Comparison of the effects of controlled-rate cryopreservation and vitrification on 2-cell mouse embryos and their subsequent development

Hiroto Uechi1,1, Osamu Tsutsumi,32, Yutaka Morita1,1, Yasushi Takai1,1 and Yuji Taketani1,1

1 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo and 2 CREST, Japan Science and Technology, Kawaguchi, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of two cryopreservation procedures (conventional slow controlled-rate freezing using a programmable freezer and vitrification by direct plunging into liquid nitrogen) were compared on 2-cell embryos and their subsequent development to blastocysts, fresh or cryopreserved 2-cell mouse embryos were developed into blastocysts in vitro. The percentage of vitrified embryos which developed into blastocysts was significantly lower than that of fresh and slow controlled-rate frozen embryos. Although blastocysts from each cryopreservation procedure appeared morphologically normal and neither number of cells in the blastocysts nor in-vitro trophoblast spreading differed significantly, there were significant differences in their functional viability. First, the glucose incorporation activity in terms of [3H]2-deoxyglucose (2-DG) uptake in vitrified and thawed 2-cell embryos significantly decreased compared with fresh or slow controlled-rate frozen and thawed 2-cell embryos. Second, 2-DG uptake by blastocysts developed in vitro from fresh 2-cell embryos and from slow controlled-rate frozen or vitrified 2-cell embryos was 105 ± 75, 43.0 ± 28.3 and 22.0 ± 11.4 fmol/embryo/h respectively. Third, the implantation rate of blastocysts developed in vitro from vitrified 2-cell embryos (10.2%) was significantly lower than that from fresh 2-cell embryos (30.8%) or slow controlled-rate frozen 2-cell embryos (22.1%). Since these data suggest that cryopreservation may have ulterior consequences on the functional development of embryos and that vitrification may exert a more harmful effect than slow controlled-rate freezing, more attention should be paid to its safety before vitrification is used routinely in a clinical programme.

Key words: glucose incorporation/implantation/mouse/preimplantation embryo/slow controlled-rate cryopreservation/ultrarapid cryopreservation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subsequent to the first report of successful cryopreservation of mouse embryos (Whittingham et al., 1972Go), live offspring have been produced from frozen–thawed embryos in many other mammalian species, including humans (Trounson and Mohr, 1983Go). Cryopreservation has become increasingly important in assisted reproductive techniques since it offers the potential advantages of reducing the risk of multiple births, while increasing the number of embryo transfers and hence pregnancies per retrieval (Van Steirteghem et al., 1992Go). However, cryopreservation of human embryos significantly reduces their capacity for implantation (Levran et al., 1990Go). Successful cryopreservation depends at least in part on the mode of the freezing–thawing procedure. Slow controlled-rate cryopreservation is conventionally achieved via commercially available computer-controlled cell-freezing systems. This procedure requires expensive equipment and is time consuming. Vitrification was described as a simple method of directly transferring embryos into liquid nitrogen after a brief exposure to a cryoprotectant solution (Rall and Fahy, 1985Go). Although this is a very attractive alternative to conventional slow controlled-rate cryopreservation, studies should have been carried out to determine its safety before using a new procedure for human embryo preservation on a routine basis.

Vitrification involves the addition of high concentrations of cryoprotectants which at extremely low temperatures are in an amorphous state without crystallizing. The original vitrification solution consisted of permeating compounds [dimethylsulphoxide (DMSO), acetamide and propylene glycol] and a macromolecular compound (polyethylene glycol) (Rall et al., 1987Go). Later, a new cryoprotectant solution was developed (Ishida et al., 1997Go) containing 40% ethylene glycol, 18% Ficoll and 0.3 mol/l trehalose, modifying an earlier one (Kasai et al., 1990Go) containing ethylene glycol, Ficoll and sucrose. Ethylene glycol, a permeating compound, has an important role in stabilizing the cellular membrane during freezing, though it also has some harmful effects on embryo development (Kasai et al., 1990Go). Ficoll is used as a low osmotic effect macromolecule to increase the viscosity of the medium. Trehalose, providing a non-permeating solution with significant osmotic effects, is a natural cryoprotectant that can be found in yeast, fungal spores, brine shrimp cysts and some soil-dwelling nematodes (Sussman and Lingappa, 1959Go). It seems to prevent alteration to the cellular membrane during reduced water states but the mechanism is still not well understood (Rudolph and Crowe, 1985Go).

Research comparing of the effect of conventional slow controlled-rate freezing and vitrification has been focused on embryo cleavage and implantation capacity and the results were controversial. Some studies have reported no statistical difference between the two procedures in blastocyst formation and implantation capacities of mouse (Rall and Wood, 1994Go) or bovine (Van Wagtendonk-De Leeuw et al., 1995Go) embryos, while Dinnyes et al. (1995) reported that vitrification yielded significantly higher rates of implantation than those achieved after slow freezing using mouse embryos. However, little consideration has been devoted to functional or metabolic aspects of the embryo following cooling. We have recently reported that the slow controlled-rate freezing–thawing procedure of mouse embryos decreases not only development rate to blastocyst stage, but also decreases glucose incorporation of the developed blastocysts due to decreased expression of GLUT1, suggesting that cryopreservation may have ulterior consequences on the functional development of embryos (Uechi et al., 1997Go). We therefore decided to compare the efficacy of slow controlled-rate freezing and vitrification by assessing developmental potential of cryopreserved–thawed 2-cell mouse embryos into blastocysts in vitro and their ability to incorporate glucose as well as morphological features and implantation rate in recipient mice.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Embryos
Eight to 10 week old Crj; CD-1 (ICR) female mice were superovulated with 5 IU of pregnant mare's serum gonadotrophin followed 48 h later by 5 IU of human chorionic gonadotrophin (HCG). Mating with males of the same strain was verified by the presence of a vaginal plug. Two-cell embryos were obtained at 44 h after HCG administration by flushing the oviducts. The embryos were placed in 2 ml of modified Biggers–Whitten–Whittigham (mBWW) medium (Biggers et al., 1971Go), and cultured in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 48 h to obtain blastocysts in vitro. Randomly selected 2-cell embryos were simultaneously slowly frozen or vitrified as described below. They were thawed a few days later and then cultured in the same manner as fresh embryos. The percentage of blastocycts developed from 2-cell embryos was calculated after a total of 92 h in culture rather than the more usual 120 h post HCG to avoid allowing less viable embryos to `catch up' with more viable ones and thus blur the differences. In-vivo developed blastocysts were collected from the uteri as described previously (Suenaga et al., 1996Go).

Slow controlled-rate freezing and thawing procedures
Slow controlled-rate freezing and thawing of 2-cell embryos were carried out (Fugger et al., 1988Go). The embryos were placed in l.5 mol/l propanediol (PROH; Sigma, St Louis, MO, USA) in mBWW containing 0.3% bovine serum albumin (BSA) for 15 min at room temperature. The embryos were transferred to the same medium with 0.1 mol/l sucrose (Sigma) for 15 min and then loaded into 0.25 ml plastic straws filled with the same medium. Freezing was carried out in a programmed freezer (Cryoembryo-HP, Hoxan, Tokyo, Japan). Straws were cooled from room temperature down to –7°C at a rate of –2°C/min. Seeding was automatically induced during 15 min at this temperature. The straws were then slowly cooled down to –30°C at –0.3°C/min and then at –50°C/min to –140°C. After holding at –140°C for 5 min, they were plunged into liquid nitrogen for storage. A few days later, embryos were thawed by removing the straws from liquid nitrogen and keeping them at room temperature for 40 s. They were then hand-held until totally thawed. Cryoprotectants were removed stepwise at room temperature by transferring embryos successively (every 5 min) into mBWW supplemented with 0.3% BSA containing l.5 mol/l PROH + 0.1 mol/l sucrose, 1.0 mol/l PROH + 0.2 mol/l sucrose, 0.5 mol/l PROH + 0.2 mol/l sucrose, and then into 0.2 mol/l sucrose. Sucrose was finally removed by placing the embryos in mBWW.

Vitrification and thawing procedures
Vitrification and thawing of 2-cell embryos were carried out (Ishida et al., 1997Go). The embryos were placed in 40% ethylene glycol, 18% Ficoll, 0.3 mol/l trehalose in phosphate-buffered saline (PBS) containing 0.3% BSA for 5 min at 4°C. The embryos were then loaded into 0.25 ml plastic straws filled with the same medium. Straws were plunged directly into liquid nitrogen for storage. A few days later, embryos were thawed by removing the straws from liquid nitrogen and keeping them at room temperature for 40 s. They were then hand-held until totally thawed. Cryoprotectants were removed stepwise at room temperature by transferring embryos successively (every 5 min) into mBWW supplemented with 0.3% BSA containing 0.35 mol/l trehalose and then into 0.2 mol/l trehalose. Trehalose was finally removed by placing the embryos in mBWW.

Cell number of blastocysts
Blastocysts were added to Hoechst 33258 (Bisbenzimide H33258 Fluorochrome; Wako, Osaka, Japan) and left for 15 min at room temperature (Tarkowski, 1966Go). Observation was carried out under ultraviolet light using fluoroscein microscopy (Model BX50; Olympus, Tokyo, Japan) and the number of nuclei in each blastocyst was counted (Tsutsumi et al., 1998Go).

Trophoblast spreading of blastocysts
Trophoblast spreading of cultured blastocysts was quantitatively analysed as described previously (Suenaga et al., 1996Go). Blastocysts developed in vitro either from fresh, frozen–thawed, or vitrified–thawed 2-cell embryos were transferred to F0-CMRL medium (Suenaga et al., 1996Go) supplemented with fetal bovine serum at a concentration of 20% (v/v), and cultured in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 96 h. The surface areas of the trophoblast spreads were quantitatively evaluated using a digitizer tablet (Model DT1000; Watanabe Sokki, Tokyo, Japan) connected to a personal computer (Model 9801, NEC, Tokyo, Japan).

2-Deoxyglucose uptake
Measurement of [3H]2-deoxyglucose (2-DG, Amersham, Little Chalfont, Bucks, UK, 17 Ci/mmol) uptake was performed as described (Morita et al., 1992Go). Fresh, frozen–thawed, and vitrified–thawed 2-cell embryos and blastocysts developed in vitro were incubated in 15 µl of mBWW solution containing 25 µmol/l 2-DG instead of glucose. They were incubated for 60 min at 37°C under an atmosphere of 95% air and 5% CO2 with 100% moisture. Each embryo was then washed five times with 100 µl of glucose-free mBWW solution. The uptake of 2-DG into each embryo was counted in a Beckman scintillation counter with 1 ml of Aquasol solution.

Embryo donation model
Seven blastocysts of each experimental group, i.e. blastocysts developed in vitro either from fresh, frozen–thawed, or vitrified–thawed 2-cell embryos were transferred surgically to the tip of one or the other uterine horn in the recipient mice on day 3 of pseudopregnancy as described previously (Morita et al., 1994Go). On day 9 of gestation, the recipients were killed and autopsied and the implantation rate was calculated as the ratio of implanted embryos to transferred blastocysts.

Statistical analysis
Statistical analysis was performed using Student's t-test and the {chi}2-test. Statistical significance was established at the P < 0.05 level.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
After slow controlled-rate freezing and vitrification, 81.6% (746/914 embryos) and 77.4% (230/297) of 2-cell embryos were recovered with normal morphology respectively. In-vitro developed blastocysts were obtained by explanting either fresh, frozen–thawed, or vitrified–thawed 2-cell embryos and allowing them to develop in vitro for 48 h. The percentage of normally developed blastocysts from slowly frozen–thawed 2-cell embryos (32.8%; 198/603 embryos) was significantly lower compared with that of fresh embryos (47.1%; 248/526 embryos) (Figure 1Go). The control blastocyst formation rate starting from 2-cell embryos (47.1%) was rather low and may have been related to the mouse strain used (see below). It may also have been caused by us making our observations at 92 h post HCG rather than at 120 h which is more usual. The lowest blastocyst formation rate (22.3%; 124 in 556 embryos) was observed when vitrified–thawed 2-cell embryos were cultured in vitro, which was significantly lower than those of fresh and slowly frozen–thawed embryos (Figure 1Go). Morphologically, those blastocysts developed in vitro with or without cryopreservation could not be distinguished from those developed in vivo obtained 92 h following HCG administration from the mouse uteri. In addition, the number of cells in these blastocysts developed from frozen– and vitrified–thawed 2-cell embryos did not differ significantly from those developed in vitro from fresh 2-cell embryos (Table IGo). On the other hand, blastocysts from fresh embryos exhibited a significantly faster rate of trophoblast spreading after 96 h culture (Figure 2AGo) than those from frozen–thawed (Figure 2BGo) and vitrified–thawed (Figure 2CGo) 2-cell embryos, but there was no significant difference between the two cryopreservation procedures (Table IIGo).



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Figure 1. Development rates of fresh, slowly frozen–thawed, and vitrified–thawed 2-cell mouse embryos to blastocysts. Two-cell embryos were cultured in vitro for 48 h to yield blastocysts.*P < 0.001 compared with development rate of fresh 2-cell embryos. #P < 0.05 compared with that of slowly frozen–thawed 2-cell embryos.

 

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Table I. The number of cells in the blastocysts developed in vitro from fresh, slowly frozen–thawed, and vitrified–thawed 2-cell mouse embryos
 


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Figure 2. Representative photomicrographs showing the trophoblast spreading after 96 h culture of blastocysts developed from fresh (A), slowly frozen–thawed (B), and vitrified–thawed (C) 2-cell embryos. Bar = 100 µm.

 

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Table II. Comparison of the surface areas of the trophoblast spreading after 96 h culture of blastocysts with or without cryopreservation procedure
 
2-Deoxyglucose (2-DG) uptake in 2-cell embryos and blastocysts developed in vitro from 2-cell embryos with or without cryopreservation is shown in Figure 3. GoSlow controlled-rate freezing–thawing procedure itself did not alter the uptake of 2-DG in 2-cell embryos. However, 2-DG uptake by 2-cell embryos after vitrification was significantly lower than that of fresh 2-cell embryos and that after slow controlled-rate cryopreservation (Figure 3Go). There was significant increase in the uptake of 2-DG in the blastocysts developed in vitro compared with the corresponding 2-cell embryos irrespective of presence or absence of cryopreservation (Figure 3Go). 2-DG uptake increased >6-fold in the blastocysts developed in vitro compared with the fresh 2-cell embryos. An ~3-fold significant increase in 2-DG uptake was observed in blastocysts developed from slowly frozen–thawed embryos. However, glucose incorporation of the blastocysts developed from slowly frozen–thawed embryos was significantly lower than that developed from fresh 2-cell embryos. Significant increase was observed in 2-DG uptake also in blastocysts developed from vitrified–thawed embryos, but the glucose incorporation of these blastocysts was significantly lower than those blastocysts that had not undergone the freezing–thawing procedure and those that experienced the slow controlled-rate freezing–thawing procedure (Figure 3Go).



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Figure 3. 2-Deoxyglucose (2-DG) uptake in mouse 2-cell embryos and blastocysts developed from fresh (open bars), slowly frozen–thawed (hatched bars), and vitrified–thawed (closed bars) 2-cell embryos. The `T' on the top of the bars indicates SD. Numbers of samples of embryos and blastocysts appear in parentheses. There was significant increase in the uptake of 2-DG in the blastocysts developed in vitro compared with the corresponding 2-cell embryos, P < 0.01 for fresh and slowly frozen–thawed embryos,P < 0.05 for vitrified–thawed embryos. Significant differences:*P < 0.05, **P < 0.001 respectively.

 
The highest rate of implantation was observed when blastocysts developed in vitro from fresh 2-cell embryos were transferred to recipient mice; 41/133 embryos (30.8%) implanted successfully (Table IIIGo). The implantation rate of blastocysts developed from slowly frozen–thawed embryos was lower than that from fresh ones but no significant difference was observed between these two groups of blastocysts. The lowest implantation rate was observed when blastocysts developed from vitrified–thawed 2-cell embryos were transferred, which was significantly lower compared with that of blastocysts developed in vitro from fresh or slowly frozen–thawed embryos (Table IIIGo).


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Table III. Implantation rates of blastocysts developed in vitro from 2-cell embryos with or without cryopreservation
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The rate of embryonic development differs between cryopreserved embryos and fresh embryos as shown here (Figure 1Go) and previously (Fugger et al., 1988Go; Selick et al., 1995Go; Uechi et al., 1997Go). In the present study, the percentage of development to blastocysts from vitrified–thawed 2-cell embryos, after 48 h culture, was significantly lower compared with that from fresh and slowly frozen–thawed 2-cell embryos (Figure 1Go). These data confirm that cryopreservation of early embryos is lethal to some embryos and suggest that vitrification, though simple and less time consuming, is more detrimental than conventional slow controlled-rate cryopreservation, when performed on mouse 2-cell embryos.

In contrast with our present study, there have been many studies so far reporting good results with vitrification. Two main reasons may explain our obviously poor rates of blastocyst development, i.e. mouse strain and the developmental stage at which mouse embryos undergo cryopreservation. The Crj; CD-1 (ICR) mouse strain we used is inbred, showing a developmental rate of ~50% as shown in our present study and recent publication (Tsutsumi et al., 1998Go). Our vitrification protocol is almost the same as that previously reported (Ishida et al., 1997Go), but they used a B6C3F1 hybrid mouse strain. Ethylene glycol-based cryoprotectant solution similar to that in our present study had previously been used (Kasai et al., 1990Go) but with morula stage embryos. Using a later developmental stage such as 8-cell and morula caused a higher proportion of vitrified embryos to develop to blastocysts (Miyake et al., 1993Go). 8-cell embryos of an ICR mouse strain were used (Mukaida et al., 1998Go) and they reported that ethylene glycol-based cryoprotectant solution was more suitable for vitrification than PROH-, DMSO-, acetamide-, or glycerol-based solution. The reason that we used 2-cell embryos of ICR mouse strain is that we assumed that any difference in detrimental effect between the two cryopreservation procedures may be emphasized if `suboptimal' developmental stage embryos of a `suboptimal' strain is used instead of optimal stage embryos of an optimal strain, as previously shown (Kasai et al., 1990Go; Ishida et al., 1997Go). Moreover, we believe that our data obtained using 2-cell embryos of ICR mice are helpful for the improvement of the freezing–thawing procedure for human embryos in clinical practice which sometimes do not have optimal quality.

These data concerning the rate of embryonic development to blastocysts also raise a question whether or not slow controlled-rate freezing and vitrification procedures exert an `all or nothing' effect on these mouse embryos. Therefore, we focused on the comparative effects of these cryopreservation procedures on the quality and viability of those embryos that survived and developed into blastocysts. However, blastocysts developed in vitro from slowly frozen–thawed or vitrified–thawed 2-cell embryos could not be morphologically distinguished from blastocysts developed from fresh 2-cell embryos and the number of cells in the blastocysts did not differ significantly between the three groups (Table IGo). It has been reported that ultrarapid freezing of mouse oocytes lowers the cell number in the inner cell mass of day 5 blastocysts (96 h after 2-cell embryos) (Van der Elst et al., 1998Go). Similarly, in the present study, there was a slight decrease in the surface area of trophoblast spreading after 96 h culture (120 h after 2-cell embryos) of blastocysts developed from cryopreserved 2-cell embryos compared with fresh 2-cell embryos. However, there was no significant difference between the two cryopreservation procedures (Figure 2Go and Table IIGo). Thus, we attempted to assess the viability of embryos by measuring their glucose uptake because current experimental data indicate that there is an alteration in the uptake or metabolism of glucose in early stage embryos (Leese and Barton, 1984Go; Khurana and Wales, 1987Go; Butler et al., 1988Go; Brison and Leese, 1991Go; Morita et al., 1992Go). Indeed, it has been shown that glucose uptake can be used to select prospectively viable blastocysts immediately after thawing (Gardner et al., 1996Go).

In the present study, it is of interest to note that 2-DG uptake in 2-cell embryos was significantly decreased by vitrification compared with those of fresh or slowly frozen–thawed 2-cell embryos (Figure 3Go). It is postulated that vitrification itself causes an alteration in embryonic quality by affecting functional integrity of the 2-cell embryos. Cellular glucose uptake is dependent upon a family of glucose transporter proteins that contain multiple membrane-spanning domains (Birnbaum et al., 1986Go; James et al., 1989Go; Orci et al., 1989Go). A more plausible explanation for this decrease in glucose incorporation activity is that during the vitrification–thawing procedure, glucose transporters in the membrane of the blastomeres of embryos are damaged, resulting in a decreased 2-DG uptake in the embryos.

The 2-DG uptake in blastocysts was significantly higher than the respective 2-cell embryos in all three groups. However, 2-DG uptake in the blastocysts developed in vitro from cryopreserved–thawed 2-cell embryos was significantly lower than that of the blastocysts developed in vitro from fresh 2-cell embryos (Figure 3Go). As we have already reported (Uechi et al., 1997Go), this decrease in 2-DG uptake of morphologically normal embryos may reflect a delayed effect of cryopreservation, suggesting that it may have ulterior consequences on the functional development of embryos. Moreover, 2-DG uptake of the blastocysts developed from vitrified–thawed 2-cell embryos was significantly lower than that of the blastocysts developed from slowly frozen–thawed 2-cell embryos. These data indicate that different types of freezing–thawing procedure have different degrees of delayed effect as detected by 2-DG uptake, and that it is not an `all-or-nothing' type of effect that is assessed by 2-DG uptake assay. Since glucose incorporation activity is dependent on glucose transporter GLUT1 expression in early embryos (Morita et al., 1994Go) and impaired GLUT1 expression is reported in slowly frozen–thawed embryos (Uechi et al., 1997Go), further investigation into mechanisms responsible for the gene expression may help to understand the impact of cryopreservation on the metabolic activity of embryos.

The viability of each group of embryos was assessed also by an embryo donation model that provides a way of determining whether a developmental failure occurs due to a defect in the embryo or in the environment. The implantation rate of blastocysts developed from vitrified–thawed 2-cell embryos was significantly diminished compared with that of blastocysts developed from fresh and slowly frozen–thawed 2-cell embryos (Table IIIGo). This suggests that the quality of the blastocysts developed after vitrification might be impaired as a result of delayed effects or consequences and thus implantation capacity might be reduced although they were morphologically indistinguishable from those obtained after slow controlled-rate freezing in terms of cell number and trophoblast spreading. This has implications for the application of cryopreservation technology as well as for cryobiology. Empirical studies are necessary for continued development of techniques that will maximize success rates and minimize time and expense of cryopreservation procedures and thus mechanisms of cryoinjury and its prevention may be understood.

Studies on perinatal outcome and follow-up of babies conceived from cryopreserved embryos have shown no pathological features (Wada et al., 1994Go; Olivennes et al., 1996Go). However, embryos that survive to blastocysts following vitrification may have some cryoinjury not in an `all or nothing' way since they appear to be normal in morphology and are capable of further development. It was reported recently that slow controlled-rate freezing is more efficient than ultrarapid cooling, not vitrification, for human embryos (Van den Abbeel et al., 1997Go). It remains to be determined whether offspring from vitrified embryos are phenotypically and genetically normal in all regards. Before vitrification is used routinely in clinical in-vitro fertilization programmes, its safety must be convincingly demonstrated.


    Acknowledgments
 
We would like to thank Dr Siya S.Sharma (Assistant Professor, Department of Obstetrics/Gynaecology, Kasturba Medical College, Manipal, India) for critical reading of our manuscript. This work was supported by grants from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare, Japan.


    Notes
 
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7–3–1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on March 9, 1999; accepted on July 22, 1999.