Thawing Protocol Research Articles

Cryo-damaged single blastomeres may be used efficiently for preimplantation genetic diagnosis (PGD) for the purpose of screening for single gene defect

Application of preimplantation genetic diagnosis (PGD) is rapidly growing and provides important advantages compared to the alternative method of the prenatal diagnosis. The cases performed in the world have been for the indication either of aneuploidy or for preventing the inheritance of commonly occurring single gene defects (such as cystic fibrosis). Today the standard method of PGD requires the removal of a cell from a day 3, 6–8 cell cleavage stage IVF embryo, followed by transferring the cell into a PCR tube, performing the PCR reaction and analyzing the results. Although there are some minor variations in the different PGD protocols, all of them require the use/biopsy of an intact and living cell. The objective of the present study was to investigate if it was possible to apply the PGD protocol in a way that it can be used for PCR analyses using lysed embryonic cells instead of intact cells. Experimental research protocol. A total of 6 embryos donated to research were involved in this study. A standard slow freezing and rapid thaw protocol was applied. Both intact and degenerated cells were biopsied from thawed, disaggregated and transferred to a PCR tube. A total of 24 blastomeres were removed from the embryos; and they were separated according to intactness. PCR tubes were marked correspondingly if they contained a single living or a single degenerated/lysed blastomere. Both live and degenerated cells were submitted to fluorescent polymerase chain reaction (PCR) and subsequent restriction enzyme digestion and fragment analysis on an automated sequencer. The test was designed to analyze the region corresponding to the most frequently occurring mutation represented by deletion of 3 basepairs, at the amino acid position 508 of the putative product of the CF gene. A total of 24 blastomeres were separated from the frozen-thawed embryos. Twelve of them were completely intact and therefore considered to be alive while the other 12 blastomeres were displaying all the signs of cryodamage therefore were considered as degenerated. All the 12 intact blastomeres provided clear signals (100%) on the gene region that was amplified corresponding to a non-mutated sequence, as expected. Equally, all the 12 degenerated blastomeres provided clear signals (100%) to the same region corresponding to a non-mutated sequence. The results of the present study demonstrate that it is possible to use degenerated/lysed blastomeres for the purpose of preimplantation genetic diagnosis to detect alterations in the gene sequences. It has also been shown that amplification of genetic material obtained from a single cell is similarly efficient when performed on degenerated cells compared to live/intact cells using a non-altered protocol. As the results demonstrated, fragmentation of nuclear DNA in the degenerated cells may not present a high risk (that would interfere with amplification efficiency) depending perhaps on the timing of blastomere separation relative to the thaw procedure. This new approach is especially important for cryo-ET cycles, because it does not require the removal of live blastomere(s) from the embryo which in return would assist to preserve a higher viability and developmental potential of the embryo. Extension of the present study to test different gene sequences is warranted in order to demonstrate that this new approach is well applicable for screening various types of single gene defects.

Improved Outcome of Frozen-Thawed Blastocyst Transfer with Menezo's two-step Thawing Compared to the Stepwise Thawing Protocol

To compare the effects of two different blastocyst thawing protocols on implantation, pregnancy, and live birth rates. Ninety four consecutive frozen-thawed blastocyst transfers from 1996 to 2002 were retrospectively analyzed. Blastocysts were cryopreserved using Menezo's two-step slow freezing protocol. Frozen blastocysts were thawed for transfer according to Menezo's stepwise or two-step protocol. Immediately after thawing, assisted hatching was performed and all embryos were cultured for 3 to 5 h before transfer. Only viable embryos were selected for transfer. Implantation, pregnancy (determined by the presence of fetal cardiac activity), and live birth rates were significantly higher with two-step (25, 45.7, and 42.9%) than with stepwise thawing protocol (9.2,18.6, and 16.9%, P < 0.01). The percentage of gestational sacs resulting in live babies was higher with two-step thawing (76.6%) than with stepwise thawing (50%, P = 0.04). We conclude that the two-step thawing protocol offers advantages over the stepwise method. The two-step thawing protocol dramatically increased embryo implantation potential, resulting in higher pregnancy rate, and subsequent live birth rate, after frozen blastocyst transfer.

Potential factors affecting embryo survival and clinical outcome with cryopreserved pronuclear human embryos

Objective This study was undertaken to determine whether the method of fertilization has a significant impact on survival and/or clinical pregnancy rates of cryopreserved human pronuclear (2PN) stage embryos. Design A retrospective analysis of cryosurvival and clinical pregnancy rates after thawing of 2PN stage embryos from January 2000 through December 2002 in a private Assisted Reproductive Technology (ART) center. Material and methods A total of 1408 human 2PN embryos were cryopreserved using a Planer Kryo 10 Series III freezing unit (TS Scientific, Perkasie, Pa) after dehydration/equilibration through Propanediol (Sigma Chemical, St. Louis, Mo) and sucrose. On thawing, embryos were cultured in vitro with P-1 medium with 10% Serum Substitute Supplement (Irvine Scientific, Santa Ana, Calif). Embryo transfer was performed at 40 to 48 hours from time of thaw into a recipient uterus after standard estradiol/progesterone preparation. Results In 2000, 78% of all frozen 2PN embryos survived and were transferred in 181 cycles producing a delivery rate of 26% per transfer. However, 59% of these cycles were intracytoplasmic sperm injection (ICSI), and the survival of frozen 2PN from these cycles (72%) was lower than the respective survival of frozen 2PN embryos from in vitro fertilization (IVF) (81%; P<.025). Changes to protocols for thawing frozen 2PN embryos were therefore explored and implemented during 2001, resulting in equivalent survival rates of frozen 2PN embryos from IVF and ICSI during 2001 (78% and 80%, respectively) and 2002 (73% and 74%, respectively). Coincidentally, the proportion of all cycles that were performed with ICSI increased (73% in 2001 to 78% in 2002; P<.01) and pregnancy rates after transfer of frozen/thawed 2PN embryos from ICSI increased from 15% in 2000 to 30% in 2002. Conclusion 2PN stage embryo cryosurvival may be negatively affected by ICSI, possibly caused by disruption of the zona pellucida and vitelline membrane before cryopreservation, and/or because ICSI promotes fertilization of some compromised eggs (producing compromised 2PN embryos) that would not have fertilized by conventional IVF. Without close attention to embryo freezing and thawing protocols relative to outcome, lower cryosurvival of unselected ICSI-produced embryos can negatively impact pregnancy outcomes.

New approach to improving endothelial preservation in cryopreserved arterial substitutes

The endothelial loss provoked by the methods of vascular cryopreservation used at most human vessel banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vessel substitutes. This study evaluates the effects of the storage temperature and thawing protocol on the endothelial cell loss suffered by cryopreserved vessels, and optimises the thawing temperature and protocol for cryopreserving arterial grafts in terms of that producing least endothelial loss. Segments of the common iliac artery of the minipig ( n=20) were frozen at a temperature reduction rate of 1 °C/min in a biological freezer. After storing the arterial fragments for 30 days, study groups were established according to the storage temperature (−80, −145 or −196 °C) and subsequent thawing procedure (slow or rapid thawing). Fresh vessel segments served as the control group. Once thawed, the specimens were examined by light, transmission, and scanning electron microscopy. The covered endothelial surface was determined by image analysis. Data for the different groups were compared by one way ANOVA. When cryopreservation at each of the storage temperatures was followed by slow thawing, the endothelial cells showed improved morphological features and viability over those of specimens subjected to rapid thawing. Rapidly thawed endothelial cells showed irreversible ultrastructural damage such as mitochondrial dilation and rupture, reticular fragmentation, and peripheral nuclear condensation. In contrast, slow thawing gave rise to changes compatible with reversible damage in a large proportion of the endothelial cells: general swelling, reticular dilation, mitochondrial swelling, and nuclear chromatin condensation. Gradually thawed cryopreserved arteries showed a lower proportion of damaged cells identified by the TUNEL method compared to the corresponding rapidly thawed specimens ( p<0.05, for all temperatures). In all the groups in which vessels underwent rapid thawing (except at −145 °C), significant differences ( p<0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at −80 °C followed by rapid thawing led to greatest endothelial cell loss (61.36 ± 9.06% covered endothelial surface), while a temperature of −145 °C followed by slow thawing was best at preserving the endothelium of the vessel wall (89.38 ± 16.67% surface cover). In conclusion, storage at a temperature of −145 °C in nitrogen vapour followed by gradual automated thawing seems to be the best way of preserving the endothelial surface of the arterial cryograft. This method gives rise to best endothelial cell viability and cover values, with obvious benefits for subsequent grafting.

Functional assessment of cryopreserved pig aortas for pharmaceutical research.

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached. After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.

105VITRIFICATION OF BOVINE EMBRYOS USING THE CLV METHOD

Vitrification has become the preferred method for cryopreserving in vitro-produced bovine embryos (IVP). Here we introduce a technique for vitrification developed at CryoLogic (the CLV Method), in conjunction with a study comparing the post-thaw viability of IVP embryos frozen by the widely used open pulled straw method (OPS—Vajta et al., 1997 Cryo-Letters 18, 191) and the new CLV Method. Vitrification on thin metal surfaces has been explored and demonstrated previously (Le Gal &amp;amp; Massip 1999, Cryobiology 38, 290), and Dinnyes presented a Solid Surface Vitrification (SSV) (Dinnyes et al., 2000, Biol. Reprod. 63, 513). The CLV Method utilizes vitrification on the surface of a solid metal block. This surface has been custom shaped and treated to enhance vitreous formation. The method also includes handling, storage and thawing protocols designed to avoid damage from crystallization of the unstable glass. Briefly, the block is precooled in LN2 to −196°C. Up to 10 embryos are collected into a droplet of medium (3μL), on the end of a fibre carrier attached to a handle. The droplet is presented to the vitrification surface, where it is converted into a glass bead by cooling rates comparable to that of plunging into solid/liquid phase nitrogen (−210°C) (Arav et al., 2001 Theriogenology 55, 313). The glass bead, fibre and handle are transferred quickly into a half-sealed, precooled straw, and the handle seals the open end. A bead is thawed very rapidly by removing the handle, fibre and bead from the straw and transferring the bead into washing medium. COCs collected from bovine ovaries obtained from abattoirs were matured, fertilized and cultured for 6 days (Fry et al., 2003 Theriogenology 59, 446). Embryos reaching the blastocyst or expanding blastocyst stage of development were graded (Grades 1, 2, and 3), equilibrated for 5min in HEPES-199 medium with 20% FCS (HFm), placed in HFm with 10% EG, and 10% DMSO (VS1) for 2min, and then transferred to HFm with 20% EG, 20% DMSO (VS2) for 30s (Vajta). Between 5 and 10 IVP embryos were processed and collected for vitrification, either in an OPS plunged into LN2, or in a 3μL droplet vitrified by the CLV Method. The two sets of specimens were stored in LN2, and later thawed. Both OPS tips and beads were thawed in 0.5mL of HFm with 0.2M sucrose at 39°C. Embryos were maintained at 39°C, examined after 5min for contraction, and again after 6 h for re-expansion. They were then transferred to culture medium, incubated and examined at 24 and 48h to assess hatching. As shown in Table 1, the CLV method appears to be satisfactory for maintaining membrane integrity (expansion) and developmental potential (hatching) for even poorer grade embryos, that might be more sensitive to the stresses of cryopreservation. Table 1 Re-expansion and hatching rates of graded thawed bovine embryos vitrified by OPS or CLV methods

Quantitative RT-PCR reveals tuberous sclerosis gene, TSC2, mRNA degradation following cryopreservation in the human preimplantation embryo.

The use of cryopreserved human embryos in gene expression studies provides an additional source to the scarce embryos available for research. To validate their use we have implemented a quantitative RT-PCR to characterize the levels of the tuberous sclerosis, TSC2 gene in fresh and frozen-thawed human embryos. Frozen embryos were thawed using two different clinical protocols. In fresh embryos 9.95 fg of TSC2 cDNA was present in the unfertilized oocyte, which was comparable to the level on day 2 of preimplantation development. On day 3 there was a significant drop (P<0.001) to 6.8 fg, followed by an increase in cDNA levels to 10.8 fg (P<0.01) on day 6 at the expanded blastocyst stage. Day 2 frozen embryos possessed 50% less (P<0.001) TSC2 mRNA in comparison to the fresh embryos using thawing protocol one (from frozen to 37 degrees C) and 25% less TSC2 mRNA (P<0.01) with thawing protocol 2 (from frozen to room temperature). After culturing day 2 frozen embryos for an additional day they showed mRNA levels comparable with fresh day 3 embryos. There was no significant difference in the levels of TSC2 mRNA between fresh and frozen day 3 human embryos with either thawing protocol. This study demonstrates that cryopreservation does affect the normal pattern of gene expression during human preimplantation development, and that intact frozen-thawed embryos are not equivalent to their non-frozen counterparts. Furthermore human embryos frozen on day 2 appear to be more susceptible to temperature change than embryos frozen on day 3.

Effects of thawing procedure on postthawed in vitro viability and in vivo fertility of red deer epididymal spermatozoa cryopreserved at -196 degrees C.

In this study, we have determined the effects of individual factor and thawing procedure on in vitro viability and in vivo fertility of frozen-thawed red deer epididymal spermatozoa. The spermatozoa that were collected from 13 Iberian deer stags were diluted at room temperature in a Triladyl-20% egg yolk medium and frozen in nitrogen vapors. In the first experimental series, sperm samples were collected from 10 mature stags. For thawing, the frozen straws were subjected to 3 different procedures: I (37 degrees C for 20 seconds), II (60 degrees C for 8 seconds) and III (70 degrees C for 5 seconds). Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SM) and of plasma membrane and acrosome (NAR) integrities. Statistically significant variations were found (P <.05) between stags for most of the seminal parameters evaluated. The thawing procedure did not have an effect on the seminal characteristics evaluated after this process, except for SM (P <.05), with the best overall recovery rates after freezing and thawing found with the use of protocol I. Our results also show a differential resistance to return to isosmotic conditions of spermatozoa thawed using the different thawing protocols. In the second experimental series (insemination artificial trial), with spermatozoa from 3 stags, results of fertility were statistically higher (69.7% vs 42.4%, P =.014) when spermatozoa were thawed at 37 degrees C for 20 seconds than were warmed at 60 degrees C for 8 seconds. Therefore, thawing protocol I, which provides slow thawing rates, was the most beneficial for epididymal spermatozoa thawing of the cervid subspecies analyzed in this study. In summary, high in vitro survival and in vivo fertility of frozen-thawed deer epididymal spermatozoa were dependent on warming rates, but each stag exhibited its own sensitivity to cryopreservation.

Improved outcome of frozen-thawed blastocyst transfer with Menezo’s 2-step thawing protocol

Improved outcome of frozen-thawed blastocyst transfer with Menezo’s 2-step thawing protocol

Timing of blastocyst transfer relative to the thaw: a critical factor in outcome following FET

Objective: To evaluate effects of different blastocyst freezing and thawing protocols on pregnancy and implantation rate. Design: Retrospective cohort study. Setting: Multi-site, private practice. Patients: Infertile women ages between 27-51 years (n 95) undergoing IVF. Interventions: Women undergoing in vitro fertilization (IVF) between January and December 2002 had surviving expanded supernumerary blastocysts cryopreserved 5 and 6 days following fertilization. All embryos were cultured in P1 (Irvine Scientific) medium until day 3 and thereupon in Blastocyst medium (Irvine Scientific) until day 5 or 6. Patients were allocated to Group 1 or Group 2 based on the method of cryopreservation and the time interval between the thawing process and the frozen embryo transfer (FET). In Group 1, the cryopreservation process involved treatment with 5% and 9% Glycerol 0.2 M sucrose for 10 minutes followed by freezing through exposure of the blastocysts to 7°C for 17 min, and thereupon lowering the temperature by 0.3°C/min to 38°C. Prior to the FET the embryos were thawed in a water bath at 33°C, treated with 0.5 M and 0.2 M sucrose for 10 min and then immediately transferred to the uterine cavity. In Group 2, the same method of exposure of the blastocysts to glycerol and sucrose as described above was employed at 20°C. Thereupon the temperature was progressively reduced by 2°C/min to 6.5°C and maintained at this point for 17 min. Thereafter the temperature was lowered at a rate of 0.3°C/min to 38°C. However, in contrast with Group 1 patients whose blastocysts were transferred immediately upon thawing, the timing of FET’s in Group 2 was predicated upon whether day 5 or day 6 blastocysts were being thawed; whereas day 5 blastocysts were thawed, then treated with 0.5 and 0.2 M sucrose for 10 min, cultured overnight and transferred the following afternoon. Blastocysts frozen on day 6 were thawed similarly on the morning of the scheduled FET, cultured for at least 4 hours and then transferred. Primary measures of outcome: Ongoing pregnancy rate (beyond 12 weeks) and implantation rate (number of viable gestations per transferred blastocyst). Results: The ongoing pregnancy and implantation rates were 16% and 11% for Group 1 as compared to 32% and 20% for Group 2 (P 0.05). Conclusions: Blastocyst transfer has been shown to be associated with improved pregnancy and implantation rates. Recent advances in embryo culture media and techniques have brought about improved embryo quality and survival to the blastocyst stage in the IVF setting. The resulting increase in availability of blastocysts has allowed for an ever increasing degree of discretion in the selection of only the best quality supernumerary blastocysts for cryopreservation. Our data strongly suggests that while the protocol of blastocyst cryopreservation does not affect FET outcome, the timing of the thaw, relative to the transfer could be a critical consideration.

Cryopreservation of oocytes from pre-antral follicles.

Cryobiology is a very important tool in reproductive biology. Research in this area focuses on the possibility of restoring fertility in women with reproductive problems or after cancer treatments. Another goal is to establish a genetic resource bank for endangered or commercially important animal species. Cryopreservation of oocytes from pre-antral follicles has been studied during the past decade. Procedures can be divided between the cryopreservation of either ovarian tissue or isolated follicles. Most studies describe a slow freezing/rapid thawing protocol to cryopreserve ovarian fragments. Histology shows that the follicles maintain their morphological integrity, and transplantation of ovarian tissue demonstrates that the follicles can restart their growth and eventually ovulate. Some research groups have obtained offspring using this procedure in mice and sheep. With regard to the cryopreservation of isolated follicles, the few studies reported in this area used the same freezing protocol, and some of them described follicular growth using in-vitro culture. The best result was obtained in mice, with animal birth after follicular cryopreservation and culture. However, additional studies are necessary for a better understanding of the events during follicular cryopreservation and to establish a standard protocol for ovarian transplantation or follicle culture.

Effect of freezing–thawing protocols on the performance of semen from three rabbit lines after artificial insemination

The effect of different freezing and thawing protocols on the results observed after artificial insemination with semen from three different rabbit lines (two maternal lines selected for litter size at weaning, lines A and V, and one line selected for growth rate from weaning to slaughter, line R) was studied. The sperm were frozen with a Tris–citric acid–glucose extender which included 1.75 M DMSO and 0.05 M sucrose as cryoprotectants. The straws were cooled to 5 °C for 45 min and then some of them were frozen in a freezer at −30 °C for 30 min, whereas the other group of straws were frozen in liquid nitrogen vapor (LNV, 5 cm above the liquid nitrogen level) for 10 min. Straws were thawed at two different temperatures: 50 or 70 °C for 10–12 s. Significant differences were observed between freezing–thawing protocols, obtaining better results in fertility rate (percentage of pregnant females) when sperm had been frozen in LNV (fertility rate increased between 30 and 50 points in all the lines); the best prolificacy was observed when sperm had been frozen in LNV and thawed at 50 °C (70% versus 32% fertility rate, P<0.01 and 7.4 versus 5.9 total number of young born, P<0.01 when sperm had been frozen in LNV or at −30 °C and thawed at 50 °C, respectively). As for the rabbit line, significant differences were observed between lines in fertility rate (62 and 68% versus 45% fertility rate for lines A, V and R, P<0.01), and total number of young born (5.8 versus 6.9 versus 4.6 total number of young born for lines A, V and R, P=0.02). The best results for all lines in both fertility and total number of young born were observed when sperm had been frozen in LNV and thawed at 50 °C (85% versus 84% versus 50% fertility rate and 6.7 versus 8.3 versus 7.3 total number of young born for lines A, V and R, respectively), when compared to the results of the control group, frozen at −30 °C and thawed at 50 °C (30% versus 52% versus 19% fertility rate and 6.7 versus 6.4 versus 4.5 total number of young born for lines A, V and R, respectively). In conclusion, the best results (fertility rate and prolificacy) for all the rabbit lines were obtained after freezing in liquid nitrogen vapor and thawing at 50 °C, being more pronounced in the line selected for high growth rate (line R).

Membrane changes during different stages of a freeze–thaw protocol for equine semen cryopreservation

Many theories have been postulated concerning the possible effects of cryopreservation on spermatozoa, including suggestions the freeze–thawing process produces membranes that have greater fluidity and are more fusogenic, thus inducing changes similar to those of capacitation. The main objectives of this study were to determine at what stage of the freeze–thaw process membrane changes occur and whether evaluation with chlortetracycline (CTC) stain could predict the freezability of stallion sperm. Sperm viability and state of capacitation were simultaneously evaluated using CTC and Hoechst 33258 (H258) techniques. Membrane function was evaluated using the hypo-osmotic swelling test (HOS) and progressive motility (PM) was evaluated under light microscopy at each stage of a freeze–thaw protocol. Evaluated were raw semen; after dilution and centrifugation; after redilution and equilibration at room temperature; after cooling to 5°C; after super cooling to −15°C; and after thawing. The most pronounced functional damage to membranes and the greatest decrease in PM occurred in samples of all stallions after thawing (P<0.05). Cryopreservation, as evaluated by CTC/H258 staining, significantly (P<0.05) affected sperm membrane integrity after centrifugation, after redilution and equilibration at room temperature and after cooling to 5°C. The HOS and H258 tests gave similar results (R values of approximately 0.75) and correlated inversely with the number of live noncapacitated sperm cells (R values of approximately −0.75). Remarkably, the subpopulation of capacitated live cells was unaffected in all freeze–thawing steps and the number of live acrosome reacted cells increased by a factor of 4. However, it was not possible to determine whether the changing CTC patterns reflect a true capacitation phenomenon or an intermediate destabilized state of the sperm cell membrane. This increase may indicate that the subpopulation of functional sperm cells capable of binding to the zona pellucida increases after freeze–thawing despite the deteriorative effect of this procedure for the entire live sperm population.

Evaluation of meiotic spindles in thawed oocytes after vitrification using polarized light microscopy

Objective: Meiotic spindles are crucial for normal chromosome alignment and separation of chromosomes during meiosis I and meiosis II. Traditional evaluation of the integrity spindles has been the immunofluorescent staining of tubulin. Recently, Polscope has emerged as a tool to view spindles in living oocytes. In this study we assess the spindle status of thawed oocytes after vitrification using Polscope and investigate their feasibility to replace immunofluorescent staining. Design: The Polscope was used to image spindles in living mouse oocytes before vitification. After thawing, the spindles of oocytes were imaged again and classified according to the spindles images. The results were compared with those by immunofluorescent staining of tubulin. Materials/Methods: The oocyte-cumulus complexes were collected from ICR mice superovulated by PMSG and hCG injections. The cumulus cells were removed by hyaluronidase. The MII oocytes were imaged by Polscope. Only oocytes with good spindle morphology were subsequently undergoing vitrification thawing protocol. The oocytes were vitrified with 5.5 M Ethylene Glycol- and 1 M sucrose-containing Dulbecco phosphate buffered saline. They were loaded into thin walled pulled straw and were stored at liquid nitrogen till thawing. After thawing, the oocytes were evaluated by Polscope again. The spindleimages were saved in computer for later analysis. The survived thawed oocytes were then fixed and incubated with anti-tubulin monoclonal antibody. Tubulin was stained by fluorescein isothiocyanate (FITC) conjugated anti-mouse IgG and chromatin was stained with Hoechst 33258. Results: Spindle images by Polscope were calssified as three groups (Wang et al. ASRM, 2001). Group I oocytes had normal spindles. Group II oocytes had abnormal spindles. Group III oocytes had no image of spindles. Spindle morphology by immunofluorescent staining was classified as normal, reduced or disrupted. Totally, 100 MII oocytes were vitrified. After thawing, 79 oocytes were morphologically intact. Images by Polscope were classified as 26 in group I, 28 in group II and 25 in group III. With immunofluorescent staining, 25 were classified as having normal spindle morphology, 30 as having abnormal spindle morphology and 24 as having disrupted morphology. The chromosome pattern was compact in 50 oocytes, dispersed in 24 oocytes and absent in 5 oocytes. Conclusions: The spindles of frozen thawed oocytes can be assessed using Polscope and the image is well correlated to the results by traditional immunofluorescent staining. After vitrification and thawing, only 26 (33%) of 79 oocytes appear good and have good morphology of spindles. A portion (32%) of oocytes has relative good appearance but absent spindle images. Without Polscope or immunofluorescent stain to microtubulin, these cannot be selected out. It reflected the unavoidable cryo-injury to meiotic spindle and the imperative improvement of the protocol. Whether the spindles of the thawed oocytes can recover after some period of incubation will need further investigation. Supported by: This study was supported by grants from Chang Gung Memorial Hospital CMRP 891 and CMRP 949.

Factors relating to a successful cryopreserved blastocyst transfer program

Objective: To review the authors’ experience in a successful frozen blastocyst program. Design: Retrospective study. Setting: University IVF program. Patients: Women of all ages undergoing 64 frozen blastocyst nondonor thaw cycles. Intervention(s): Thaw cycles with day 5 or day 6 frozen blastocysts replaced into luteal day 5 endometrium in natural or programmed cycles; cryopreservation of blastocysts by Menezo two-step protocol and Testart slow cool program; thawing by two step thaw protocol. Main Outcome Measure(s): Implantation, clinical pregnancy, and delivery rates. Result(s): The implantation rate was 16% and was similar with day 5 frozen blastocysts and day 6 frozen blastocysts cycles. The clinical pregnancy rate was 36% and the delivery rate was 27%, with no significant difference between day 5 and 6 blastocysts. Conclusion(s): Blastocyst cryopreservation is a viable option for patients of all ages and complements fresh blastocyst culture and transfer. The presence of good quality blastocysts for freezing on day 5 and day 6 yields comparable results and is critical for the success of thaw cycles.

Effects of supplementation with free radical scavengers on the survival and fertilization rates of mouse cryopreserved oocytes.

This study was conducted to investigate the effects of supplementation with free radical scavengers on the survival and fertilization rates of freeze-thawed mouse oocytes. Superovulated oocytes with cumulus cells were cryopreserved by slow freezing in propanediol combined with a rapid thawing protocol. The cryopreservation medium was supplemented with the antioxidant enzymes superoxide dismutase (SOD) and catalase, and with the nitric oxide (NO) scavenger, haemoglobin (Hb). The addition of 50 IU/ml SOD showed significantly higher survival and fertilization capabilities compared with control (P < 0.01). Oocyte survival was greatly increased by concomitant addition of SOD with 10 IU/ml catalase (P < 0.01). On the other hand, the NO donor (sodium nitroprusside) inhibited survival and fertilization rates (P < 0.05). Significantly decreased survival and fertilization rates were also observed following the addition of high concentrations (10(-3) to 10(-6) nmol/l) of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). In contrast, significantly better oocyte survival and fertilization rates were detected with low concentrations (10(-7) nmol/l) of L-NAME. Oocyte survival potential was significantly increased by addition of Hb (1 microg/ml, P < 0.05). Moreover, oocyte survival and fertilization rates were significantly promoted by the concomitant addition of SOD with Hb (P < 0.01). These results suggest that supplementation of free radical scavengers, particularly combinations of SOD with NO scavengers in freezing and thawing media, improved the post-thaw survival and fertilization rates of cryopreserved mouse oocytes.

Increased Clinical Pregnancy Rates from Frozen Thawed Human Blastocysts Using a Rapid Dilution and Transfer Protocol

Objectives: Day 5 blastocyst transfers has increased the implantation and pregnancy rates of human in vitro fertilization however the use frozen-thawed blastocysts (FBET) has not been reported to be as efficient. This report is a retrospective analysis of the data from 3 non-contemporaneous phases of a blastocyst freezing-thawing program using 3 thawing protocols.

The Effect of Increased Interval Between Embryo Thaw and Transfer on Success in a Frozen Embryo Transfer Program

Objective: This study was performed in an effort to increase success in a frozen embryo transfer program. The changes described were introduced after a major change of laboratory personnel. However, the study represents changes in timing of embryo thaw only as cryopreservation and thaw protocols were constant over the course of the study. Design: This study was a retrospective analysis of 144 frozen embryo cycles performed between January 1998 and February 2000. Treatment groups were constructed chronologically at the time point where a thaw protocol changed in concert with major laboratory personnel changes (July 1999). The first group (prior to protocol change) was comprised of 110 thaw cycles where embryos frozen at the pronuclear stage were thawed 1 day (approximately 24hr) prior to embryo transfer (PN Day1) and multi-cell embryos were thawed the day of the embryo transfer (Multi Day0). The second group (after protocol change) was comprised of 34 thaw cycles where embryos frozen at the pronuclear stage were thawed 2 days (approximately 48hr) prior to embryo transfer (PN Day2) and multi-cell embryos were thawed 1 day (approximately 24hr) prior to embryo transfer (Multi Day1). Endpoints evaluated included clinical pregnancy rates and implantation rates. Materials and Methods: All embryos were cryopreserved using propanediol and were frozen and thawed using conventional protocols. Embryo recipients were prepared for transfer utilizing Estrace and Progesterone administration. All embryos were transferred to recipients on day 4 following Progesterone administration. Results: Post thaw embryo survival rates were similar prior to and after the changes described for both pronuclear stage and multi-cell stage embryos. Tabled 1PN Day1PN Day2Multi Day0Multi Day1N89102124Embryos trans.2.62.32.93.2Clin. preg. rate16/89 (18%)6/10 (60%)3/21 (14.3%)9/24 (37.5%)Implant rate19/231 (8.2%)6/23 (26.1%)4/60 (6.7%)11/77 (14.3%) Open table in a new tab Conclusions: These data demonstrate that the interval between thaw and embryo transfer is critical to the success of a frozen embryo transfer program. These data also suggest that transferring embryos at a more advanced developmental stage is preferable than a retarded stage, when compared to uterine receptivity.

Zur Fertilisation kryokonservierter humaner Oozyten (Kryo-Oo) durch intrazytoplasmatische Injektion von Spermatozoen (ICSI) - Fertilisation of Cryopreserved Human Oocytes by Intracytoplasmatic Injection of Spermatozoa -

Objective: Intracytoplasmatic sperm injection (ICSI) has improved fertilization and pregnancy rates in assisted reproduction programs. We studied the fertilization of cryopreserved oocytes with ICSI. Methods: A long protocol (GnRH analogues/FSH, hMG) was generally used for ovarian stimulation. Oocytes from 57 patients were cryopreserved using modified standard freezing and thawing protocols. 16 patients became pregnant after the first treatment cycle with native oocytes so that 31 remained for ICSI with cryopreserved oocytes. 38 cycles with cryopreserved oocytes were carried out in 26 of these patients. Results: After thawing 24 of 161 oocytes (15%) were degenerated. The degeneration rate after ICSI was a further 13%. Thus the total degeneration rate was higher than that with pronuclear stages (28% vs. 22%). 36 embryo transfers resulted in 7 clinical pregnancies. One pregnancy ended in spontaneous abortion, 2 in the delivery of healthy twins, and 4 are ongoing. Conclusion: Fertilization of cryopreserved oocytes by ICSI is associated with a good fertilization rate (48%) and a good pregnancy rate (24% per thawing cycle). The procedure is indicated if freezing of embryos or pronuclear stages is not an option. Furthermore, it appears to be a potential alternative for young patients scheduled for chemotherapy or radiation therapy.

Germinal Vesicle Transfer Between Fresh and Cryopreserved Immature Mouse Oocytes

Objective: Germinal vesicle (GV) transfer offers the potential to assess and rescue genomic maturation in mammalian oocytes. However, as a clinical or research procedure, this technique presents a major logistic problem to ensure that germinal vesicle oocytes are obtained in the same cell cycle stage. This problem could be eliminated if cryostored, immature oocytes are shown to be appropriate for use as GV donors in the transfer procedure. Design: Genomic maturation, as judged by first polar body extrusion, was assessed in mouse oocytes reconstructed by GV transfer using nuclear and/or cytoplasmic components from cryopreserved oocytes. Materials and Methods: Female mice (CD-1 strain) were treated with PMSG (10 IU ip). The mice were killed at 36–48 h post injection and the eggs harvested by follicular puncture. Oocytes were frozen in propanediol-sucrose using routine slow embryo freezing and rapid thawing protocols. GV transfer was performed to exchange nuclei between fresh and frozen oocytes; the transfer and electrofusion protocols have been described previously (Liu et al, Human Reproduction 14:2357, 1999). Throughout these procedures, the oocytes were treated with isobutyl-methylxanthine to block meiotic progression. Following electrofusion the eggs were cultured in HTF containing 10% fetal bovine serum and monitored between 16–20 h for first polar body extrusion. Staining with Hoechst 3342 revealed DNA in both oocyte and polar body. Results: legendPreparationlegendThe survival rate for the freeze-thaw procedures was 76% (679/890).Electrofusion rateMaturation rateFresh GV-fresh cytoplasm41% (55/135)80% (44/55)Frozen GV-frozen cytoplasm41% (57/138)91% (52/57)Fresh GV-frozen cytoplasm51% (73/143)86% (63/73)Frozen GV-fresh cytoplasm40% (49/124)78% (38/49)Fresh control (no micromanipulation)–77% (167/218)Fresh control (electrofusion pulses only)–86% (36/42)Frozen control (no micromanipulation)–79% (216/275)legend The survival rate for the freeze-thaw procedures was 76% (679/890). Open table in a new tab Conclusions: Immature oocytes reconstructed by GV transfer with nuclei or cytoplasm from frozen eggs show comparable electrofusion and maturation rates as non-manipulated fresh oocytes. These results suggest cryobanked immature oocytes may be suitable for use in research or clinical GV transfer procedures.