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

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 [(3)H]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.     

Evidence that glucose is not always an inhibitor of mouse preimplantation development in vitro

A factorial experimental design was used to examine the effects of 16 combinations of four concentrations of glucose (0.20, 0.60, 1.8, 5.4 mmol/l) and four concentrations of potassium dihydrogen phosphate (KH(2)PO(4); 0.05, 0.15, 0.45, 1.35 mmol/l) on the development in vitro of outbred CF1 mouse zygotes. Three responses were measured: (i) the number of zona-enclosed blastocysts; (ii) the number of blastocysts that started to hatch; and (iii) the total cell counts in the blastocysts. General linear modelling was used to estimate the most parsimonious two-dimensional concentration-response surfaces that represent the three responses to the different concentrations of glucose and KH(2)PO(4). There were no significant interactions between the effects of glucose and KH(2)PO(4) in all cases. Thus, the effects of glucose and phosphate are independent. No significant effects of glucose on blastocyst formation and the initiation of hatching were observed. Increasing the concentration of KH(2)PO(4) inhibited slightly (相似文献   

Energy substrate requirements for in-vitro development of hamster 1- and 2-cell embryos to the blastocyst stage

Energy substrate requirements (pyruvate, lactate and amino acids) were determined for in-vitro development of hamster 1- and 2-cell embryos to blastocysts, using a chemically defined, protein free medium (hamster embryo culture medium, HECM). One-cell embryos were very sensitive to energy substrate type and concentration. Pyruvate alone could not support development of 1-cell embryos to greater than 4-cells, whereas lactate as sole energy substrate supported 14% development into morulae/blastocysts. Pyruvate, with lactate and 20 amino acids, inhibited 1-cell embryo development into blastocysts relative to lactate and 20 amino acids. The highest development of 1-cell embryos to blastocysts (up to 27%) occurred with reduced lactate concentration (less than 10 mM) and either 20 amino acids or 0.2 mM glutamine. Hamster 2-cell embryos were much less sensitive to energy substrates, requiring only lactate for development to blastocysts (53%). Lactate with 20 amino acids supported 70-75% of 2-cell embryos to blastocysts. Glutamine as sole energy and nitrogen source supported development to morulae and blastocysts of some 2-cell, but not 1-cell, embryos. Pyruvate did not enhance development of 2-cell embryos. We conclude that (i) altering the types and concentrations of available energy substrates drastically changes the developmental responses of 1-cell hamster embryos in vitro and (ii) energy substrate requirements for hamster embryo development in vitro are markedly different from those of mouse embryos, the standard model for studies on preimplantation development. This is the first report of successful in-vitro culture of hamster 1-cell embryos to the blastocyst stage.     

The effects of cryopreservation and thawing on the development in vitro and in vivo of biopsied 8-cell mouse embryos.

The possible impact of cryopreservation on biopsied 8-cell mouse embryos was investigated. Biopsied and control 8-cell embryos were cryopreserved using a slow freezing and quick thawing protocol with 1,2-propanediol as a cryoprotectant. The cryopreservation process did not affect either the recovery or the survival of biopsied embryos, when compared with intact controls; however, sham controls survived significantly better than biopsied 8-cell embryos (88.6 versus 74.2%, P less than 0.001). When fully and partially intact surviving embryos were cultured in vitro to the blastocyst stage, there was no difference in the proportions of embryos which formed blastocysts (biopsy 97.2%, intact control 98.4% and sham control 93.7%). The developmental potential and fetal development in vivo following embryo transfer were not impaired when assessed on day 17 of pregnancy. Cryopreservation of biopsied 8-cell mouse embryos is therefore a feasible approach to storing embryos while analysis of the biopsied material is carried out.     

Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage

Normally fertilized human embryos biopsied 3 days after in-vitro fertilization (IVF) have been examined for effects on viability and development in vitro after removal of one or two cells at the 8-cell stage (1/8 and 2/8) from each embryo. A high proportion of 7/8 and 6/8 biopsied and unmanipulated embryos developed to the blastocyst stage between days 5 and 6 (79, 71 and 59%, respectively), and many biopsied embryos (56%) hatched from the zona pellucida in vitro. The viability of biopsied embryos which developed to the blastocyst stage was assessed by daily non-invasive measurement of the uptake of two energy substrates, glucose and pyruvate. Uptake of both substrates was generally lower in 7/8 and 6/8 biopsied embryos but only in proportion to the reduced cellular mass. The total cell number and the numbers of both trophectoderm (TE) and inner cell mass (ICM) cells in biopsied embryos at the blastocyst stage, counted by differential labelling of their nuclei, were also reduced in proportion but the ratio of ICM to TE cells was maintained in both 7/8 and 6/8 biopsied embryos. We conclude that removal of one or two cells at the 8-cell stage, while reducing the cellular mass, does not adversely affect the preimplantation/development of biopsied embryos in vitro and suggest that this approach could be used for preimplantation diagnosis of genetic defects.     

The formation of mesoderm and mesectoderm in presomite rat embryos cultured in vitro, using WGA-Au as a marker

Summary Presomite rat embryos cultured in vitro were injected with the cell marker wheat germ agglutinin-gold in order to find out whether the ectoderm already formed mesodermal cells. These labelled so-called mesectodermal cells were found in all embryos studied, ranging in age from 8.7 to 9.3 days post coitum. In embryos younger than 9.0 days, the entire head fold ectoderm produced mesectodermal cells. From 9.0 days onwards, the neural crest and surface ectoderm placodes were recognizable as separate entities, both producing mesectodermal cells. The early onset of mesectodermal cell formation and the numerous and continuous manufacture led us to the conclusion that mesectodermal cells are deposited at their definitive location and that subsequent long-distance migration is unnecessary.     

The formation of mesoderm and mesectoderm in 5- to 41-somite rat embryos cultured in vitro, using WGA-Au as a marker

Summary The formation of mesectodermal cells by the neural crest in 5- to 41-somite stage embryos was investigated experimentally in rat embryos cultured in vitro, using lectincoated colloidal gold as a probe. This method labelled all ectodermal cells, among them neural crest, surface ectodermal placodal and epiblastic (primitive streak) cells. The neural crest provides the mesodermal compartment of the entire head region with cells, including the primitive cranial ganglia and the branchial arches. In the head region migration of neural crest cells over a great distance (long-distance migration) was not observed. In the trunk region neural crest derived cells were mainly found to form the primitive spinal ganglia and the sympathetic trunk, once again without long-distance cell migration. Structures and tissues that supposedly were derived from the primitive streak were hardly labelled with colloidal gold. Surface ectodermal placodes were not only found at the expected sites (e.g. epibranchial placodes) but also in the ectoderm covering the transverse septum and lateral abdominal walls.     

A study of diclofenac-induced teratogenicity during organogenesis using a whole rat embryo culture model.

BACKGROUND: Diclofenac is a non-steroidal anti-inflammatory drug, commonly used by reproductive age women for the treatment of a variety of conditions. However, there is limited information regarding the teratogenic effects of this drug. METHODS: The effect of diclofenac on the developing embryo during the critical period of organogenesis was investigated by using a whole rat embryo culture model. Embryos were exposed to various concentrations of diclofenac and scored for growth and differentiation at the end of the culture period. RESULTS: Total developmental score and score for caudal neural tube, flexion and hindlimb were significantly lower in embryos exposed to high concentrations of diclofenac (7.5 and 15.0 microg/ml), but no difference in these parameters was observed when embryos were exposed to low concentration of diclofenac (1.5, 2.5 and 5.0 microg/ml). No significant differences in yolk sac diameter, crown-rump length and number of somites was found between embryos in the experimental and the control group. CONCLUSIONS: Our study has demonstrated that diclofenac exerts direct teratogenic effects on rat embryos. Until more is known about the effects of diclofenac (especially in moderate to high doses) in women of reproductive age, we suggest its use should be treated with caution.     

Effects of cryopreservation on survival and development of interphase- and mitotic-stage 1-cell mouse embryos.

The effects of cryopreservation with 1,2-propanediol on two groups of 1-cell mouse embryos were studied in terms of survival after thawing, growth in vitro until the blastocyst stage and development in vivo assessed by the number of implantations and living fetuses. The two groups were divided according to different stages in the cell cycle: cells in (i) interphase with two distinct pronuclei or (ii) mitosis just prior to the first cleavage division. Zygotes in the interphase stage proved to be more resistant to freezing and thawing procedures, showing a significantly higher survival rate after thawing than zygotes in mitosis (78.5 versus 61.3%, P < 0.05). Blastocyst formation was similar in the two experimental groups: 72.7% for interphase and 60.8% for mitosis (P = 0.06), but for both groups fewer blastocysts formed when compared with the control group (86.7%) (P < or = 0.01). The implantation rates were not statistically different: 54.2% for the interphase cells and 47.4% for the control group and 44.0% for the mitotic cells and 49.4% for the control group. The formation of living fetuses was similar between the experimental and control groups: 36.5% for the interphase group (40.0% for its control group) and 22.6% for the mitotic group (38.8% for its control group). We conclude that freezing embryos during nuclear division is detrimental for their survival after thawing.     

Effects of supplemental L-methionine on E-64 [trans-epoxysuccinyl-1-leucyl-amido (4-guanido) butane]-induced dysmorphology in rat embryos cultured in vitro

ABSTRACT  E-64 [trans-epoxysuccinyl-1-leucyl-amido (4-guanido) butane] is teratogenic, inducing a spectrum of malformations in vivo and producing similar effects in vitro. Numerous studies support the concept that E-64-induced malformations result from embryonic nutritional deficiency, without affecting the maternal nutritional status. This has provided a useful model with which to investigate the nutritional requirements of the early embryo, as well as the role of various nutrients in the etiology of congenital defects.
In the current investigation, we examined effects of L-methionine on E-64-induced embryotoxicity in vitro. For these experiments, we cultured rat embryos 9.5 days postconcep-tion (p.c.) for 48 hours with E-64 and/or L-methionine. We found that the addition of L-methionine to E-64-exposed cultures reduced optic abnormality and increased embryo protein. These results suggest that embryopathy largely results from a deficiency of L-methionine although E-64 limits the supply of all amino acids to the embryo. Furthermore, although endocytosis and degradation of proteins by the visceral yolk sac (VYS) supply most amino acids to the embryo, free amino acids may be compensatory when this source is reduced. These results support those of previous investigations that suggest L-methionine is a limiting nutrient for embryonic development.     

Inhibitory effect of glucose and phosphate on the second cleavage division of hamster embryos: is it linked to metabolism?

Hamster embryos cannot complete the second cleavage divisionin vitro in the presence of glucose and inorganic phosphate(Glu/Pi). Embryos can recover from an 8 h (12: 30–20:30 h) exposure to Glu/Pi, although cleavage is retarded by 10h and continued development to the blastocyst stage is rare(2%). The vulnerability of 2-cell embryos to Glu/Pi changesduring the cell cycle, being greatest just prior to cleavagewhich occurs at –21: 00 h: exposure to Glu/Pi from 12:30–14: 30 h allowed 94% development to morulae/blastocystswhile exposure from 18: 30–20: 30 h decreased morula/blastocystdevelopment to 48%. This inhibitory effect is also modulatedby the length of exposure and energy substrate composition ofthe culture medium. It is unclear how Glu/Pi interferes withcleavage, but a metabolic explanation is likely because (i)metabolic inhibitors L-glyceraldehyde, iodoacetate and cystamineexacerbated the effect and (ii) the organization of active mitochondriaobserved with Rhodamine 123 and confocal microscopy is disturbedby Glu/Pi.     

Co-culture of 1-cell outbred mouse embryos on bovine kidney epithelial cells: effect on development, glycolytic activity, inner cell mass: trophectoderm ratios and viability

In an attempt to enhance embryo development, we have co-cultured1-cell OF1 mouse embryos on bovine kidney epithelial (Madine-Darbybovine kidney; MDBK) cells in a complex medium called complexmouse tubal fluid (cMTF; based on the energy substrate levelsfound in the mouse oviduct, containing non-essential amino acids,glutamine and EDTA). To determine the quality of the blastocystsobtained, we examined several parameters: morphology, totalcell numbers, inner cell mass (ICM): trophectoderm (TE) ratio,glycolytic activity and viability after transfer. A significantlylower number of blastocysts developed on MDBK cells comparedwith cMTF medium. cMTF blastocysts had a significantly higherglycolytic activity and a lower blastocyst cell number thanthose grown in co-culture, while both in-vitro groups had higherICM: TE ratios compared with in vivo. Blastocysts grown on MDBKcells displayed an elevated ICM number compared with those grownin cMTF medium alone. However, the percentage of fetuses aftertransfer remained drastically low in both culture groups comparedwith in-vivo blastocysts. In conclusion, co-culture did notincrease the number of zygotes reaching the blastocyst stage.Although co-culture blastocysts show some similarities to in-vivoembryos in cell number and glycolytic activity, no enhancementin viability was observed.     

Fertilization and early embryology: Alleviation of the '2-cell block' and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters

The role of amino acids, ethylenediaminetetraacetic acid (EDTA),transferrin, oxygen, glucose, glutamine, taurine and ammoniumin CF1 mouse zygote development in culture was examined. Non-essentialamino acids and glutamine were shown to alleviate the 2-cellblock in culture, and acted in synergy with EDTA to facilitatedevelopment to the blastocyst stage. In the presence of aminoacids and EDTA, transferrin conferred no beneficial effect Developmentof zygotes was significantly impaired if amino acids were removedfrom the collection medium, even when they were subsequentlycultured in the presence of amino acids. Zygote developmentto the blastocyst stage was significantly improved when modularincubator chambers were used compared to using a conventionalincubator, and when an oxygen concentration of 7% was used asopposed to 20%. Addition of taurine to medium containing non-essentialamino acids had no effect on embryo development, whereas theremoval of glutamine and/or glucose from the culture mediumsignificantly reduced blastocyst cell number. Removal of glucosefrom the culture medium also resulted in a significant decreasein implantations. Ammonium, generated from the breakdown ofamino acids, significantly reduced blastocyst development EDTAwas found to confer its beneficial effects during the first48 h of culture, and indeed was inhibitory during the second48 h, resulting in loss of subsequent viability. In summary,the data demonstrate that development of CF1 zygotes to theblastocyst stage is readily achievable. In the presence of non-essentialamino acids and glutamine the removal of glucose is detrimentalto CF1 mouse embryo development in culture and reduces subsequentviability. Optimal development and maintenance of viabilityrequires more than one culture medium to support the preimplantationperiod.     

Improved development of human embryos in vitro by a human oviductal cell co-culture system.

This study was undertaken to determine the effect of co-culture with human oviductal cells on human embryos. Spare embryos from gamete intra-Fallopian transfer (GIFT), pronuclear stage transfer (PROST) and in-vitro fertilization/embryo transfer (IVF/ET) programmes were either cultured in serum-supplemented Earle's balanced salt solution alone, or co-cultured in the same solution with oviductal cells from the pronuclear stage (day 1 post-insemination) or two- to four-cell stage (day 2 post-insemination). The co-cultured embryos appeared to have a higher developmental potential (higher rate of blastocyst formation and lower fragmentation rate), although there was no statistical difference in their rate of development, degree of fragmentation and stages attained, when compared with conventionally cultured embryos. The percentage of hatching blastocysts was significantly higher (P less than 0.05, Fisher's exact test) for embryos co-cultured from day 1 post-insemination (38%) than for embryos which had not been co-cultured (7%). The blastocyst hatching rate for embryos co-cultured from day 2 post-insemination was 15%. It was therefore concluded that co-culture of human embryos with oviductal cells could improve the development of the embryos in vitro. The degree of improvement was more pronounced when the co-culture started at an earlier stage.     

Similar delivery rates in a selected group of patients, for day 2 and day 5 embryos both cultured in sequential medium: a randomized study

BACKGROUND: The existence of a real benefit of blastocyst transfer is still a matter of debate. The aim of this study was to compare, in a prospective randomized trial, the outcome of day 2 and day 5 transfer of human embryos cultured in an 'in-house' sequential medium. METHODS: A total of 193 cycles from 171 patients with less than four previous IVF cycles, <39 years old and with four or more zygotes on day 1, were randomly allocated to day 2 (94 cycles) or day 5 (99 cycles) transfer. Zygotes were kept in fertilization medium until 18 h post-fertilization and then placed in a 'glucose-free' cleavage medium. Embryos allocated for day 5 transfer were placed in a blastocyst medium 66 h post-fertilization. Two or three embryos were replaced according to the morphology. RESULTS: A mean (+/- SEM) number of 2.1 +/- 0.4 and 1.9 +/- 0.3 embryos were replaced on day 2 and day 5 (P < 0.001) respectively. Delivery rates per transfer were 44.1 and 37.1% [P = not significant (NS)], implantation rates were 31.4 and 29.4% (NS) and multiple delivery rates 22 and 36% (NS) for day 2 and day 5 groups respectively. Ten patients (10.1%) had no blastocysts available for transfer. CONCLUSIONS: No clear benefits were observed using blastocyst transfer for patients aged <39 years who had had less than four previous IVF cycle attempts.     

Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos.

BACKGROUND: The effects of exposure time (20 versus 45 s) to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) and freezing rates (1200-10 300 degrees C/min) during rapid freezing of mouse pronuclear stage embryos on survival and development to blastocysts were investigated. Different freezing rates were achieved by directly plunging the straws (rapid freezing) and open pulled straws (OPS) in liquid nitrogen (OPS freezing) and by plunging the straws (super rapid) and OPS (super OPS) in a super cooled liquid nitrogen chamber (at -212 degrees C) before storage in liquid nitrogen. METHODS: Morphologically intact mouse zygotes (n = 891) pre-equilibrated in 1.5 mol/l ethylene glycol for 5 min were either loaded in 0.25 ml straws containing cryoprotectant or loaded in OPS with 2 microl cryoprotectant. After 20 or 45 s of loading the straws or mixing in cryoprotectant and loading in OPS, they were plunged either directly in to liquid nitrogen or were plunged first in to liquid nitrogen in a super cooled chamber and then stored in liquid nitrogen. Zygotes were thawed and intact embryos cultured in vitro. RESULTS: The rate of survival was higher (91%, P < 0.01) when zygotes were frozen with rapid freezing compared with super rapid, OPS and super OPS freezing rates with an exposure time of 20 s (70, 65, and 76% respectively). When zygotes were exposed to cryoprotectant for 45 s and frozen with rapid freezing rates, the survival was higher (86%, P < 0.01) compared with those frozen with OPS (62%) but was not different from those frozen with super rapid and super OPS freezing rates (81 and 75%). A higher rate of survival was observed when zygotes were exposed to cryoprotectant for 45 s and frozen with super OPS than with OPS freezing (75 versus 62%; P < 0.05). The rate of cleavage and development of intact zygotes to blastocysts was not different among the different groups. CONCLUSION: Exposure of zygotes to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) for 20 or 45 s did not influence their survival and development and increasing the freezing rate from 1200-10 300 degrees C/min was of no advantage when using a rapid freezing procedure for freezing mouse pronuclear stage embryos.     

Effect of damage to the zona pellucida on development of preimplantation embryos in the mouse

The effect on development of early mouse embryos of making ahair-line slit in the zona pellucida of approximately one-thirdits diameter was investigated. The rate of development to mid-gestationof operated zygotes and2-cell embryos transferred directly tothe oviduct was significantly lower than that of sham-operatedor unoperated controls. However, the operation had no discernibleeffect on the development of 2-cel embryos that were culturedfor 2 days prior to transfer to the uterus, or on embryos composedof 8 or more cells transferred directly to the oviduct. Zonaslit zygotes and 2-cell embryos exhibited a significantly higherrate of anomalous development to the morula or blastocyst stagethan controls following short-term transfer to the adult orimmature oviduct. Such anomalies could not be attributed todamage of the embryos by leucocytes or bacteria entering throughthe wound in the zona. Rather, the typically non-spherical shapeof slit zonae, together with the fact that some were empty onrecovery, was consistent with operated embryos having been damagedby compression during passage through the oviduct. This suggeststhat, providing it is intact, the zona pellucida protects theearly embryo from contraction of the oviductual musculaturewhich is sufficient to lyse, arrest or extrude blastomeres priorto the formation of intercellular junctions. Hence, in experimentalmanipulations entailing damage to the zonae of early embryos,there may be a case for allowing them to form morulae in vitroprior to transfer, rather than returning them directly to theoviduct     

The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo: BAT6 a new medium for improved culture of mouse embryos in vitro

The effect of iron and iron chelators on the development of the mouse embryo in vitro from the 1-cell stage to the blastocyst has been investigated. An adverse effect of iron was found. The high affinity iron chelator, desferal, also blocked development, whilst transferrin (whether as apoprotein or saturated with iron), DETAPAC and EDTA promoted development. The addition of transferrin permitted development to the blastocyst stage of embryos from stains normally exhibiting the 2-cell block. Under such circumstances both the rate of embryonic development and the proportion of embryos reaching the blastocyst stage approached levels found in vivo. Based on these results, a new medium, BAT6, is described for the optimal in-vitro culture of mouse embryos.     

An in-vitro study of ginsenoside Rb1-induced teratogenicity using a whole rat embryo culture model

BACKGROUND: Ginseng is a commonly used herbal medicine worldwide. However, there is limited information regarding its effects on the developing embryo. METHODS: The effect of ginsenoside on the developing embryo during the critical period of organogenesis was investigated using a whole rat embryo culture model. Embryos were exposed to various concentrations of ginsenoside Rb(1) and scored for growth and differentiation at the end of the culture period. RESULTS: Median total morphological scores in embryos exposed to 30 micro g/ml of ginsenoside Rb(1) was significantly lower (P < 0.05) than that in control embryos (35 versus 45). Morphological scores for flexion, forelimb and hindlimb were also significantly reduced. The median total morphological scores further decreased to 28 when the concentration of ginsenoside Rb(1) was increased to 50 micro g/ml. At this concentration, the embryonic crown-rump length and somite number were also significantly reduced compared with control embryos (2.8 versus 3.0 mm and 16.0 versus 21.0, respectively). CONCLUSIONS: Our study has demonstrated that ginsenoside exerts direct teratogenic effects on rat embryos. Until more is known about the effects of ginsenoside in women of reproductive age, we suggest its use should be treated with caution.