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PubMed1990

Successful ultrarapid freezing of unfertilized oocytes.

Successful application of ultrarapid freezing techniques to unfertilized murine oocytes has not been reported. In an effort to improve results, preovulatory murine oocytes were exposed to three ultrarapid freezing protocols involving varying sucrose concentrations (0.25, 0.5, and 1.0 M) and 3.5 M dimethyl sulfoxide (DMSO) as cryoprotectants prior to direct immersion in liquid nitrogen. Post-thaw morphology and rates of in vitro fertilization and embryo development were compared with those obtained after freezing oocytes employing two established programmed cooling techniques. The rates of fertilization and development to the blastocyst stage in vitro of oocytes undergoing ultrarapid freezing after exposure to 3.5 M DMSO and 0.5 M sucrose were similar or superior to those obtained with programmed cooling techniques. Of oocytes which appeared morphologically normal post-thaw, only those which underwent ultrarapid freezing with 0.25 or 0.5 M sucrose and 3.5 M DMSO reached the blastocyst stage at rates similar to those of controls. Ultrarapid freezing may represent a viable option for successful murine oocyte cryopreservation.

Animals↗
PubMed1996

Serotonin reuptake inhibitors reduce conditioned fear stress-induced freezing behavior in rats.

Conditioned fear stress (CFS)-induced freezing behavior has been proposed as an animal model of anxiety. In the present study, freezing was used to determine the anxiolytic activity of selective serotonin reuptake inhibitors (SSRIs), which are reported to be clinically effective in anxiety disorders. The duration of freezing behavior was reduced by acute treatment with the SSRIs citalopram (1-10 mg/kg) and fluvoxamine (3-30 mg/kg). Acute treatment with the serotonin (5-HT)/noradrenaline (NA) mixed reuptake inhibitor milnacipran (3-30 mg/kg) also attenuated CFS-induced freezing, while acute treatment with the NA reuptake inhibitors maprotiline and ORG4428, and the dopamine (DA) reuptake inhibitor GBR12909 failed to alter CFS-induced freezing. These results indicate that facilitation of 5-HT availability in the brain produced by 5-HT reuptake inhibition reduces CFS-induced freezing behavior. CFS may be a useful model for detecting the anxiolytic potential of 5-HT reuptake inhibitors.

Animals↗
PubMed1990

Footshock-induced freezing behavior in rats as a model for assessing anxiolytics.

A number of chemically distinct anxiolytics were examined for effects on defensive behavior (foot-shock-induced freezing) in rats. Central nervous system acting drugs which are not anxiolytics were also studied. Animals were injected with a drug or vehicle (IP) prior to being placed in a chamber with a grid floor through which two footshocks were delivered. Behavior was observed during the pre-shock period (2 min) and for 4 min after the second footshock. The effects of the following drugs on the duration of footshock-induced freezing were studied: diazepam (DZP); 2-amino-4,5-(1,2-cyclohexyl)-7 phosphonoheptonic acid (NPC 12626); 3-((+/-)-2-carboxypiperazine-4-yl)-propyl-l-phosphonic acid (CPP); [(+)-5-methyl-10-11,dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10- imine (MK-801); buspirone hydrochloride (BUS); DL-amphetamine sulfate (AMP); haloperidol (HAL); ethyl-beta-carboline-3 carboxylate (beta-CCE). Compounds which reduced the duration of footshock-induced freezing included DZP, BUS, and the competitive NMDA antagonists NPC 12626 and CPP. The non-competitive NMDA antagonist, MK-801, had no effect on the response. The highest dose of amphetamine tested also reduced footshock-induced freezing. However, amphetamine-treated animals did not locomote or rear after footshock, suggesting fear of the environment. Animals injected with DZP, NPC 12626, CPP or buspirone spent at least 1.4 of the 4 post shock minutes locomoting. Haloperidol had no effect on freezing at the doses tested. beta-CCE tended to increase the duration of footshock-induced freezing.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Isolation of freeze-tolerant laboratory strains of Saccharomyces cerevisiae from proline-analogue-resistant mutants.

Since some amino acids, polyols and sugars in cells are thought to be osmoprotectants, we expected that several amino acids might also contribute to enhancing freeze tolerance in yeast cells. In fact, proline and charged amino acids such as glutamate, arginine and lysine showed a marked cryoprotective activity nearly equivalent to that of glycerol or trehalose, both known as major cryoprotectants for Saccharomyces cerevisiae. To investigate the cryoprotective effect of proline on the freezing stress of yeast, we isolated proline-analogue-resistant mutants derived from a proline-non-utilizing strain of S. cerevisiae. When cultured in liquid minimal medium, many mutants showed a prominent increase, two- to approximately tenfold, in cell viability compared to the parent after freezing in the medium at -20 degrees C for 1 week. Some of the freeze-tolerant mutants were found to accumulate a higher amount of proline, as well as of glutamate and arginine which are involved in proline metabolism. It was also observed that proline-non-utilizer and the freeze-tolerant mutants were able to grow against osmotic stress. These results suggest that the increased flux in the metabolic pathway of specific amino acids such as proline is effective for breeding novel freeze-tolerant yeasts.

Amino Acids↗
PubMed1996

Serotonergic activation reduces defensive freezing in the conditioned fear paradigm.

Our previous study showed that conditioned fear stress (CFS) increased serotonin (5-HT) metabolism in the medial prefrontal cortex and induced freezing behavior. Although these results could support the 5-HT hypothesis of anxiety, the functional significance of the 5-HT response to stress is unclear. In this study, the effects of 5-HT reuptake inhibitors, agonists, antagonists, and diazepam on freezing behavior induced by CFS were examined using a time-sampling procedure. Various doses of test compounds were administered subcutaneously to rats 24 h after the last session of repeated foot-shock for 5 days. Rats were again placed in the shock chamber without shocks 20 min after injections of drugs, and observed. Diazepam (1 mg/kg) and the 5-HT1A agonist ipsapirone (0.5-10 mg/kg) significantly inhibited freezing behavior. L-5-Hydroxytryptophan (with benserazide) and the selective 5-HT reuptake inhibitor citalopram (10 mg/kg) reduced freezing behavior. The 5-HT2 antagonists ICI169,369 and ketanserin failed to change freezing behavior. p-Chlorophenylalanine (200 mg/kg) administered 15 h before the test did not affect freezing. The effect of ipsapirone was not modified in rats with lesions of 5-HT neurons, produced by p-chloroamphetamine (2 x 10 mg/kg). In conclusion, these results suggest the anxiolytic potential of ipsapirone and citalopram, and support the hypothesis that the facilitation of 5-HT neurotransmission decreases anxiety.

Animals↗
PubMed1987

Freezing bovine embryos with a portable liquid nitrogen freezer requiring no external seeding.

One-hundred and twenty excellent morula to blastocyst stage bovine embryos were obtained nonsurgically from superovulated Holstein heifers. Completely portable, nonelectric (manual) liquid nitrogen (LN) freezers combined with simplified freezing curves utilizing self-seeding were compared to a programmable LN freezer (Planner-R204) following the conventional freezing rate for freezing embryos. Seeding was self induced in ampules at -6.8 degrees C and at -5.5 degrees C in straws in the manual freezers. Glycerol was used as the cryoprotectant at 1.5 M concentration. Post-thaw appearance, fluorscein diacetate testing (FDA), and growth after 12 and 24 h incubation were used as indicators of embryo viability. There were no significant differences between embryos frozen in the two types of freezers in terms of the viability tests used. Pregnancy rates resulting from transfer of embryos frozen in the two types of freezers will be determined in subsequent field trials. The manual LN freezers used in this study are capable of successfully freezing bovine embryos. The simplified nature of these freezers and the freezing procedures used with them greatly decreases the complexity and expense of freezing embryos.

Journal Article↗
PubMed2005

Airborne PCB as a contamination risk on freeze-drying of sediment.

In a study of polychlorinated biphenyls (PCB) in freeze-dried sediments from Swedish lakes, the samples were found to contain unexpectedly high concentrations of PCBs. In one of the lakes (Björken), e.g. the concentration of PCB-28 was found to be 189 ng/g dw after freeze-drying compared to 7 ng/g dw after air-drying of the sediment and therefore our hypothesis was that the contamination had occurred during the freeze-drying procedure. Hence, a dry reference sediment (RS) was used in order to identify potential sources of PCB contamination. The investigation included freeze-drying of the dry RS in five different laboratories, exposure to the air in the freeze-drying room, storage at room temperature and air-drying in a certain fume hood and in a laminar flow clean bench. The pattern and concentrations of PCBs detected in the exposed RS were strongly influenced by low chlorinated PCB congeners under all of these conditions with exception for air-drying in the fume hood. Storage in the laboratory air resulted in a slight elevation in the concentrations of low chlorinated PCB congeners, whereas higher concentrations of all PCB congeners were observed after freeze-drying. In all experiments the contamination appeared to be due to adsorption of PCB from the laboratory air. On the basis of these findings we recommend that reference material be subjected to the entire procedure, including sample preparation, in order to monitor contamination and that a procedure involving solvent extraction of wet sediment samples is used, whenever possible, instead of procedures involving dry samples.

Adsorption↗
PubMed2005

Freezing of gait after bilateral subthalamic nucleus stimulation for Parkinson's disease.

OBJECTIVE: To determine the long-term effect of bilateral subthalamic nucleus (STN) deep brain stimulation (DBS) on freezing of gait in Parkinson's disease patients. PATIENTS AND METHODS: Sixty-seven patients were followed 12 months after bilateral STN DBS and 32 patients were followed 24 months post-surgery. Patients were evaluated with the Unified Parkinson's Disease Rating Scale (UPDRS) with medications on and off at baseline and again in both conditions with stimulation on at follow-up. Specific attention was paid to the freezing item of the ADL section. RESULTS: Freezing scores in the medication off state improved significantly with stimulation at 1 and 2 years. In the medication on state there were no significant improvements in freezing. CONCLUSION: Off period freezing of gait is significantly improved with STN DBS up to 24 months; however, on period freezing is not affected by STN DBS.

Adult↗
PubMed2005

Pharmacological dissociation of moderate and high contextual fear as assessed by freezing behavior and fear-potentiated startle.

The amplitude of the whole-body acoustic startle response is reliably enhanced when elicited in the presence of foreground signals, such as light, previously paired with footshocks. It has been shown that this enhancement is evident by moderate fear levels, but is less affected by high fear levels. Potentiation of the acoustic startle reflex has also been reported in the presence of background cues previously associated with footshocks. However, the effects of anxiolytic drugs on different levels of fear elicited by moderate and intense contextual fear conditioning associated with startle reflex have not been examined yet. To approach this issue, we examined the effects of the anxiolytic, midazolam, on two intensities of contextual fear; freezing behavior and the startle response to loud noise. First, we compared the magnitude of the freezing behavior and the startle amplitude during the testing sessions in groups of rats submitted to fear conditioning using 0.3 and 0.6 mA as unconditioned stimuli (10 stimuli of 1 s each, intertrial interval from 60 to 180 s). Afterwards, the effects of midazolam (0.5 and 1.0 mg/kg) were assessed in these two conditions. Rats showed a potentiated startle reflex and a significant freezing behavior to moderate fear conditioning, which were both attenuated by midazolam. Higher levels of fear conditioning caused more intense freezing behavior without enhancing the startle reflex. Whereas midazolam reduced this freezing response, the startle response was unaffected. These results are indicative that anxiolytic-sensitive freezing and fear-potentiated startle are triggered by moderate contextual fear conditioning, while contextual conditioning with the use of high footshocks causes a distinct pattern of behavioral responses, which is only partially affected by midazolam. Due to the differential sensitivity to midazolam of these two patterns of startle responses generated as a function of the intensity of contextual fear conditioning, it is proposed that they represent moderate and intense aversive states that may be related to anxiety or panic/phobic conditions, respectively.

Acoustic Stimulation↗
PubMed2005

Cryopreservation of boar semen: equilibrium freezing in the cryomicroscope and in straws.

Supercooling causes very abrupt temperature and osmotic changes and can thus lead to freezing damage. Supercooling can be prevented by seeding, using a sample volume and geometry that allows rapid spreading of the ice throughout the sample. In a split-sample comparison of such samples on the cooling stage of a cryomicroscope and seeded at -5 and -15 degrees C, respectively, the percentages of membrane-intact sperm and sperm with acrosomes with a 'normal apical ridge' (NAR) were 72.5+/-3.8 and 75.8+/-2.0 versus 46.3+/-4.8 and 36.0+/-3.7 (means+/-S.E.M., n=4). In ejaculates of 15 unselected AI boars, after seeding at -5 degrees C, the post-thaw % live and % NAR were 66.3+/-10.4 and 74.8+/-7.5, respectively. Our present research is aimed at translating these findings to freezing in straws and at a high sperm concentration. We have designed a novel type of freezing apparatus for controlled-rate freezing of straws, in which supercooling can be effectively prevented in the entire straw. In a split-sample comparison of semen frozen in straws at a sperm concentration of 1.5 x 10(9) cells/ml with nine ejaculates from eight unselected AI boars, we found 54.8+/-1.9% versus 40.7+/-1.7% (means+/-S.E.M.) membrane-intact sperm for the new apparatus and a conventional freezing apparatus, respectively. With bull semen (eight ejaculates from six bulls), we obtained 67.3+/-3.0% versus 59.3+/-2.9% (means+/-S.E.M.) membrane-intact sperm for the new apparatus and conventional freezing, respectively. Additionally, the temperature curve after ice nucleation is of great importance. We have developed a model that allows us to predict that optimal cryopreservation requires a non-linear cooling curve in which the cooling rate varies as a function of subzero temperature.

Animals↗
PubMed2005

Freezing of stored, chilled dog spermatozoa.

The aims of this study were to find out if dog spermatozoa can be stored chilled for 1 or 2 days prior to freezing without a deterioration in post-thaw vitality and longevity, and to compare two extenders; the Uppsala Equex-2 (UE-2) and a TRIS egg yolk extender (EYT). Pooled dog semen was frozen immediately after collection, or was extended and stored at 4 degrees C for 1 or 2 days before freezing. Sperm motility and acrosome integrity were evaluated before freezing and for 6h post thaw at 38 degrees C, while sperm plasma membrane integrity was evaluated post thaw. There were no effects of pre-freeze storage time or extender on post-thaw motility or plasma membrane integrity, but a significant effect of extender (P < 0.0153) on post-thaw acrosomal integrity was found, UE-2 being better than EYT. There was a significant (P < 0.0001) negative effect of post-thaw storage time on acrosome integrity, but this was not influenced by pre-freeze storage time or extender. In conclusion, we found that dog spermatozoa can be frozen after 1 or 2 days of cold storage without significant deterioration in post-thaw motility, acrosome integrity or sperm plasma membrane integrity compared to when frozen immediately after collection. The UE-2 extender was superior to the EYT extender for freezing of cold stored dog spermatozoa.

Acrosome↗
PubMed2007

Dialysis of boar semen prior to freezing-thawing: its effects on post-thaw sperm characteristics.

Low-molecular weight components of the seminal plasma have a detrimental effect on sperm function. The present study was undertaken to evaluate the effect of the removal of low-molecular weight components by dialysis on sperm characteristics prior to and after freezing. Semen, collected from 5 boars, was extended in Kortowo-3 extender (K-3, Poland) and cooled for 3h (control non-dialysis) or dialyzed for 5h in semi-permeable dialysis bags of 12-14kDa molecular weight cut-off prior to freezing. The semen samples were diluted in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or lactose-lyophilized lipoprotein fractions-glycerol extender (lactose-LPFo-G), packaged into aluminum tubes and frozen in a controlled programmable freezer. Pre-frozen and frozen-thawed spermatozoa were evaluated for motility, plasma membrane (SYBR-14 and propidium iodide) and acrosome integrity, mitochondrial function (Rhodamine 123) and ATP content. The results of the study showed that dialysis significantly improved the sperm characteristics prior to freezing. Dialysis enhanced (P<0.05) post-thaw sperm motility, plasma membrane integrity and mitochondrial function, but had no significant effect (P>0.05) on recovery of spermatozoa with intact acrosomes. Furthermore, dialyzed spermatozoa exhibited higher (P<0.05) ATP content compared with the control after freezing-thawing. Consistent inter-boar variability was detected mainly in dialyzed semen following freezing-thawing. These results indicated that the improvement in sperm quality characteristics prior to freezing and the post-thaw sperm recovery were due to the removal of low-molecular weight components from the seminal plasma. It can be suggested that dialysis is effective in improving the post-thaw quality of boar spermatozoa and has also great practical importance in improving the protocols for cryopreservation of semen. Dialysis may also contribute to a better understanding of different mechanisms underlying cryo-induced damage to boar spermatozoa.

Acrosome↗
PubMed1999

Behavioral analysis of the freezing phenomenon in Parkinson's disease: a case study.

The "freezing" phenomenon was examined in a 77 yr old male patient suffering from Parkinsons's disease (PD). Motor blocks occurred several times a day, mainly affected walking, and lasted mostly between 2 and 5 min. Behavioral analysis showed that freezing was elicited and intensified not only by external factors (limited space and room to move), but also by negative cognitions, negative emotions and tension. The most effective behavior to overcome freezing was a combination of relaxation and loud rhythmic counting. Effectiveness in overcoming freezing correlated positively with self-rated mobility before trials. Relaxation training improved self-rated mobility. It is suggested that relaxation training and cognitive techniques should be used to reduce tension, negative thoughts and negative emotions contributing to freezing and to improve the ability to cope with freezing.

Aged↗
PubMedDate not supplied

Flash freezing of erythrocyte suspensions.

Freezing whole blood in bulk usually results in severe cellular destruction through the action of ice crystals and osmotic effects in the freezing liquid. The potential of flash freezing blood aerosols onto a liquid nitrogen surface as a means of inhibiting cellular damage was studied in this work. Three commercial spraying devices were employed to spray-freeze either whole blood or concentrated erythrocyte suspensions, using hydroxyethyl starch (HES) as a cryoprotectant. The integrity and viability of the processed cells were assessed by measuring gross rheological properties and the extent of hemolysis. Cells were found to be susceptible to the very high shear stresses imposed by some of the spraying devices. Bulk freezing of blood, even in the presence of cryoprotectant, resulted in complete cellular destruction. Whereas flash freezing was capable of substantially reducing the level of hemolysis to 12.6% and preserving the cellular deformability.

Aerosols↗
PubMed2002

Lipid and protein changes due to freezing in Dunning AT-1 cells.

Defining the process of cellular injury during freezing, at the molecular level, is important for cryosurgical applications. This work shows changes to both membrane lipids and protein structures within AT-1 Dunning prostate tumor cells after a freezing stress which induced extreme injury and cell death. Cells were frozen in an uncontrolled fashion to -20 or -80 degrees C. Freezing resulted in an increase in the gel to liquid crystalline phase transition temperature (T(m)) of the cellular membranes and an increase in the temperature range over which the transition occurred, as determined by Fourier transform infrared spectroscopy (FTIR). Thin layer chromatography (TLC) analysis of total lipid extracts showed free fatty acids (FFA) in the frozen samples, indicating a change in the lipid composition. The final freezing temperature had no effect on the thermotropic response of the membranes or on the FFA content of the lipid fraction. The overall protein secondary structure as determined by FTIR showed only slight changes after freezing to -20 degrees C, in contrast to a strong and apparently irreversible denaturation after freezing to -80 degrees C. Taken together, these results suggest that the decrease in viability between control and frozen cells can be correlated with small changes in the membrane lipid composition and membrane fluidity. In addition, loss of cell viability is associated with massive protein denaturation as observed in cells frozen to -80 degrees C, which was not observed in samples frozen to -20 degrees C.

Adenocarcinoma↗
PubMed2003

Spawn viability in edible mushrooms after freezing in liquid nitrogen without a cryoprotectant.

Five strains of edible mushrooms (Lentinula boryana, Lentinula edodes, Pleurotus djamor, Pleurotus pulmonarius, and Volvariella volvacea) were studied. Spawn were prepared from sorghum seeds and then incubated for 14 days under optimum conditions for each species. Once covered by mycelia, the sorghum seeds were placed in polycarbonate vials for freezing in liquid nitrogen. The effect of adding a cryoprotective solution before freezing (either 10% glycerol v/v or 5% dimethylsulfoxide v/v) was evaluated as a function of mycelial growth and percent viability. Three main treatments were undertaken: (1) freezing with a glycerol or dimethylsulfoxide cryoprotectant, (2) freezing with water and (3) freezing without cryoprotectant or water. Samples were maintained frozen for a week, after which time they were thawed (10 min at 30 degrees C) and the seeds placed in Petri dishes with a culture medium. A recovery rate of 96.8% was obtained for the total number of samples summed over all strains and treatments. In contrast, 99.2% of the samples frozen without cryoprotectant were recovered. The recovery of frozen mycelia was delayed with respect to a control group, which was not frozen. However, no difference was observed in percent recovery and mycelial diameter when a new series of spawn was prepared from mycelia that had been previously frozen. Results obtained from this experiment demonstrate that an adequate recovery of mycelia can be obtained without using a cryoprotectant. This capacity might enable large quantities of commercial mushroom strains to be handled at reduced production costs. It is suggested that the mycelia survived freezing without cryoprotectants because they were embedded and protected within the sorghum seeds used to elaborate the spawn.

Agaricales↗
PubMed2000

Monoamine oxidase inhibitors reduce conditioned fear stress-induced freezing behavior in rats.

The present study examined the acute anxiolytic effects of monoamine oxidase inhibitors on freezing behavior, a putative index of anxiety induced by conditioned fear stress. The selective serotonin 1A receptor agonist tandospirone (0.1-10 mg/kg) inhibited freezing dose dependently. The irreversible, non-selective monoamine oxidase inhibitors tranylcypromine (3 and 15 mg/kg) and phenelzine (30 and 80 mg/kg) reduced freezing significantly. Clorgyline (10 mg/kg, irreversible selective monoamine oxidase A inhibitor), N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide (Ro 41-1049) (30 mg/kg, reversible selective monoamine oxidase A inhibitor), selegiline (3 mg/kg, irreversible selective monoamine oxidase B inhibitor) and lazabemide (10 mg/kg, reversible selective monoamine oxidase B inhibitor) had no effect on freezing behavior. However, combined administration of clorgyline (10 mg/kg) and selegiline (3 mg/kg) reduced freezing significantly, as well as combined administration of clorgyline (10 mg/kg) and lazabemide (10 mg/kg), Ro 41-1049 (30 mg/kg) and selegiline (3 mg/kg), or Ro 41-1049 (30 mg/kg) and lazabemide (10 mg/kg). These effects of monoamine oxidase inhibitors on freezing were not due to non-specific motor effects. These results suggest that acute inhibition of both monoamine oxidase A and B reduced anxiety or fear, while inhibition of monoamine oxidase A or B alone failed to reduce anxiety or fear.

Animals↗
PubMed2003

Transfer of frozen-thawed blastocysts that underwent quarter laser-assisted hatching at the day 3 cleaving stage before freezing.

OBJECTIVE: To examine the pregnancy potential of frozen-thawed blastocysts that underwent quarter laser-assisted hatching (AH) at the cleaving stage before freezing and to compare clinical and embryo characteristics between the groups that succeeded in and failed to achieve pregnancy. DESIGN: Prospective observational study. PATIENT(S): Thirty-four of 112 patients with frozen blastocysts requiring transfer of thawed embryos between January 2000 and December 2001. SETTING: Assisted reproductive technology unit in a tertiary medical center. INTERVENTION(S): Embryos of patients undergoing blastocyst transfers routinely underwent quarter AH using a nontouch 1.48-microm diode laser. Blastocysts not transferred were cryopreserved using a six-step freezing protocol with glycerol as the cryoprotectant. MAIN OUTCOME MEASURE(S): Postthaw embryo survival and zona pellucida (ZP) maintenance, implantation rate, and clinical pregnancy rate per transfer of thawed blastocysts. RESULT(S): A total of 118 frozen blastocysts was thawed. Of these, 89 (75.4%) embryos survived and were transferred in 35 cycles. Assisted hatching-manipulated ZP tolerated the freeze-thaw procedures without shape distortion in surviving embryos. Eleven (31.4%) clinical pregnancies with 15 intrauterine gestational sacs occurred, for an implantation rate of 16.9%. Those who succeeded in pregnancy tended to have more embryos available before freezing, more original blastocysts of good quality for freezing, and more surviving blastocysts after thawing for transfer than did those who failed to achieve pregnancy. CONCLUSION(S): An acceptable clinical pregnancy rate was obtained from transfer of thawed blastocysts that underwent laser AH on the ZP at the day 3 cleaving embryo stage in fresh cycles in selected patients. Embryo characteristics before freezing played major roles in determining implantation potential of thawed embryos.

Adult↗