastocoelic cavity, an increase in the survival rate of up to 70.6% was noted. The pregnancy rates were improved after the artificial shrinkage procedure (20.5%) compared with the control intact blastocyst group (4.5%) (not significant). After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4% P < 0.01). CONCLUSIONS: Our results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. This was presumably because mechanical damage caused by ice crystal formation was avoided. These observations should be considered when establishing a strategy and a protocol for cryopreservation of day 5 embryos.

Key words: blastocoele/blastocysts/cryopreservation/embryo culture/vitrification


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Discussion
 References
 
Following the development of commercial sequential media (Gardner et al., 1998Go), in accordance with the physiology of the genital tract, we started in 1996 to apply a prolonged culture programme of preimplantation embryos until day 4 or day 5. In spite of the fact that high implantation and pregnancy rates were obtained after transfer of fresh blastocysts, we rapidly recognized that this technique had some inconveniences. One of them was the relatively poor survival rate after cryopreservation of blastocysts, which reduced the chances for transferring supernumerary viable embryos in a subsequent frozen embryo transfer cycle.

Our first freezing protocol consisted of a controlled slow-freezing procedure using a permeable cryoprotectant such as glycerol (Cohen et al., 1985Go) at a relatively low concentration (1.5 mol/l), generally associated with a non-permeable cryoprotectant such sucrose. However, it became clear that the efficiency of blastocyst freezing was lower than that of zygote or cleavage stage embryo freezing, and in order to obtain the best results after extended culture technique and blastocyst transfer programme with a maximum of two embryos, a reliable cryopreservation procedure must be ascertained. Consequently, the alternative approach by vitrification procedure was considered (Fahy et al., 1984Go).

This alternative procedure consists of increasing the concentration of cryoprotectant simultaneously with the cooling rates. The concentration of the cryoprotectant is so high that during the ultra-rapid freezing until –196°C, the viscosity of the solution increases and forms a glass-like solid. This procedure allows the embryo to be plunged directly into the liquid nitrogen, avoiding crystallization during the freezing and warming steps. As a consequence, the physical injuries caused by the formation of extracellular as well as intracellular ice crystals during the controlled slow-freezing procedure are eliminated. However, the toxicity caused by the high concentration of cryoprotectant is a drawback of this technique (Fahy et al., 1984Go; Rall et al., 1987Go).

Based on our previous experiences in the animal model (Massip et al., 1986Go; Scheffen et al., 1986Go; Vanderzwalmen et al., 1988Go), we decided in 1996 to apply vitrification as an alternative technique for handling day 4 and day 5 embryos (Vanderzwalmen et al., 1997Go).

After application of this technique on day 4 morula, day 5 early blastocyst, blastocyst and expanded blastocyst, our preliminary results showed that the efficiency of this vitrification method was dependent on the stage of embryo development. The percentage of blastocysts that remained intact and produced a pregnancy was much lower compared with those of morula and early blastocyst. After vitrification of day 4 morula or day 5 early blastocyst, an ongoing pregnancy rate per transfer of 29 and 25% was obtained respectively. On the other hand, after vitrification of blastocysts and expanded blastocysts only one ongoing pregnancy out of 13 transfers was achieved (8%) (Vanderzwalmen et al., 1999Go).

Among the various hypotheses that can explain the differences between the cryopreservability of the different stages of embryo development, the structural difference between morula and blastocyst is one parameter to consider. Compared morphologically with early stage embryos, blastocysts and expanded blastocysts show a fluid-filled cavity, i.e. the blastocoele. A large proportion of the water content of embryos at the morula stage is present inside the cells, while in the case of blastocysts and expanded blastocysts the blastocoele contains the largest amount of water.

Such marked reduction in the survival rate as the blastocoele enlarges is in agreement with previous reports demonstrating the developmental stage-dependent survival rate of mice (Scheffen et al., 1986Go; Vanderzwalmen et al., 1988Go; Valdez et al., 1990Go; Zhu et al., 1993Go) and bovine (Vanderzwalmen et al., 1989Go; Tachikawa et al., 1993Go) embryos after vitrification. One study reported that when mouse blastocysts with various blastocoele volumes were vitrified or underwent very rapid freezing, survival rates decreased as the volume of the blastocoele cavity increased (Miyake et al., 1993Go). However, to our knowledge, no report has examined, in the human, the effect of the blastocoele during cooling protocols.

Many reports related to the cryopreservation of mammalian embryos analysed the best procedures for the pre-treatment and exposure of the blastocyst to the vitrification solution in order to minimize the chemical toxicity of the solution (reduce the time and the temperature of equilibration with cryoprotectant) (Vanderzwalmen et al., 1988Go; Zhu et al., 1993Go), intracellular ice formation (different mixtures of cryoprotectant) (Ali and Shelton, 1993Go), fracture damage (pre-equilibration of the straw in the liquid nitrogen vapour) (Kasai et al., 1996Go) and osmotic swelling during the removal of the cryoprotectant (increase the equilibration steps) (Kuwayama et al., 1992Go).

We postulated that the loss of viability after vitrification of blastocysts could be attributed to physical damage resulting from ice formation during the cooling procedure. It is probable that inadequate permeation of the cryoprotectants (ethylene glycol–Ficoll–sucrose) or a too slow cooling rate leads to intra-blastocoelic ice formation during freezing, damaging the embryos. In order to improve the efficiency of vitrification of blastocysts and expanded blastocysts, we suggest reducing the blastocoele by removing artificially the fluid in order to decrease ice crystal formation at low temperature.

The objective of the present study was: (i) to present the survival rates of vitrified embryos according to their developmental stage after 4 or 5 days of culture, and (ii) to study the consequence of removing artificially the fluid from the blastocoele cavity (artificial shrinkage) before vitrification.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Discussion
 References
 
Patients selection
From March 1996 until October 1999, 100 patients had their embryos cryopreserved at the morula (day 4) or blastocyst (day 5) stages by the use of a two-step vitrification protocol (see below). Women aged 26–47 years (median 34.6) undergo fresh embryo transfers after a short culture period of 2 or 3 days or following culture in sequential medium for 4 or 5 days. Vitrification was applied to supernumerary embryos that developed to day 4 or 5. The couples entered the IVF programme for different causes of infertility: male factor (with ejaculate, epididymal or testicular sperm) and/or female factor (tubal, endometriosis, idiopathic). For male factor couples, ICSI was used.

Ovarian stimulation, oocyte retrieval, sperm preparation and embryo transfer procedures
Women undergoing assisted reproduction procedures received the combination of gonadotrophin-releasing hormone analogue (Suprefact, SP; Hoechst, Belgium) in association with human menopausal gonadotrophin (HMG, Humegon; Organon, Oss, The Netherlands) or pure FSH (Metrodin; Serono Laboratories Inc., Brussels, Belgium). HCG (Pregnyl; Organon Profasi; Serono Laboratories Inc.) was administered when the cohort of follicles reached a diameter of ~20 mm. Luteal phase support consisted of administering 5000 IU HCG on days 4 and 8 after embryo transfer or 600 mg Utrogestan (Piette, Belgium) daily vaginally until HCG assay.

Sperm preparation was carried out using discontinuous Percoll (Sigma Chemical Co., St Louis, MO, USA) or Pure sperm (Nidacon international AB) gradient.

Oocytes were collected 34–35 h after administering 10 000 IU of HCG and were incubated in 0.5 ml IVF 20 medium (Scandinavian Science, AB products, Gothenburg, Sweden). When sperm exhibited normal sperm parameters, insemination was performed after a minimal delay of 3 to 4 h. In case of patients entering the IVF programme for male or unexplained infertility problems, cumulus and corona cells were removed from the oocytes 2–3 h after oocyte retrieval by incubation in 25 IU/ml hyaluronidase (Type VIII; Sigma) before performing ICSI as described previously (Vanderzwalmen et al., 1996Go).

In-vitro culture of embryos to the morula–blastocyst stage and classification of blastocysts
All gamete and embryo cultures were performed in 4-well multi-dishes (Nunc; Invitrogen, Belgium) containing each 500 µl. of the sequential media in a humidified gas incubator at 37°C (5% CO2 in air). Injected oocytes or classical inseminated oocytes were incubated in IVF 100–20 medium (Scandinavian Science). Sixteen to 20 h after insemination or ICSI, all ova were checked for the presence of two pronuclei. After rinsing the zygotes, they were transferred into S1 or G1-2 medium (Scandinavian Science) for an additional period of 48 h.

After evaluation of the embryo cell number and morphology they were transferred on day 3 to 500 µl of S2 or G2-2 or CCM medium (Scandinavian Science) for further culture to the morula (day 4) or the blastocyst stage (day 5).

On day 5, the percentage of blastocysts formation was recorded and classified under an inverted microscope (magnification x200) according to the degree of expansion of the blastocoele, the quality of the inner cell mass (ICM) and the trophectoderm.

The blastocysts on day 5 were classified into three different categories according to the degree of expansion of the blastocoele: the early blastocyst with a blastocoele being less than half the volume of the embryo, the blastocyst with a blastocoele being greater than half of the volume of the embryo, the expanded blastocyst with a blastocoele larger than the volume of the blastocyst and with a thin zona pellucida (Schoolcraft et al., 1999Go).

Vitrification: selection of morula and blastocysts
Vitrification was applied to supernumerary embryos that developed to day 4 (morula compact) or day 5 (early blastocysts, blastocysts, expanded blastocysts). After a short culture period and transfer on day 2 or 3, some of the remaining embryos were selected for slow-freezing protocol at the 4- or 8-cell stage and the others were left in the appropriate culture medium until day 4 or 5. Morulae and blastocysts were also obtained from a group of patients designed for a prolonged culture with a transfer on day 4 or 5. The patients undergoing our blastocyst culture programme were informed about the poor results obtained after cryopreservation of blastocysts. For this reason, when enough zygotes were available, some of them were cryopreserved by the conventional slow-freezing method using 1,2-propanediol–sucrose and the others were left in sequential medium for an extended culture period of 5 days.

Before application of the vitrification technique in our clinical programme, the in-vitro survival of vitrified blastocysts was evaluated on day 5. The viability of the blastocysts was assessed after 24 h of culture in G2-2 medium.

For this study, we used blastocysts considered not good enough for conventional freezing, or blastocysts obtained from culture of frozen–thawed day 1 or 2 embryos donated by patients. In-vitro assessment of survival was performed in a sibling study. Before vitrification, artificial shrinkage was performed on some blastocysts, the others were left intact as controls.

Vitrification: preparation of the solution
The vitrification method used in this study was based on a published method (Kasai et al., 1990Go; Mahmoudzadeh et al., 1994Go).

The solutions for equilibration, vitrification and dilution were prepared using Dulbecco's phosphate-buffered saline (Sigma) plus 20% human serum albumin (PBS–HSA) (Irvine Scientific, Santa Ana, CA, USA). Two solutions were prepared for equilibration and vitrification: (i) an equilibration solution designated EG20 containing 20% ethylene glycol (v/v) in PBS–HSA and (ii) a second solution (EFS) containing 40% (v/v) ethylene glycol (Fluka, Sigma Aldrich, Belgium), 18% (w/v) Ficoll (mol. wt 70 000; Sigma) and 0.3 mol/l sucrose (Sigma) in PBS–HSA. The EFS solution had to remain transparent during the cooling and the thawing procedure. The solution for dilution after thawing designated S05 and S025 was made of 0.5 and 0.25 mol/l sucrose in PBS–HSA.

Vitrification: preparation of the 0.25 ml French straw and vitrification of the embryos
The 0.25 ml French straw (Cryo Bio System, l'Aigle, France) is loaded in two intervals. Before equilibration of the embryos in EG20 and EFS, a 5 cm column of S05 solution and two columns of ~5 and ~15 mm of EFS solution were aspirated into the straw and separated by air. The straw was kept horizontal on the microscope plate at room temperature.

The embryos were equilibrated according to their developmental stage and were then exposed for exactly 3 min to 500 µl EG20 at room temperature before being exposed to the EFS solution. After a short exposure of 15 s to EFS, they were loaded with the aid of a mouth glass pipette directly into a 0.25 ml French straw in the part containing EFS medium. This portion was separated by 8 mm air bubbles and the two remaining extremities were filled with 0.5 mol/l sucrose in PBS. The time elapsed between embryo introduction into the EFS and plunging them into a container filled with liquid nitrogen was kept as brief as possible (maximum 60 s).

Vitrification: thawing procedure
After storage for several days or months, the straws were taken out of the liquid nitrogen tank and warmed rapidly in water at 40°C. As soon as the crystallized SO5 medium in the straw began to melt, the content was expelled into a Petri dish containing 3 ml of 0.5 mol/l sucrose solution at room temperature. Five minutes after being flushed out, the embryos were transferred to 1 ml of 0.25 mol/l sucrose solution before washing them three times in a solution of PBS–HSA. The embryos were then incubated for 24 h in S2 or G2-2 medium before embryo transfer or for in-vitro survival assessment. Embryo transfers were performed using a Wallace or Cook catheter on day 4 or 5 after ovulation in the spontaneous cycles. Vitrified day 5 embryos were thawed in the afternoon of the fourth day.

Artificial shrinkage procedure
Artificial shrinkage of blastocysts and expanded blastocysts was performed before vitrification and also before a fresh transfer in order to examine if the artificial shrinkage technique had no harmful effect on the further developmental capacity of the blastocysts.

Ten to 15 min before the vitrification assay or transfer, the blastocysts and expanded blastocysts were dropped into a Petri dish containing 50 µl. drops of pre-equilibrated S2 or G2-2 medium covered with mineral oil. After holding the blastocyst with a holding pipette and placing the ICM at the 12 or 6 o'clock position, a glass micro-needle was pushed through the cellular junction of the trophectoderm into the blastocoele cavity until it shrank (Figure 1Go). After removing the pipette, contraction of the blastocyst was observed after 30 s to 2 min. If shrinkage was not observed, the needle was re-introduced at the opposite pole of the ICM and a slit was made in the trophectoderm cells and the zona pellucida by rubbing the needle on the holding pipette. Directly after complete shrinkage of the blastocoele, the blastocysts were vitrified or loaded in a catheter for a fresh transfer in order to assess the capacity of further development of artificially shrunk blastocysts.



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Figure 1. Artificial reduction of the blastocoelic volume: (A) immobilization of the blastocyst with the inner cell mass at 12 o'clock;(B) insertion of a needle inside the blastocoele; (C) shrinkage after 15 s; (D) shrinkage after 1 min.

 
Statistics
{chi}2-Analysis was used to determine if differences in pregnancy rate were significant between the different categories of embryos. The percentage of blastocysts that re-expanded with or without artificial shrinkage was also compared using the same test.

Results

The survival rate, the ongoing pregnancy rate and the implantation rate after two-step vitrification in straws containing only one developmental stage of embryos are summarized in Table IGo.


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Table I. Results after vitrification of intact embryos (no artificial shrinkage) in relation to the stage of embryo development
 
A total of 61 vitrification–thawing cycles, containing 167 embryos, were performed. Twenty-two, 17 and 22 vitrification–thawing cycles were performed with straws containing respectively 55 compacted morulae (day 4), 41 early blastocysts (day 5) and 71 blastocysts–expanded blastocysts (day 5).

Twenty-four hours after thawing, the percentages of blastocysts and expanded blastocysts that showed signs of degeneration or arrested development was 79.7%. Out of the 22 vitrification–thawing assays of 71 blastocysts, only 14 (20.3%) re-expanded and seven transfers (14.3%) occurred. For morulae and early blastocysts respectively 54.5 and 58.5% continued their development and transfers were achieved in 81.8% with morula (18 transfers) and 94.1% with early blastocysts (16 transfers).

A total of 12 patients became pregnant resulting in 10 deliveries. The percentage of deliveries per vitrification–thawing cycles was respectively 22.7, 23.5 and 4.5% for morulae, early blastocysts and blastocysts–expanded blastocysts. The implantation rate in terms of babies per vitrified embryos was respectively 10.9, 9.8 and 1.4% for morulae, early blastocysts and blastocysts–expanded blastocysts.

A total of 86 blastocysts and expanded blastocysts were obtained from in-vitro development of frozen–thawed two-pronuclear (2PN) zygotes or cleaved embryos donated by couples who no longer wished to store their own embryos in liquid nitrogen (n = 45) and from blastocysts considered as poor quality according to the criteria of Schoolcraft (n = 33).

Table IIGo shows the effect of the artificial shrinkage procedure on the in-vitro development of blastocysts that were not cryopreserved. No harmful effect of the procedure was noted. Fifty-one and 69% (not significant) of the blastocysts hatch on day 6 respectively in the control or in the artificial shrinkage group.


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Table II. Effect of the artificial shrinkage procedure of blastocysts (B) and expanded blastocysts (ExB) on in-vitro embryo development
 
In a total of 12 patients, artificial shrinkage was performed before transfer by pushing the needle trough the trophectoderm of 24 blastocysts and expanded blastocysts. Immediately after reduction of the volume of the blastocoele, 12 transfers were performed and resulted in five ongoing pregnancies with six fetuses. The pregnancy and implantation rates after artificial shrinkage was respectively 41.7 and 25%.

Table IIIGo showed the effect of the artificial shrinkage procedure on the in-vitro development of blastocyst after vitrification. Compared with blastocysts without artificial shrinkage, higher survival rates were obtained when blastocysts had been pretreated with the micro-needle in order to collapse the blastocoele. Out of 20 blastocysts and expanded blastocysts vitrified after artificial shrinkage, 12 (60%) seemed intact after thawing compared with five out of 16 without artificial shrinkage (31%). The rate of re-expansion was 19 and 40% and the percentage of hatching on day 6 was 6 and 25% respectively in the control and artificial shrinkage groups. There was a higher expansion and hatching rate after artificial shrinkage. However, this was not statistically significant due to the low number of embryos present in the study.


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Table III. In-vitro development after vitrification of blastocysts (B) and expanded blastocysts (ExB) with or without artificial shrinkage procedure
 
The clinical application of artificial shrinkage was detailed in Table IVGo. Out of 39 vitrification–thawing assays, 35 transfers (89%) of 46 blastocysts and expanded blastocysts were performed resulting in a pregnancy rate of 20.5% per vitrification assay and 22.9% per transfer. Compared with the results obtained without shrinkage (see Table IGo), we observed an increase in the number of transfers, and consequently, an increase in the rate of pregnancy per vitrification–thawing attempt. We noticed an apparently higher pregnancy rate after the artificial shrinkage procedure (20.5%) (Table IGo) compared with control intact blastocyst group (4.5%) (Table IVGo) but not reaching the level of statistical difference due to the small number of vitrification assays. After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4%, P < 0.01).


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Table IV. Outcome of vitrified blastocysts (B) and expanded blastocysts (ExB) transfers after artificial shrinkage
 
In order to analyse the efficacy of the shrinkage and vitrification procedures, the implantation rates of fresh embryo transfers were compared with that obtained after transfers of vitrified day 4 and day 5 embryos.

We took into account fresh transfers that were performed with only one category of embryo in terms of developmental stage. Between March 1998 until April 2000, 41, 79 and 192 transfers were achieved with respectively 101 day 4 morulae, 166 early blastocysts and 413 blastocysts and expanded blastocysts. The implantation rates after transfers of fresh embryos were 23% for morulae, 19% with early blastocysts and 29% with blastocysts and expanded blastocysts.

Compared with fresh transfers, the implantation rates of intact vitrified blastocysts and expanded blastocysts is low (29% for fresh versus 7% for intact vitrified). An increase in the implantation rates up to 18% is observed after artificial shrinkage. But the efficacy of the procedure (artificial shrinkage–vitrification–warming) has to be optimized, seeing that the implantation rates are >10% lower compared with the fresh transfer group.

Concerning day 4 morulae and day 5 early blastocysts, the vitrification procedure seems more efficient, seeing that only a slightly increase in the implantation rates is observed after fresh transfers (23 and 19% for fresh morulae and early blastocysts versus 20 and 17% after vitrification of morulae and early blastocysts).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Discussion
 References
 
Two basic approaches were developed to cryopreserve mammalian embryos: `controlled slow freezing' (Whittingham et al., 1972Go) and `vitrification', developed for mouse embryos (Rall and Fahy, 1985Go).

It has been reported that blastocysts produced via a co-culture procedure could be successfully cryopreserved using the classical slow-freezing protocol with glycerol (Kaufman et al., 1995Go). On the other hand, our first experience in 1996, after application of this slow protocol on day 5 embryos, was disappointing. This poor result was confirmed by other groups (Ludwig et al., 1999Go; Plachot et al., 2000Go) who observed a low efficiency of blastocyst freezing after extended culture in sequential medium compared with frozen early cleavage stage embryos and blastocysts obtained after co-culture. The decrease of blastocysts available after the slow-freezing procedure would result in a reduction in the cumulative pregnancy rate per retrieval following the transfer of both fresh and frozen blastocysts. Consequently, the ability to successfully cryopreserve human blastocysts is a requirement for the overall success of prolonged culture of embryos until day 5. Hence, we adopted in 1996 the other alternative for cryopreservation of blastocysts.

Our results showed that application of the vitrification method using ethylene glycol–Ficoll–sucrose solution and loading the embryos in a 0.25 ml French straw was effective for the cryopreservation of day 4 morulae and day 5 early blastocysts. To date, several live births after vitrification of day 4 morulae or day 5 early blastocysts have been obtained. However, it was still questionable whether this two-step simple method was applicable to blastocysts and expanded blastocysts. Only one liveborn had resulted from application of this vitrification protocol.

Our results, attesting an influence of the embryo stage development, correlated with the results obtained with mice embryos (Massip et al., 1984Go; Scheffen et al., 1986Go; Vanderzwalmen et al., 1988Go; Valdez et al., 1990Go; Zhu et al., 1993Go), bovine embryos (Vanderzwalmen et al., 1989Go; Tachikawa et al., 1993Go), sheep embryos (Ali and Shelton, 1993Go) and equine embryos (Hochi et al., 1995Go). All observed that cryopreservation methods developed for mice, bovine and sheep morula or early blastocysts were less effective for blastocysts. It was concluded from all these previous reports on mammalian embryos that every stage of development had its own mechanism relative to the permeation of cryoprotectants and the extent of dehydration during the addition of the cryopreservation solution.

Mechanisms responsible for the post-thaw low survival rate of blastocysts and expanded blastocysts are not yet fully understood. After changing variables such as type (Kasai et al., 1990Go), concentration (Ali and Shelton, 1993Go) or combination of cryoprotectants, the number of equilibration steps (Kuwayama et al., 1992Go; Ali and Shelton, 1993Go), the temperature (Vanderzwalmen et al., 1988Go) as well as the time of equilibration (Vanderzwalmen et al., 1988Go; Kasai et al., 1990Go; Nakamichi et al., 1993Go) in the cryoprotectant, an increase in the survival rate was obtained. However, this remained low compared with cases involving an early stage of embryo development.

In order to apply extended culture in sequential media in a clinical setting, it is essential to investigate causes responsible for the low survival rate of blastocysts after cryopreservation. It is difficult to explain discrepancies in survival rate. The difference in volume of the cells between morulae and the blastocysts and the presence of the blastocoelic cavity are two factors to consider.

If we consider only the volume of the blastomeres constituting the morula and the blastocyst, we can expect a better prevention of ice crystal formation in the more advanced stage. Due to the small volume of the blastomeres forming the blastocysts, the concentration of the cryoprotectant (ethylene glycol) normally increases faster inside the cells, allowing a sufficient permeation of the cryoprotectant and a more rapid equilibration before freezing. Furthermore, smaller blastomeres are less sensitive to osmotic stress and, consequently, less osmotic injury when the cryoprotectant is removed (Tachikawa et al., 1993Go).

However, another factor that can affect the survival rate is that the blastocyst consists of a fluid-filled cavity called the blastocoele. The likelihood of ice crystal formation is directly proportional to volume and inversely proportional to viscosity and the cooling rate. A decrease in survival rate after vitrification was noticed when the volume of the blastocoelic cavity increased. We suggest that an insufficient permeation of ethylene glycol inside the cavity might cause ice crystal formation during the cooling step, reducing the post-warming survival. Intra-blastocoelic water, which is detrimental to vitrification, may remain in the cavity after a 3 min exposure to EG20 solution.

The inclusion of a macromolecule such as Ficoll, present outside the trophoblast and ICM cells, protects the outer part of the embryo against crystallization. The cytoplasm of the blastomeres contains various intrinsic macromolecules that increase during equilibration and favour the amorphous state. Inside the blastocoele however, there must be few macromolecules. Due to the short exposure time to the EG20 and EFS solution, a low concentration of permeable cryoprotectant is present inside the cavity, probably not sufficient to protect the blastocysts against formation of ice crystals inside the blastocoele. Early blastocysts can survive after the vitrification procedure probably because the initial amount of liquid is reduced.

According to such an hypothesis, the present study investigated the effectiveness of reducing artificially the fluid from the blastocoelic cavity when touching the trophectoderm cells with a glass pipette. After the artificial shrinkage of the blastocyst, the post-thaw survival rate increased dramatically, suggesting the negative influence of the cavity using the two-step vitrification procedure with ethylene glycol as the main permeating cryoprotectant. The beneficial effect of removing the blastocoelic fluid is also manifest when blastocysts are artificially shrunk before slow freezing procedure (personal observation). The present study showed that decreasing artificially the volume of the blastocoele has a beneficial effect on the post-thaw survival rate of blastocysts vitrified inside a 0.25 ml French straw. We can therefore suggest that the blastocoelic fluid was the source of injury probably due to the presence of ice crystals inducing a mechanical damage. The technique of performing the artificial shrinkage procedure before vitrification has resulted, since 1998, in the birth of several babies.

If the lower viability of blastocysts was related to an insufficient permeation of cryoprotectant inside the cavity, alternatives other than the artificial shrinkage can be advocated in order to reduce the negative effect of the blastocoele. Increasing either the time of exposure or temperature to EG20 and/or EFS solutions or a stronger dehydration by increasing the sucrose concentration are alternative options. Using a solution of cryoprotectant containing 1 mol/l sucrose, it was suggested (Ali and Shelton, 1993Go) that the prior removal of the blastocoelic fluid could be beneficial and could enhance the survival of vitrified sheep blastocysts.

In our protocol of vitrification, the embryos were inserted in a 0.25 ml French straw and the rate of cooling bordered –2000°C/min. Under insufficient permeation of cryoprotectant, the amorphous state could be reached by increasing the speed of cooling. Therefore, in recent years, high speed vitrification methods, allowing cooling rate up to 20 000°C/min were developed. This requires loading of the blastocyst in a small volume of solution and increasing the contact between the cryoprotectant solution and liquid nitrogen. (Vatja et al., 1997Go; Hamawaki et al., 1999Go; Lane et al., 1999Go; Choi et al., 2000Go). Using the same principle, our preliminary results showed an increase in the survival rate and an acceptable pregnancy and implantation rate after loading the blastocysts on the open wall of a hemi-straw (Vanderzwalmen et al., 2000Go). After 28 vitrification assays of blastocysts and expanded blastocysts, we observed a clinical pregnancy rate and an implantation rate of 28.6% (eight pregnant out of 28 transfers) and 21.5% (11 fetuses out of 51 transferred blastocysts) respectively. However, when intact, very expanded blastocysts are vitrified in this way, the post-thaw survival rate is still lower as compared with less expanded blastocysts. Therefore, we suggest that a combination of artificial shrinkage with very fast vitrification protocol, as in the hemi-straw system, would be useful for the cryopreservation of very expanded blastocysts.

Immediately after warming and dilution of the cryoprotectant, it is difficult to assess exactly the viability of the embryos at the stereomicroscopic level. Under high magnification (x200), if only minor morphological changes are detectable, a prolonged culture period would be necessary to evaluate the re-expansion and the viability of the embryos. The beneficial effect of the artificial shrinkage, assessed by the rate of blastocysts that survived after thawing and that re-expanded, is encouraging. However, considering the satisfactory level of re-expansion and the optimal morphological aspect of the blastocysts before transfer, we found that the pregnancy and implantation rates of blastocyst and expanded blastocyst after artificial shrinkage were below our expectation.

Independent factors that are not linked to the vitrification process, such as the effect of the artificial shrinkage procedure and the in-vitro culture conditions, may also explain the lower effectiveness achieved after vitrification of artificially shrunk blastocysts.

Our results of blastocoele shrinkage achieved before a fresh transfer showed no harmful effect of the artificial shrinkage procedure. A comparable pregnancy rate was achieved after transfers of re-expanded blastocysts.

Another factor that can affect the viability of blastocysts to implant resides in the in-vitro culture conditions. The introduction of sequential culture media allows the production of blastocysts without the need for co-culture with feeder cells. Notwithstanding the fact that fresh blastocysts give good implantation rates, it has been shown (Dumont et al., 1999Go) that cryopreserved IVF human embryos obtained in sequential media are less viable than cryopreserved blastocysts after co-culture. According to one study (Massip et al., 1995Go), inappropriate in-vitro culture conditions can also explain the low pregnancy rates obtained after blastocyst freezing. They report that a high survival rate of cryopreserved, in-vitro cultured, embryos requires improvement in the techniques of maturation and culture, rather than simple changes in cryopreservation methods.

In conclusion, this vitrification procedure using a solution of ethylene glycol–sucrose–Ficoll solution as cryoprotectant is simple and efficient for the cryopreservation of day 4 morulae or day 5 early blastocysts in sealed 0.25 ml French straws. Current results strongly support the concept outlined above, namely that the results were dependent upon the structural differences between earlier stage embryos and blastocysts. In the case of blastocysts exhibiting a large fluid-filled cavity, artificial reduction of the blastocoele has a beneficial effect on their post-thaw survival rate.

In order to improve blastocyst culture, efforts have to focus on better in-vitro culture conditions and to adopt specific cryopreservation methods for each developmental stage of the embryo, especially for expanded blastocysts. Recent attention has been focused on extremely rapid cooling as a more successful method for the cryopreservation of blastocysts. Our preliminary results with the extremely high-speed cooling of blastocysts are encouraging. A combination of this technique associated with the artificial shrinkage of very expanded blastocysts seems promising.

Compared with the conventional vitrification procedure, the artificial shrinkage of blastocysts or expanded blastocysts improves the results after vitrification, but the efficacy of the method compared with the rate of implantation after fresh blastocysts transfers is still not optimal and needs further investigation. Therefore, in order not to reduce the patients' chances, we recommend the cryopreservation of some zygotes with conventional freezing procedures. The remaining embryos should be left in culture until the blastocyst stage for transfer and vitrification.


    Notes
 
4 To whom correspondence should be addressed. E-mail: pierrevdz{at}hotmail.com Back

Submitted on June 28, 2001


    References
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 Abstract
 Introduction
 Materials and methods
 Discussion
 References
 
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accepted on November 5, 2001.