Pregnancies achieved after frozen-thawed pronuclear oocytes obtained by intracytoplasmic sperm injection with spermatozoa extracted from frozen-thawed testicular tissues from non-obstructive azoospermic men. (49/2910)

The use of frozen-thawed testicular tissue as a source of spermatozoa for intracytoplasmic sperm injection (ICSI) in non-obstructive azoospermia yields favourable fertilization and pregnancy rates while avoiding both repetitive biopsies and unexpected cycle cancellations. Spermatozoa were obtained from frozen-thawed testicular biopsy specimens from 67 non-obstructive azoospermic men. Following fertilization, supernumerary two pronuclear (2PN) oocytes were frozen. After thawing, 17 cycles of embryo transfer were carried out with a mean number of 2.7 embryos and a mean cumulative embryo score (CES) of 18.3 per transfer. The clinical pregnancy and implantation rates per transfer in these cycles (23.5 and 8.3% respectively) were comparable to those of fresh embryo transfers (35.7 and 12.7% respectively) with a mean number of 2.7 embryos and a mean CES of 28.7 per transfer. Abortion rates, although higher with cryopreserved 2PN oocytes were not significantly different. With this approach, cryopreservation of supernumerary 2PN oocytes can be used to improve the cumulative pregnancy rates in a severely defective spermatogenetic population. To our knowledge, these are the first pregnancies reported which have been obtained by the transfer of cryopreserved pronuclear oocytes obtained from ICSI using cryopreserved testicular spermatozoa.  (+info)

Light and electron microscopic analysis of human testicular spermatozoa and spermatids from frozen and thawed testicular biopsies. (50/2910)

The morphological changes caused by freezing and thawing human testicular spermatozoa have been assessed here. Retrieval of testicular biopsies was carried out on six patients with obstructive azoospermia preparatory to intracytoplasmic sperm injection (ICSI). Light microscope analysis was carried out on testicular cells and ultrastructural analysis was carried out on spermatozoa and different spermatid stages before and after the freezing procedure. Upon examination under light microscopy, all germ cells presented increased vacuolization in their cytoplasm and shrinkage or swelling of the nuclei and cytoplasmic membranes. These altered structures were accentuated in the spermatocyte I cell which often presented disrupted membranes. The ultrastructural findings under transmission electron microscopy demonstrated that after freezing and thawing the major types of cryoinjury were the swelling and rupture of inner and outer acrosomal and plasma membranes. The acrosome material often appeared as dispersed material or as condensed spots or was even lost. Such damage was observed mainly at the spermatozoa and late spermatid stages. We conclude that the freezing and thawing of testicular biopsies causes similar morphological damage to testicular spermatozoa and frozen-thawed ejaculated spermatozoa. It is still unclear whether these changes in testicular spermatozoa after freezing and thawing may compromise its use in the ICSI procedure.  (+info)

Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1, 2-propanediol. (51/2910)

Thin slices of human ovarian cortex were evaluated following cryopreservation in 1,2-propanediol (PROH)/sucrose under various conditions. Following rapid thawing, 1 microm sections were assessed by light microscopy and oocyte abnormalities were further examined by electron microscopy. Follicles (n = 503) were predominantly primordial (91%), with no follicles larger than the proliferating primary stage. Proportions of intact pre-granulosa cells and oocytes (expressed as percentages of the total numbers observed) were significantly reduced following cooling at three different rates with the highest levels of intactness (55 and 85% respectively) being achieved with slow cooling. The frequency of oocyte abnormalities [loss of organelles (mitochondria), organelle-free areas, and/or cytoplasmic vacuolation] was significantly increased at all cooling rates with slow cooling resulting in the highest proportion (56%) of normal oocytes. With slow cooling, increasing dehydration time increased the proportions of intact pre-granulosa cells and oocytes (maximum 74 and 91% respectively after 90 min dehydration). Under these conditions, the highest proportion of follicles with all pre-granulosa cells intact (44%) was observed, as was the highest proportion of 'normal' oocytes (85%). In this study, single step dehydration in PROH/sucrose for 90 min and slow cooling/rapid thawing results in the highest proportion of intact human primordial and primary follicles.  (+info)

Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. (52/2910)

Freezing ovarian cortex is a new option to preserve the fertility of young patients undergoing cancer treatment or in women facing premature menopause. However, the best way to use this banked tissue remains unclear. The function of heterotopic and orthotopic autografts of frozen-thawed ovarian cortex of sheep was compared in the present study. Fresh and frozen-thawed fragments of ovarian cortex were autografted on the uterine horn of six ewes (orthotopic grafts) and under the skin of the belly in nine ewes (heterotopic grafts). In both orthotopic and heterotopic grafts, the resumption of follicular growth and ovulation was monitored. In orthotopically grafted ewes, fertility was recorded. Oocytes from both types of grafts were collected, matured and fertilized in vitro. In both fresh and frozen-thawed grafts follicular growth resumed normally; preantral and antral follicles were first detectable 4 and 10 weeks respectively following grafting but only 5% of the primordial follicles appeared to have survived. This confirms that grafting procedures are more deleterious for follicle survival than cryopreservation. Although ovulation resumed in most ewes, none of the ewes grafted orthotopically became pregnant at a synchronized mating. Seven months following grafting, oocytes could be collected from heterotopic and orthotopic grafts, matured and some of them fertilized, but none developed to the blastocyst stage. Heterotopic grafting may be an alternative to orthotopic grafting to preserve fertility provided follicle survival in the grafts is markedly improved.  (+info)

Subzero water permeability parameters of mouse spermatozoa in the presence of extracellular ice and cryoprotective agents. (53/2910)

Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse sperm cell was modeled as a cylinder (122-microm long, radius 0.46 microm) with an osmotically inactive cell volume (V(b)) of 0.61V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L(pg) = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp) = 94.1 kJ/mole (22.5 kcal/mole) (R(2) = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp)[cpa] = 122.2 kJ/mole (29.2 kcal/mole) (R(2) = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 0.68 x 10(-15) m(3)/Ns (0.004 microm/min-atm) and E(Lp)[cpa] = 63.6 kJ/mole (15.2 kcal/mole) (R(2) = 0.99). These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPAs. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5 degrees C/min; "fast," or 20 degrees C/min) in both the low and high CPA media. The greatest sperm motility and viability was found with the low CPA media under fast (20 degrees C/min) cooling conditions.  (+info)

Comparison of pregnancy outcome of pronuclear- and multicellular-stage frozen-thawed embryo transfers. (54/2910)

PURPOSE: Our purpose was to determine if supernumerary embryos generated by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) should be frozen (using 1,2-propanediol) at the pronuclear or multicellular stage. METHODS: The study was a retrospective analysis conducted at the Dubai Gynaecology & Fertility Centre of the Department of Health & Medical Services, Dubai, U.A.E. One hundred forty-one women undergoing frozen-thawed embryo replacement cycles with IVF generated embryos and 84 women undergoing the same with ICSI generated embryos. RESULTS: Supernumerary, IVF-generated embryos frozen at the multicellular stage had a significantly higher rate of survival on thawing (73.9%) than embryos frozen at the pronuclear stage (64.4%). The morphological grades of the embryos in the two groups were similar, but a significantly higher pregnancy rate was obtained with embryos frozen at the multicellular stage (22.8%) than with pronuclear-stage embryos (14.8%). Similarly, with ICSI-generated embryos, significantly higher survival was seen with multicellular-stage frozen embryos (74.8%) than pronuclear-stage embryos (64.4%). The morphological grades of the embryos and pregnancy outcomes of the two groups were similar. CONCLUSIONS: Supernumerary embryos generated by IVF and ICSI should be frozen at the multicellular stage so as to allow selection of the best embryos for transfer and embryo freezing of only robust embryos.  (+info)

Effect of Vero cell coculture on the development of frozen-thawed two-cell mouse embryos. (55/2910)

PURPOSE: Our purpose was to evaluate the beneficial effects of long-term coculture of Vero cells on the development of frozen-thawed two-cell mouse embryos. METHODS: Two-cell mouse embryos were frozen slowly with 1,2-propandiol and sucrose as cryoprotectants and thawed rapidly, followed by stepwise dilution. Vero cells were cultured in drops of RPMI 1640 to establish monolayers. Frozen-thawed embryos were cultured alone (control) or cocultured with Vero cells. The rate of development in both groups was compared. RESULTS: After 4 days of culture, significantly more embryos in coculture were developed to expanded blastocysts (61 vs 37% for controls; P < or = 0.0001). In addition, on the fifth day of cultivation, more embryos in coculture showed the potential of hatching from the zona pellucida (26 vs 7% in controls; P < or = 0.0001). The rate of degeneration in coculture was also much lower than in controls (6 and 15%, respectively). CONCLUSIONS: Coculture of cryopreserved preimplantation-stage embryos with Vero cells seems to be a useful tool to eliminate the postthaw deleterious effect of freezing and also to obtain better-quality embryos appropriate for transfer.  (+info)

Cryopreserved aortic homograft replacement in a patient with Takayasu's arteritis. (56/2910)

A patient with severe long-standing Takayasu's arteritis underwent successful replacement of the aortic root and ascending aorta with a cryopreserved aortic homograft. Her postoperative course was uneventful and echocardiography demonstrated evidence of neither aortic regurgitation nor graft detachment more than 2 years after the operation. Magnetic resonance image demonstrated no signs of graft enlargement.  (+info)