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PubMed2006

[Ovarian cryopreservation: clinical and ethical problems arisen further to study on cases of ovarian cryopreservation].

OBJECTIVE: An early ovarian failure and sterility, on patients often still young, may result from some of the anticancerous treatments. Ovarian cryopreservation is a research way for fertility restoration on patients who will have gonadotoxic treatment. The aim of our work was to study the cases of ovarian tissue preserved in our Department, enabling us to assess the clinical and ethical problems of this technique. PATIENTS AND METHODS: A retrospective study was conducted from January 2001 onwards up to October 2005, on 14 patients, whose ovarian cortex was frozen our the University Fertility Center (Hôpital de la Conception, Marseille, France). RESULTS: In our study, the patients' average age was 17+/-5.5 and the median was 14 years [13-24]. Eight patients were under 18 (11+/-3-years-old). The cryopreservation indications were especially haematological (N = 9). More than half of the patients (N = 8) had undergone a gonadotoxic treatment before ovarian cryopreservation. DISCUSSION AND CONCLUSION: Ovarian cryopreservation is still a recent technology in reproductive medicine. The future of ovarian tissue after thawing, with its different techniques of autograft, is still not consensual. Experimental research remains essential to improve the freezing protocols and ovarian transplant in human medicine.

Adolescent↗
PubMed2003

Safety of cryopreservation straws for human gametes or embryos: a study with human immunodeficiency virus-1 under cryopreservation conditions.

BACKGROUND: The possibility of assisted reproductive technology (ART) for couples carrying viruses, especially HIV-1, necessitates consideration of the safety of cryopreserving human gametes or embryos in liquid nitrogen tanks. Following our evaluation of the safety of three kinds of straws containing HIV-1 at 37 degrees C, we have now examined the HIV-1 imperviousness of the same straws under cryopreservation conditions. METHODS: Polyvinyl chloride (PVC), polyethylene terephthalate glycol (PETG) and high-security ionomeric resin (IR) straws (24 each) were tested. Each straw was filled with 100 microl of HIV-1-containing supernatant [reverse transcriptase (RT) activity: 15 000 c.p.m./50 microl]. Then PVC and PETG straws were sealed ultrasonically only at their free-end, and IR straws were thermosoldered at both ends. Each straw was put in a 15 ml Falcon tube which was capped and submerged in a liquid-nitrogen tank for 7 days. After bleach decontamination or not, the outside of each end of the straw was rinsed with RPMI medium (1 ml) before cryopreservation and after thawing. Viral RNA was extracted from the medium and then amplified by RT-polymerase chain reaction (PCR) followed by nested-PCR using HIV-1 protease-specific primers. RESULTS: HIV-1 RNA was detected in some PVC and PETG rinse media, probably resulting from splashing during ultrasonic sealing, but not in the rinse media of thermosoldered IR straws. CONCLUSION: Under cryopreservation conditions, IR straws would appear to be safe for HIV-1 storage in ART. For PVC and PETG straws, as highlighted in this study, the ultrasonic sealing could be the weak safety link.

Composite Resins↗
PubMed1995

Cryopreserved cord blood myeloid progenitor cells can serve as targets for retroviral-mediated gene transduction and gene-transduced progenitors can be cryopreserved and recovered.

To determine future possibilities for gene transfer, we evaluated whether myeloid progenitor cells from human umbilical cord blood (CB) could be frozen, thawed in viable form and transduced with a Neomycin resistance (NeoR) gene using retroviral vectors, and if fresh progenitor cells transduced with a Neo gene could be cryopreserved and recovered. Fresh and thawed cryopreserved nonadherent low-density T-lymphocyte depleted (NALT-) CB cells were assayed before and after gene transduction for colony formation in the presence of multiple growth factors in the absence and presence of G418. The results demonstrate that the NeoR gene could be introduced into thawed cryopreserved myeloid progenitor cells at an efficiency similar to that of fresh cells and that fresh cells transduced with the NeoR gene could be frozen in a cryopreserved state and recovered after thawing. Proviral integration, as assessed by PCR/Southern Analysis, confirmed the G418R colony data. Proviral integration was detected not only in primary G418R-colonies, but also in replated colonies in secondary dishes derived from G418R-multipotential progenitor cells (CFU-GEMM) suggesting stable integration of the transduced gene into early subsets of replatable progenitors. This information may be of use clinically.

3T3 Cells↗
PubMed1988

Platelet avidity of cryopreserved veins: thrombogenicity of cryopreserved veins.

Prior work has suggested that cryopreserved venous allografts may serve as an effective arterial substitute. To determine the relative thrombogenicity of this material, platelet uptake of cryopreserved canine jugular veins (CJV) before and after deendothelialization was compared to fresh CJV before and after deendothelialization, and to PTFE. CJV were frozen in Medium 199 (M199) with 10% dimethylsulfoxide to -70 degrees C. CJV were deendothelialized with collagenase (165 u/mg Worthington type II in phosphate-buffered saline) for 15 min at 37 degrees C. Canine platelets were harvested, labeled with indium-III-oxine, and suspended in 1 liter of M199. Labeled platelets were used to perfuse each graft in a nonpulsatile flow loop for 120 min. Deendothelialization led to a significant increase in platelet uptake (P less than 0.05). Frozen deendothelialized CJV showed the highest affinity for platelets. PTFE, cryopreserved, and fresh CJV showed similar affinity for platelets. Cryopreservation alone did not seem to inhibit the ability of endothelial cells to act as a nonthrombogenic surface and did not alter the histologic structure of the veins.

Animals↗
PubMed1990

[Application of experimental findings of cryopreservation of animal tissue to results of cryopreservation of human tissue].

In order to compare the results after experimental corneal cryopreservation of porcine corneas to human tissue we cryopreserved several human corneas that were not suitable for transplantation. The corneas were frozen in a medium containing 2% chondroitin sulfate, 15% DMSO, and 20% fetal calf serum in MEM-medium to -196 degrees C and thawed at 37 degrees C in a waterbath. Subsequently they were kept in organ culture for 24 h. After vital staining with trypan blue and alizarin red S they were evaluated morphometrically. Endothelial cell densities after the experiment ranged between 47% and 64% of the endothelial cell counts revealed before cryopreservation. Human corneal endothelium showed enlargement of single nucleated cells, and large numbers of giant cells with one or two nuclei but only few multinucleated giant cells and only few necrotic spots compared to porcine tissue in which multinucleated giant cells and large necrotic areas were a frequent finding. We conclude that human corneal endothelium is less sensitive to the freeze-thaw trauma than porcine tissue. Porcine tissue can be used as a susceptible model for the development of new methods of corneal cryopreservation.

Adult↗
PubMed2004

In-vitro maturation of germinal-vesicle oocytes and cryopreservation in metaphase I/II: a possible additional option to preserve fertility during ovarian tissue cryopreservation.

This study describes the possibility of combining two options in order to preserve female fertility: cryopreservation of human ovarian tissue and in-vitro matured germinal vesicle (GV) oocytes retrieved during tissue dissection. In contrast to ovarian tissue cryopreservation, the cryostorage of in-vitro matured unfertilized metaphaseI/II oocytes could be a more realistic option. This concept of preserving fertility before chemotherapy and/or radiotherapy without a long time delay could be an additional reason for favouring ovarian tissue cryopreservation. This concept is discussed in regard to two cases.

Adult↗
PubMed2000

Is a review of standard procedures for cryopreservation needed?: safe and effective cryopreservation-should sperm banks and fertility centres move toward storage in nitrogen vapour?

A recent consultation documentation by the Human Fertilisation and Embryology Authority (HFEA) which focused on the safe cryopreservation of gametes and embryos highlighted the need for a review of the way that fertility clinics in the UK store potentially infective material. The main points for consideration were to: (i) ensure containers used for cryopreservation are guaranteed by manufacturers to withstand low temperatures; (ii) use secondary containers, i.e. 'double bagging' of samples if stored in the liquid phase; and (iii) store in nitrogen vapour as a 'safer' alternative. In this article we examine a number of issues related to vapour storage which need careful consideration, including safety, cost and the effectiveness of various storage techniques in maintaining gamete and embryo viability. We also discuss the effectiveness of vapour storage in comparison with current liquid nitrogen storage techniques. In conclusion, we propose that fertility clinics should be compelled to review their cryopreservation procedures, not just because of new legislation or indeed fear of litigation but by a moral obligation.

Bacterial Infections↗
PubMed1992

The contribution of the toxicity of a glycerol-egg yolk-citrate cryopreservative to the decline in human sperm motility during cryopreservation.

The contribution of the toxicity of glycerol-egg yolk-citrate (GEYC) cryopreservative medium to the loss of function of human spermatozoa during cryopreservation was determined by investigating the effect of mixing semen with the medium on sperm motility. The percentage of progressively motile spermatozoa, velocity (micron s-1) and lateral head displacement (micron) (mean +/- SEM, n = 28) were 55 +/- 4.1, 47 +/- 2.7, 4.4 +/- 0.2 and 32 +/- 3.8, 40 +/- 2.5, 3.6 +/- 0.25 and 15 +/- 2.5, 28 +/- 1.1, 2.8 +/- 0.15 in suspensions of washed spermatozoa prepared from fresh, GEYC-treated and frozen-thawed semen, respectively. The variables changed only slightly after incubation for 3 h. The toxicity of GEYC did not vary significantly between samples which survived the complete freeze-thaw cycle well or very poorly. The toxicity of GEYC is responsible for about 50% of the loss of progressively motile spermatozoa during the complete cryopreservation process, but has little effect on the quality of motility. Susceptibility to GEYC does not explain observed differences in the ability of semen samples to survive freezing.

Citrates↗
PubMed2005

Apoptosis in cryopreserved human ovarian tissue obtained from cancer patients: a tool for evaluating cryopreservation utility.

Fertility preservation is of major importance for women with cancer in whom ovarian function may be disturbed by the use of potentially sterilizing chemotherapeutic drugs and/or pelvic irradiation. Cryopreservation of ovarian cortical tissue is one of the potential options for preserving fertility among these women. Cryopreserved thawed human ovarian tissue can be autografted either orthotopically or heterotopically, but may also be transplanted first into an animal host with subsequent maturation and collection of oocytes. The objective of this study was to investigate the prevalence of ovarian follicular apoptosis in fresh and frozen/ thawed human ovarian tissue as a measure of follicular viability. The study group included 6 women with cancer who underwent ovarian tissue cryopreservation (OTCP). Ovarian tissue samples (n = 2) were obtained from each woman with one sample undergoing evaluation for apoptosis immediately following removal (control, group A) and the other evaluated for apoptosis following freezing/thawing (group B). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) and 4'6' diamido-2-phenylindole hydrochloride (DAPI) staining methods were used to investigate follicular apoptosis. Morphological changes in the same samples were evaluated in hematoxylin and eosin (H&E)-stained sections. In each slide, only primordial and primary follicles were evaluated for abnormal morphology and apoptosis. Abnormal morphology was demonstrated in 23.8+/-8.7% of group A follicles compared to 48.3+/-11.2% of group B follicles (p < 0.05). Apoptosis was demonstrated in 25.4+/-8.4% of group A follicles compared to 60.9+/-6.0% of group B follicles (p < 0.05). We have shown that the ovarian follicles in group B demonstrated a higher incidence of apoptosis compared to those of group A. Therefore, the data suggest that follicular apoptosis might be a consequence of the freezing and thawing procedure. This may be used as a method for evaluating and comparing the outcome of different freezing/thawing protocols.

Adult↗
PubMedDate not supplied

Cryopreservation of human semen. Comparison of cryopreservatives, sources of variability, and prediction of post-thaw survival.

Human semen was cryopreserved using Human Sperm Preservation Medium, TEST-Yolk buffer, or glycerol alone. Sperm characteristics for each specimen were measured before and after freezing to determine which cryopreservative resulted in better cryosurvival and recovery of motile sperm. Sperm frozen in Human Sperm Preservation Medium had a significantly better recovery of all semen parameters (motility, velocity, and recovery) than either TEST-Yolk or glycerol alone. Statistical analyses also were done to examine the variability between and within donor semen specimens. Differences between donors, between specimens, and measurements within donors all contributed to variability of sperm characteristics. Specimen-to-specimen variability for a given donor represented 12% to 47% of the total variability, whereas processing and measurement variability represented 12% to 41%. Donors also varied in the ability of their sperm to tolerate freezing. There was a relationship between motile count after dilution with cryopreservative and post-thaw motile count. This relationship allows the prediction of poor-thaw survival before freezing a specimen.

Cell Survival↗
PubMed1983

Transplantation of cryopreserved and fresh rat islets and canine pancreatic fragments: comparison of cryopreservation protocols.

(1) The freezing protocol we have devised for rat islets results in normal clinical indices and almost normal glucose tolerance in diabetic recipients of the same inbred strain. (2) Cryopreservation of canine islet-containing pancreatic tissue required a higher temperature than rat islets during exposure to the protective agent. (3) Because of the similar compactness of the pancreas in man and dog, we consider this canine model useful for formulating optimal cryopreservation techniques for the human pancreas. (4) Cryopreservation may partially purify islet-containing tissue of its exocrine content.

Animals↗
PubMed1997

Cryopreservation of mouse spermatozoa. I. Effect of seeding on fertilizing ability of cryopreserved spermatozoa.

To examine the effect of seeding to induce ice formation during cryopreservation on their survival, spermatozoa from B6D2F1 mice were cooled to and held at -4 degrees C for 30 min in phosphate-buffered saline (PBS) alone, in egg yolk-supplemented PBS, or in PBS with raffinose + glycerol as cryoprotective additives (CPAs). Seeding and holding spermatozoa at -4 degrees C did not affect their viability as judged by vital staining. Egg yolk protected spermatozoa against chilling injury, as cooling them to -4 degrees C in the presence of egg yolk yielded higher survivals than those cooled without egg yolk (34.4 +/- 3.4 v 9.0 +/- 1.3% in three replicates of >400 spermatozoa/replicate). To study effects of seeding on their fertilizing ability, spermatozoa in the raffinose-glycerol-egg yolk solution were frozen to -196 degrees C either without seeding or after seeding at -4 degrees C. Development of 222 oocytes into two-cell embryos after in vitro fertilization (IVF) with spermatozoa frozen without seeding was 43%; development rates of 186, 186, and 207 oocytes after IVF with spermatozoa frozen after seeding and being held at -4 degrees C for 5, 10, or 30 min were 46, 44, and 9%, respectively. In a direct comparison, after IVF with seeded or unseeded spermatozoa the respective cleavage rates into two-cell embryos were 83% of 275 oocytes and 69% of 304 oocytes, a difference that was small but significant by chi2 analysis. An additional 925 oocytes were fertilized with spermatozoa after being seeded and frozen to -196 degrees C in four separate batches of CPA solutions. Overall, after IVF with frozen sperm, 68% of those oocytes cleaved into two-cell embryos and 59% developed into 544 blastocysts. Based on these results, we concluded that embryo production by IVF seemed to be improved using spermatozoa frozen after being seeded. Mouse spermatozoa cryopreserved by the method described here are capable of fertilizing oocytes at a rather high rate.

Animals↗
PubMed2004

Is ABO group incompatibility really the reason of accelerated failure of cryopreserved allografts in very young patients?--Echography assessment of the European Homograft Bank (EHB) cryopreserved allografts used for reconstruction of the right ventricular outflow tract.

Right ventricular outflow tract reconstruction (RVOTR) with cryopreserved allograft for Ross operation and other congenital or acquired cardiac malformation has become a routine and currently, the procedure of choice for children and young patients. A tendency of accelerated degeneration in the youngest recipients has been reported. Some authors advocate the ABO group incompatibility as the main reason for such failure. This retrospective monocentric study presents the long-term outcome of the European Homograft Bank (EHB) cryopreserved allografts, used for RVOTR in Ross operation (group one) and other congenital heart malformation-s (group two). The evaluation of the allograft performance was done by means of echography, considering the allografts with the transvalvular gradient of > or =40 mmHg and/or regurgitation of > or =3+ as failed. Fifty-one patients of group one and 123 of group two were analysed after completed follow-up information. About 25.5% of patients of group one and 30.8% of group two had a compatible, whereas 74.5% of group one and 68.92 of group two an incompatible ABO group with the donor. The mean follow up was 45.77 and 68.88 months, respectively. In second group 22.76% received the aortic, while 77.24% pulmonary allograft. Only three cases of group one (5.88%) failed: one with a compatible (7.69%) and two with an incompatible ABO group (5.26%) (p=0.1), whereas 39 patients (29.4%) of group two failed between 20.1 and 120.2 months (29.73% with and 29.07% without ABO compatibility, p=0.03). Contrary, the age showed more importance in the allograft failure: out of 41 failed allografts, 24 (58.54%) were implanted in patients of 0-5 years (9 or 37.5% with compatible and 15 or 62.5% with incompatible ABO group). Generally, analysing both groups together, there was no influence of ABO mismatching on the allograft failure (p=0.79). Contrary, there was a significant difference in survival between Ross and non-Ross group (p=0.00082).

ABO Blood-Group System↗
PubMed2006

Cryopreservation of articular cartilage. Part 1: conventional cryopreservation methods.

There is increasing interest in the possibility of treating diseased or damaged areas of synovial joint surfaces by grafts of healthy allogeneic cartilage. Such grafts could be obtained from cadaver tissue donors or in the future they might be manufactured by 'tissue engineering' methods. Cartilage is an avascular tissue and hence is immunologically privileged but to take advantage of this is the graft must contain living cells. Preservation methods that achieve this are required to build up operational stocks of grafts, to provide a buffer between procurement and use, and to enable living grafts of a practical size to be provided at the right time for patient and surgeon. Review of the literature shows that it has been relatively straightforward to cryopreserve living isolated chondrocytes, but at the present time there is no satisfactory method to preserve cartilage between the time of procurement or manufacture and surgical use. In this paper, we review the relevant literature and we confirm that isolated ovine chondrocytes in suspension can be effectively cryopreserved by standard methods yet the survival of chondrocytes in situ in cartilage tissue is inadequate and extremely variable.

Animals↗
PubMed2002

Incubation at 37 degrees C prior to cryopreservation decreases viability of liver slices after cryopreservation by rapid freezing.

Precision-cut liver slices are to some extent resistant to ice formation induced by rapid freezing. Susceptibility to rapid freezing damage has been shown to be (partly) dependent on intrinsic properties of cells. In the present study an attempt was made to decrease the susceptibility of rat liver slices for rapid freezing damage: the slices were pre-incubated at 37 degrees C under oxygen, prior to cryopreservation to recover from low ATP levels, impaired ion regulation and cell swelling induced by their preparation. It was shown that, unexpectedly, recovery of cellular homeostasis prior to the cryopreservation procedure by the 37 degrees C pre-incubation markedly decreased viability of rapidly frozen slices (in which ice was formed), but not of vitrified slices (in which no ice was formed), in a time- and temperature-dependent manner. UW was found to protect slices from this 'warm pre-incubation phenomenon.' Apparently, pre-incubation prior to freezing causes certain cellular alterations that render slices more susceptible to rapid freezing damage.

Adenosine↗
PubMed2000

Cryopreservation of human germinal vesicle stage and in vitro matured M II oocytes: influence of cryopreservation media on the survival, fertilization, and early cleavage divisions.

OBJECTIVE: To study the influence of low-sodium cryopreservation media (CPM) on the survival and development of frozen-thawed germinal vesicle (GV) stage and in vitro matured human oocytes. DESIGN: Prospective experimental study. SETTING: Academic hospital-based fertility center. PATIENT(S): Experimental groups: Oocytes cryopreserved at the GV (group A, n = 63 and group B, n = 64) or M II stage (group C, n = 62) with use of conventional (group A) or low-sodium CPM (groups B and C). Control groups: Sibling GV stage oocytes subjected to in vitro maturation (IVM; control group A, n = 64; control group B, n = 64). INTERVENTION(S): IVM, intracytoplasmic sperm injection and subsequent culture. MAIN OUTCOME MEASURE(S): Rates of survival, maturation, fertilization, and cleavage. RESULT(S): The postthaw survival was significantly lower in groups A (57.1%) and B (48.4%) compared to C (84.4%). In group A, maturation and cleavage rates were significantly lower, and fertilization rate was similar to controls (GVBD: 72.2% vs. 90.6%; progression to M II: 33.3% vs. 76.6%; cleavage: 42.9% vs. 88.2%; and fertilization: 58.3% vs. 69.4% in group A vs. control group A, respectively). There was no such difference in group B. In group C, despite a slight but significant lowering of the rate of 2 PN and an increase in that of 3 PN (2 PN: 47.4% vs. 70.2% and 3 PN: 15.8% vs. 3.2% in group C vs. total controls, respectively), embryonic cleavage per GV oocyte was significantly higher (25.8%) compared to group A (4.8%) but not to group B (15.6%). The rate of maturation and cleavage per surviving GV oocyte was significantly higher in group B than group A. CONCLUSION(S): Low-sodium-based CPM is beneficial for in vitro matured M II stage oocytes and is significantly better than the conventional sodium-based media for the GV stage oocytes.

Cell Survival↗
PubMed2000

Micromanipulation of cryopreserved embryos and cryopreservation of micromanipulated embryos in PGD.

The possibility to employ cryopreservation in Preimplantation Genetic Diagnosis (PGD) should enlarge the opportunities for research and clinical activity. For these purposes, we tried three kinds of approaches on human abnormal embryos: (1) cryopreservation of biopsied embryos; (2) biopsy of thawed embryos; and (3) biopsy of embryos derived from thawed oocytes. Our preliminary results show that: (1) biopsy of thawed embryos is feasible and FISH analysis is possible on both survived and lysed cells; (2) Optimization of freezing/thawing procedures are necessary to obtain better survival rate after thawing of biopsied embryos; (3) Biopsy and FISH are feasible on embryos derived from thawed oocytes and they could be a good way to study the chromosomal arrangement of these poorly investigated embryos.

Biopsy↗
PubMed1991

[Development of an improved cryopreservation vial for cryopreservation of donor corneas].

To date, corneal cryopreservation has been performed in vials made of glass or plastic, where the cornea is placed in a large volume of culture medium. For improved cell survival, an attempt was made to define freezing curves, which are induced by variations in the temperature in the freezing chamber. Depending on the mass of the specimen to be frozen and on the material conducting the heat, the freezing vials used so far cannot be regarded as optimum. As glass and plastic do not conduct heat very well, we developed a new freezing vial that is especially suitable for corneal cyropreservation. In our experiments, the interaction of the chamber temperature and the temperature near the corneal endothelium were monitored. Additionally, endothelial cell survival was studied by postculturing the tissue and by vital staining. As a result, a cylindrical vial made of aluminum 2-4 mm thick was designed, which enables even heat transfer from the freezing chamber to the cornea. In this vial, the amount of freezing medium could be reduced to 400 microliters, so that the heat from the crystallization process was very low. In a small series the conditions that had been optimal with porcine tissue were tested on ten human donor corneas. The endothelial cell density before (2365 cells/mm2, range 1675-2800) and after cryopreservation (2199 cells/mm2, range 1600-2720) did not differ significantly.

Animals↗