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PubMed1996

Non-enzymatic formation of formaldehyde in mouse oocyte freezing mixtures.

Three cryoprotectant solvents, dimethylsulphoxide, 1,2-propanediol and glycerol, were investigated for a non-enzymatic reaction product, formaldehyde. All three cryoprotectants demonstrated a direct relationship between increasing solvent molarity and increasing formaldehyde concentration which was independent of temperature and protein (bovine serum albumin). Medium composition significantly influenced the formaldehyde concentration with HTF > T6 > M16 = M2. The formaldehyde could be effectively removed by reduced glutathione, cysteine and dithiothreitol with cysteine being the most effective scavenging agent. A reaction mechanism for this scavenging is proposed. The combination of cysteine and cryoprotectant reduced the zona pellucida 'hardening' effect in mouse oocytes.

Animals↗
PubMed1995

Cryopreservation of human oocytes and fertilization by two techniques: in-vitro fertilization and intracytoplasmic sperm injection.

Human oocyte cryopreservation results in poor survival and subsequent fertilization rates. It has been suggested that freeze-thaw-induced changes in the zona pellucida may impair sperm penetration or attachment. The aim of this study was to compare fertilization and cleavage rates in cryopreserved oocytes inseminated by conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). A total of 220 oocytes, obtained from volunteers who had undergone ovarian stimulation, were cryopreserved using a slow freeze-rapid thaw protocol with 1.5 M propanediol as the cryoprotectant. Surviving oocytes (n = 74, 34.4%) were randomly allocated for fertilization by conventional IVF (group 1) or ICSI (group 2) using cryopreserved spermatozoa from a single donor of proven fertility. Fertilization was achieved in five (13.5%) of the oocytes in group 1 and 17 (45.9%) in group 2 (P < 0.005), with only one oocyte in group 1 exhibiting normal fertilization as opposed to 16 (43.2%) in group 2 (P < 0.001). Similarly, one oocyte fertilized by IVF cleaved, while all fertilized with ICSI cleaved (P < 0.001). We conclude that although the survival of oocytes is poor following cryopreservation, fertilization and cleavage rates can be enhanced significantly using ICSI. These data also suggest that the method of cryopreservation used in this study affected the zona pellucida, such that normal sperm attachment or penetration was impaired.

Cleavage Stage, Ovum↗
PubMed1995

Effect of cryoprotectants and their concentration on post-thaw survival and development of rapid frozen-thawed pronuclear stage mouse embryos.

Experiments have been conducted to develop a simple rapid-freezing protocol for pronuclear stage mouse embryos. The effect of type of cryoprotectant (dimethyl-sulphoxide (DMSO) or 1,2-propanediol (PROH)) and concentration (ranging from 3.5 to 8.0 mol/l with 0.5 mol/l sucrose) on the post-thaw morphological survival rate, on cleavage rate on development to the blastocyst stage were studied. Further, in-vivo viability of embryos frozen using the most effective cryoprotectant concentration (PROH at 7.0 mol/l) was compared with viability of non-frozen embryos. The type of cryoprotectant and its concentration influenced the survival and development of embryos to the blastocyst stage in vitro. The best development with PROH was achieved at 7.0 mol/l (66%, 128/193), whereas with DMSO the best development was achieved at 6.0 mol/l (42%, 71/171). The rates of survival and cleavage did not differ between the two best cryoprotectant concentrations (P > 0.01) but the proportion of embryos which developed to blastocyst was higher (P < 0.001) with PROH at 7.0 mol/l compared with DMSO at 6.0 mol/l. The rates of survival and development were higher (P < 0.001) with DMSO at 3.5 and 6.0 mol/l compared with similar concentrations of PROH. The cleavage and development, however, was higher (P < 0.001) at 7.0 mol/l PROH compared with the same concentration of DMSO. At 8.0 mol/l the survival and development was not different (P > 0.01) between DMSO and PROH. The rate of implantation and the percentage of live fetuses at autopsy of the recipients receiving non-frozen embryos was higher (63 and 41% respectively) than in those receiving frozen-thawed embryos (53 and 37% respectively), but not significantly different. It may be concluded that the concentration range of cryoprotectants which allows acceptable embryo viability after freezing and thawing is very narrow. The rapid protocol using dehydration in 0.25 mol/l and 0.5 mol/l sucrose followed by exposure to 7.0 mol/l PROH and 0.5 mol/l sucrose for 45 s was the most effective for cryopreservation of pronuclear stage mouse embryos.

Animals↗
PubMed1995

The effect of equilibration temperature and time on the outcome of ultrarapid freezing of 1-cell mouse embryos.

Mouse 1-cell embryos were frozen ultrarapidly at a rate of approximately 2500 degree C/min in solutions containing 0.25 M sucrose, 0.5% (w/v) bovine serum albumin (BSA) and 3 or 4.5 M dimethyl sulphoxide (DMSO) or 3 or 4.5 M 1,2-propanediol (PROH) in HEPES-buffered modified Earle's medium. We investigated the effect of pre-freeze equilibration for 1, 3, 5 or 10 min at 22 degrees C and for 1, 3, 5, 10, 15 or 20 min at 4 degrees C. After thawing in a 22 degrees C water bath at a rate of approximately 2500 degrees C/min and dilution in 1 M sucrose in HEPES-buffered modified Earle's medium, embryos were cultured in vitro in bicarbonate-buffered modified Earle's medium with 0.5% (w/v) crystalline BSA. Embryo viability was expressed as the percentage of hatching or hatched blastocysts resulting from the initial number of frozen-thawed 1-cell embryos. To determine the toxicity of the freezing solutions, embryo viability was evaluated after equilibration without freezing. Our results demonstrated that the concentration, the equilibration temperature and time are very important factors in ultrarapid freezing of mouse 1-cell embryos. Optimal viability was found when equilibration was done in 4.5 M DMSO for 3-5 min at 22 degrees C and in 4.5 M PROH for 3-5 min at 4 degrees C. The results with regard to exposure to the freezing solutions indicated that the loss of viability beyond an optimum is not due solely to cryoprotectant toxicity, in particular not at 4 degrees C and not for DMSO.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Volumetric behaviour and survival of mouse zygotes and embryos in hyperosmotic media.

Volume changes and survival of mouse zygotes and pre-implantation embryos were studied after their exposure to hyperosmotic media. Transfer of zygotes from culture medium to medium containing 1.5 M DMSO led to a rapid volume reduction to 60% of control values. After 10 min the volume was normal again, illustrating the rapid penetration of DMSO at room temperature. Re-transfer of zygotes equilibrated in 1.5 M DMSO to culture medium in one step doubled the volume. Twelve minutes later normal diameters were observed again. Volume changes were also recorded after transfer to media rendered hypertonic by the addition of NaCl or sucrose. A proportional decrease in volume was noticed up to 1500 mOsmol. A further increase in measured osmolarity up to 3100 mOsmol led only to limited further shrinkage of the zygotes (minimal volume observed was 27% of control volume). Addition of 1.5 M DMSO or 1.5 M propanediol had no influence on the final volume reaction to NaCl and sucrose. Exposure to media with a measured osmotic value of 3200 mOsmol (NaCl added) during 10 min did not prevent further development of the embryos to the hatched blastocyst stage. A solution of 4000 mOsmol proved lethal to nearly all embryos. Addition of 1.5 M DMSO to this medium did not have a protective effect against the osmotic stress at room temperature.

Animals↗
PubMed1987

Cryopreservation of human embryos obtained after gamete intra-Fallopian transfer and/or in-vitro fertilization.

During a one-year period 636 excess embryos obtained after in-vitro fertilization and gamete intra-Fallopian transfer combined with in-vitro fertilization were cryopreserved using two different protocols. For early stage embryos including the pronucleate stage, 1,2-propanediol was used as cryoprotectant (procedure A, adapted from Renard) and for later stage embryos dimethylsulphoxide was used in protocol B, adapted from Trounson and Mohr. After thawing 288 embryos, half of them were of sufficient quality to be replaced. After cryopreservation, procedure A gave the best survival in embryos having less than or equal to 2 blastomeres; for later stage embryos best survival was obtained using the dimethylsulphoxide protocol. Survival after cryopreservation was also clearly related to the quality of the embryos prior to freezing. Embryos were replaced during endocrinologically monitored natural cycles and were transferred in synchrony between endometrial and embryonic age. After replacement of 126 embryos in 110 patients, 20 pregnancies occurred. So far six healthy children have been born, two patients aborted and 12 pregnancies are ongoing. In this series no statistical difference was observed between the implantation rate of embryos cryopreserved by procedure A or B. Six pregnancies occurred in patients from the oocyte and embryo donation programme. An adequate cryopreservation programme circumvents the difficult problem of synchronizing the ovarian cycles of donor and acceptor patients.

Embryo Transfer↗
PubMed1988

Cryopreservation of human embryos and oocytes.

The success rate of human embryo cryopreservation depends on technical and embryonic parameters. First of all, the cryoprotectant can affect embryo survival as we found by comparing two freeze-thaw procedures using propanediol (PROH) (1.5 mol) alone or with sucrose (0.1 mol). Embryo survival was significantly enhanced with sucrose (62 versus 32%). Embryo quality is another major parameter involved in the success of freezing; the rates of positive survival were found to be 67% for morphologically normal embryos versus 49% for embryos with fragments (P less than 0.001). The efficiency of embryo cryopreservation in an IVF programme could be estimated in 1986: a woman with extra embryos, stored after transfer of 3-4 fresh embryos (16% of all cycles), can expect a 22% pregnancy rate per transfer of fresh embryos and a 32% pregnancy rate per collection after transfer of the stored eggs. A comparative study of the cryopreservability of immature or mature oocytes was performed in humans. Human oocytes have a low survival rate (36%) whatever the cryopreservation protocol or the initial maturation stage. Immature human oocytes could survive freezing and thawing, mature and be fertilized in vitro, but with a very low efficiency.

Embryo Transfer↗
PubMed1989

Successful embryo transfer of cryopreserved and in-vitro fertilized rabbit oocytes.

In-vitro fertilization experiments with frozen/thawed rabbit oocytes were performed to develop an effective technique to be used for the in-vitro fertilization of cryopreserved human oocytes. Ovulatory oocytes, collected from the oviduct of virgin does 13 h after induction of ovulation by HCG injection, were cryopreserved slowly to -30 degrees C and plunged directly into liquid nitrogen. A mixture of 1.5 M 1,3-propanediol and 0.1 M sucrose was used as a cryoprotectant. After thawing, the oocytes were incubated with in-vitro capacitated sperm for 5 h in defined Brackett's medium. Fertilized ova were cultured for an additional 20 h until the 4-to-8-cell stage was reached. These embryos were transferred to pseudopregnant recipient rabbits which were 'asynchronous' in the sense that they had been given an injection of HCG 30, 24 and 18 h before starting to do the embryo transfer. A 32% survival rate of frozen/thawed oocytes was achieved. The fertilization rate was 74% (181/264) in this study. A total of 53 embryos was transferred to the oviducts of six recipients of three different asynchronicity and four young were born. The highest implantation rate (including resorptions) of 18% could be achieved in this investigation by using -6 h asynchronous recipients, while the overall implantation rate was 9.4%.

Animals↗
PubMed1989

Development of mouse embryos cryopreserved by an ultra-rapid method of freezing.

High concentrations of cryoprotectant combined with sucrose were utilized in an ultra-rapid freezing protocol for mouse preimplantation embryos. Dimethylsulphoxide (DMSO, 1.5 or 3.5 M) or propanediol (PROH, 1.5 or 3.0 M) combined with 0.25 M sucrose were used as freezing solutions. One-, 2- or 8-cell embryos were placed directly into these solutions at room temperature, loaded into straws and plunged into liquid nitrogen within 2-3 min. The straws were rapidly thawed and the embryos expelled into the solution in which they were frozen for 10 min. The cryoprotectants were then removed by single- or multi-step dilution. Survival and development of the embryos in vitro and in vivo were assessed. DMSO (1.5 M) and both concentrations of PROH were totally inadequate as a cryoprotectant in this freezing protocol. A concentration of 3.5 M DMSO gave high survival and development rates when a multi-step dilution procedure was used, but not with a single-step dilution. One-cell embryos gave 71% survival, 35% in-vitro development and 10% in-vivo viability; 2-cell embryos showed 87% survival, 77% in-vitro development and 66% in-vivo viability; and 8-cell embryos showed 97% survival, 87% in-vitro development and 62% in-vivo viability. The results for the 2- and 8-cell stages compared favourably with non-frozen controls, which had 71% in-vivo viability. This method of cryopreservation is therefore fast and viable.

Animals↗
PubMed1989

Comparative results on survival of human and animal eggs using different cryoprotectants and freeze-thawing regimens. I. Mouse and hamster.

The achievement of successful pregnancies and births after in-vitro fertilization and embryo transfer of frozen-thawed human oocytes has stimulated further work on improving the established methodology. The present investigation was conducted on 1837 mouse oocytes, 1785 mouse pronuclear stage embryos, 1400 hamster oocytes and 1024 hamster pronuclear-stage embryos. In an effort to study the advantages and disadvantages of a newly introduced, 1,2-propanediol (1,2-PROH)-based system over the conventionally used dimethylsulphoxide (DMSO)-based methodology, a direct, prospective comparison between the two cryoprotectants was undertaken in a randomized trial. The combination of 1,2-PROH and DMSO potentiates their cryoprotective effect on mouse and hamster eggs. 1,2-PROH seems to improve cryopreservation in the animal system, though not significantly. Four different protocols were used to evaluate the effects of two changing experimental parameters. These were, firstly, the intermediary temperature attained before placing the cells into liquid nitrogen and, secondly, the modification of the method of adding and removing the cryoprotectant. The morphological survival rate, fertilization rate and developmental rate were significantly better in the low intermediary temperature compared with the high intermediary temperature system of cryopreservation. In addition, the rate of zona pellucida breakdown diminishes considerably in the former compared with the latter system. The 'rapid' sucrose method of cryoprotectant equilibrium and removal showed, in most groups, results which were lower than, or equal to, the traditionally used 'multiple-step', 'slow' method.

Animals↗
PubMed1989

Comparative results on survival of human and animal eggs using different cryoprotectants and freeze-thawing regimens. II. Human.

A total of 269 human pronuclear embryos and 84 oocytes were subjected to four different protocols of cryoprotectant equilibration, washing out and freeze-thawing. The morphological survival, rate of development, fertilization in vitro and overall survival rate were estimated in the groups of fresh, aged oocytes, diploid and multipronuclear embryos used. With some restrictions, the conclusion can be drawn that slow, low intermediary temperature 1,2-propanediol (1,2-PROH) and 1,2-PROH/dimethyl sulphoxide (DMSO) systems are superior to the rapid, high intermediary temperature 1,2-PROH and the traditionally used DMSO systems. The best success rates were reached with the combination of cryoprotectants in the low intermediary temperature group. Thirty-three patients had 37 transfers of cryopreserved pronuclear embryos (n = 103), resulting in eight pregnancies (24.2% per transfer) thus doubling the pregnancy rate in the stimulation cycles for the same period (21% per transfer). Thawing at a rate of 50 degrees C/min is not incompatible with the survival of slowly frozen human oocytes and pronuclear embryos cooled to an intermediary temperature of -70 degrees C using the DMSO system.

Cell Survival↗
PubMed1990

Observations on the morphology of pronuclei and nucleoli in human zygotes and implications for cryopreservation.

The effects of cryopreservation on human zygotes at various stages between the appearance of pronuclei and their close association were investigated. Pronuclear zygotes (n = 233) from 101 patients were frozen using propanediol 21-35 h following egg collection. The incidence of implantation of thawed pronuclear zygotes frozen 29-35 h following oocyte collection was significantly higher than that of younger pronuclear zygotes (28 versus 10%, respectively). Zygote age did not affect cell survival following cryostorage. The diameter and association of pronuclei and the number and distribution of nucleoli were determined from video tape recordings of 140 fresh zygotes. Pronuclear migration continued after pronuclear enlargement. The number of nucleoli remained constant during pronuclear migration, but their random distribution within the pronucleus diminished. Strongly adhered pronuclei had significantly more aligned nucleoli on adjacent sides than pronuclei which were still visually separated by ooplasm. This equatorial distribution of nucleoli was noted in the majority of zygotes older than 26 h. The findings suggest that zygote cryopreservation should be initiated when pronuclear migration is completed. This moment can be determined accurately by studying pronuclear association and nucleolar alignment.

Cell Nucleolus↗
PubMed1990

Cryopreservation of pronucleate mouse ova: slow versus ultrarapid freezing.

The usefulness of ultrarapid freezing of mouse pronucleate ova was investigated in comparison with slow, programmed freezing. Pronucleate mouse ova were frozen using an ultrarapid method in either 3.5 M dimethylsulphoxide (DMSO), 3.5 M propanediol (PROH) or a 1:1 mixture of both. After a brief exposure to the cryoprotectant, they were plunged into liquid nitrogen. Thawing was done at 37 degrees C and the cryoprotectant was rapidly diluted in a sucrose solution. Pronucleate ova were slowly cooled in a biological freezer using 1.5 M PROH as cryoprotectant. Thawing was done at room temperature and PROH was removed by multi-step dilutions. As control groups, pronucleate ova were either given no treatment, or exposed to PROH or DMSO without freezing and cultured in vitro. Ultrarapid freezing using 3.5 M DMSO as cryoprotectant resulted in rates of survival, cleavage and post-thaw development to blastocysts of 85, 89 and 37%, respectively. PROH as cryoprotectant, however, was inadequate in the ultrarapid freezing protocol and the combination of DMSO and PROH showed no further improvement. Slow, programmed freezing (using PROH) compared to ultrarapid freezing (using DMSO) resulted in similar rates of survival, cleavage and development of 80, 84, 20% and 92, 81 and 23% respectively. In conclusion, DMSO is a better cryoprotectant than PROH for ultrarapid freezing of pronucleate mouse ova, and ultrarapid freezing with 3.5 M DMSO is as effective as slow freezing with 1.5 M PROH, as evaluated by their subsequent development in vitro.

Animals↗
PubMed1991

Clinical experience with ultra-rapid freezing of embryos.

A modified ultra-rapid freezing technique was introduced in January 1989 in our clinical in-vitro fertilization freezing programme. Thus, we wish to report our 1 1/2 year experience with ultra-rapid freezing as a simple and inexpensive method of routine cryopreservation. Of 181 frozen-thawed embryos, 110 (61%) survived with 50-100% blastomeres intact, and were used in 91 embryo replacements. Nine clinical pregnancies were established including two sets of twins. There was one miscarriage and one ectopic pregnancy. So far, five patients have delivered six normal, healthy infants and two other pregnancies are normally ongoing, including another twin pregnancy. These results were similar to those obtained by the conventional slow freezing method using propanediol. There is no doubt that there is a marked advantage of ultra-rapid freezing when compared to previous methods in terms of cost and time. We therefore believe that more clinical attention should be given to ultra-rapid freezing and it should become increasingly the method of choice for human embryo freezing in IVF programmes.

Clinical Protocols↗
PubMed1991

Pregnancy rates after transfer of embryos obtained from different stimulation protocols and frozen at either pronucleate or multicellular stages.

After in-vitro fertilization, 2161 supernumerary embryos were frozen with 1,2-propanediol and sucrose as cryoprotectants at either pronucleate or multicellular (2-6 blastomeres) stages. By the end of March 1990, 494 pronucleate stage embryos and 492 multicellular stage embryos had been thawed and 54 and 47% of them, respectively were considered suitable for transfer. Ongoing pregnancy and implantation rates were 17.9 and 10.7%, respectively for embryos frozen at the pronucleate stage and 5.5 and 4.7% for embryos frozen at the multicellular stage. Ovarian stimulation with human menopausal gonadotrophin (HMG) after pharmacological hypophysectomy with a gonadotrophin releasing hormone agonistic analogue (GnRHa) using a long protocol permitted us to freeze significantly more embryos per cycle (7.2 +/- 4.1) than stimulation with HMG and GnRHa in a short protocol (4.7 +/- 3.4) or stimulation with clomiphene citrate (CC) and HMG (2.7 +/- 1.9). Ongoing pregnancy rates after transfer during the stimulated cycles were similar for the three types of treatment (27.1, 27.3 and 32.1%, respectively). However, ongoing pregnancy rates after frozen-thawed embryo transfers were significantly higher when originating from GnRHa + HMG treatments (14.3 and 14.8%, respectively for long and short protocols) than when originating from CC + HMG treatment (5.6%). Embryo cryopreservation has permitted the ongoing pregnancy rate to increase from 28.4 to 36.9% (P less than 0.01) even though more than half of the embryos have not been thawed. We conclude that embryos obtained after stimulation with GnRHa + HMG and frozen at the pronucleate stage are more likely to result in a pregnancy.

Cryopreservation↗
PubMed1991

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.

Animals↗
PubMed1992

In vitro maturation of mouse germinal vesicle-stage oocytes following cooling, exposure to cryoprotectants and ultrarapid freezing: limited effect on the morphology of the second meiotic spindle.

Cryopreservation of germinal vesicle (GV)-stage mouse oocytes results in a developmental block. As an approach to explain the failure in development, we have investigated the morphology of the second meiotic spindle after in-vitro maturation. Fully grown GV-stage mouse oocytes were collected from the ovaries of primed mice and kept in meiotic arrest with dibutyryl cyclic AMP. These oocytes were submitted to different variables of cryopreservation: (i) cooling to 22 degrees C or 0 degrees C; (ii) exposure to 1.5 M 1,2-propanediol at 22 degrees C or 0 degrees C; (iii) exposure to 1.5 M dimethylsulphoxide (DMSO) at 22 degrees C or 0 degrees C; (iv) ultrarapid freezing with 3.5 M DMSO/0.5 M sucrose; (v) exposure to a sucrose dehydration series according to the ultrarapid freezing protocol. The morphology of the second meiotic spindle was evaluated 16 h after release from meiotic arrest. We were able to demonstrate that following cooling, exposure to cryoprotectants or ultrarapid freezing of GV-stage mouse oocytes, a normal barrel-shaped spindle with the chromosomes in midplane position is found in 79-94% of oocytes except for two conditions with great exposure to dehydration stress. Exposure to DMSO at 0 degrees C or exposure to a sucrose dehydration series resulted in significantly lower percentages of barrel-shaped spindles, respectively 64% and 62%. The effect on spindle morphology has to be put into perspective, however, since the observed abnormalities were changes of spindle shape, such as elongation or reduction, which are assumed to be restorable, and since no polar organizational defects were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗