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PubMed1996

Pore structure of hydrating cement paste by magnetic resonance relaxation analysis and freezing.

Nuclear magnetic resonance relaxation analysis has been applied to interpret the evolution of microstructure in a cement paste during hydration. A basic understanding of the wet-dry and freeze-thaw processes of cement pastes has been developed. The pore structure evolution has been studied by the suppression of the freezing temperature of water and compared with spin-spin relaxation analysis performed at room temperature. Both methods consistently show that hydrating cement pastes have two principal components in their size distribution. The NMR relaxation times provide a measure of the characteristic pore sizes. Their interpretation is made in the context of a fast exchange model. Supercooling and thawing point depression of confined water has been studied systematically. The depression of the freezing point of liquid water confined within a pore was found to be dependent on the pore size, with capillary pore water freezing at 240 K and the remaining gel pore water freezing over a temperature range extending to as low as 160 K.A modified Gibbs-Thompson analysis was used to determine pore volume distributions from the distribution of thawing temperatures.

Construction Materials↗
PubMed2002

A portable toxicity biosensor using freeze-dried recombinant bioluminescent bacteria.

A portable biosensor has been developed to meet the demands of field toxicity analysis. This biosensor consists of three parts, a freeze-dried biosensing strain within a vial, a small light-proof test chamber, and an optic-fiber connected between the sample chamber and a luminometer. Various genetically engineered bioluminescent bacteria were freeze-dried to measure different types of toxicity based upon their modes of action. GC2 (lac::luxCDABE), a constitutively bioluminescent strain, was used to monitor the general toxicity of samples through a decrease in its bioluminescence, while specific toxicity was detected through the use of strains such as DPD2540 (fabA::luxCDABE), TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2511 (katG::luxCDABE). These inducible strains show an increase in bioluminescence under specific stressful conditions, i.e. membrane-, protein-, DNA-, and oxidative-stress, respectively. The toxicity of a sample could be detected by measuring the bioluminescence 30 min after addition to the freeze-dried strains. In an attempt to enhance the sensitivity of the freeze-dried cells, glucose and Tween 80 were tested as additives. It was found that the addition of glucose had a negative effect on the viability of the freeze-dried cells, while samples having Tween 80 showed an increase in their viability. On the other hand, the addition of either Tween 80 or glucose decreased the final bioluminescent response of DPD2540 in response to 4-chlorophenol. Using these strains, many different chemicals were tested and characterized. This portable biosensor, with a very simple protocol, can be used for field sample analysis and the monitoring of various water systems on-site.

Biosensing Techniques↗
PubMed2001

Freeze-fracture studies on lipids and membranes.

Freeze-fracture electron microscopy is especially useful for investigation of lipid structures by the advantageous fracture course within hydrophobic zones. Freezing is, on the other hand, a restriction because the structures of lamellar and non-lamellar phase states with disordered acyl chains (L(alpha), H(II,) cubic) are difficult to preserve. An important aspect of this method is therefore the lipid structure of phase states with ordered acyl chains (crystal, gel), and with a different degree of hydration. Freeze-fracture of pure lipid systems creates a valid representation of the structure of non-lamellar phases and of the general structure of the "lamellar" lipid bilayer, and lamellar phases with characteristic deformations (ripples, curvatures, plane sectors) can be identified. Fracture through the hydrophobic bilayer centre of biological membranes reveals characteristic protein components, the intramembraneous particles (IMPs). The lateral distribution of the IMPs is a helpful marker for fluid and rigid phase states, also without deformation of the lamella. The overall history and the present state of knowledge concerning the different structures revealed by the freeze-fracture and freeze-etch techniques in lipid systems, and to a limited extent in biological membranes, is reviewed, taking into account studies from our own laboratory.

Animals↗
PubMed1996

Structural changes and cell viability of cultured epithelium after freezing storage.

Numerous clinical reports have shown the utility of cultured epithelial grafting in the field of plastic and reconstruction surgery. Recently, freezing storage of the cultured epithelium has been tried and has successfully grafted after thawing. It is clinically convenient if it is possible for cultured epithelium to keep its normal structure and viability. However, few papers have described the structural changes in cultured epithelium after freezing storage. In the present study, the morphological changes and cell viability of cultured mucosal epithelial sheets after freezing were studied in comparison with cultured epidermal sheets. Furthermore, we discuss the effect of storage temperature and cryoprotectants. As a result, there were some structural changes such as vacuolar degeneration in the cultured mucosal sheets using dimethyl sulphoxide (DMSO) as a cryoprotectant. Such changes were more clearly observed at -80 degrees C than at -196 degrees C with DMSO. However, little morphological change was observed in both epithelial sheets cultured with glycerin. The cell viability analysed by flow cytometry showed that more than 62% of the cells kept their viability after freezing storage. These results suggest that the optimum conditions of freezing for cultured epithelium were -196 degrees C storage by slow cooling methods with glycerin as a cryoprotectant.

Cell Survival↗
PubMed2003

Effects of liquid and freeze-dried grapefruit juice on the pharmacokinetics of praziquantel and its metabolite 4'-hydroxy praziquantel in beagle dogs.

Grapefruit juice changes the pharmacokinetic parameters of a variety of drugs metabolized primarily by cytochrome P450 3A. In a three-phase crossover study, six male beagle dogs were administered 100 ml of water (control), 100 ml of commercial liquid grapefruit juice, or 10 g of freeze-dried grapefruit juice (equivalent to 100 ml of liquid grapefruit juice) with 100 ml of water, followed after 2 h by single oral dose of praziquantel (30 mg kg(-1)). After treatment, the dogs were sampled at different times. Determination of praziquantel and its metabolite 4'-hydroxy praziquantel (identified by GC/MS) was performed by HPLC. Liquid and freeze-dried grapefruit juice preadministration increased the C(max) of praziquantel about three-fold and the AUC 2.5- and 2.3-fold, respectively. The T(max) (0.75 h) was unaffected by liquid or freeze-dried grapefruit juice, while T(1/2) was 2.3- and 1.7-fold higher compared with controls. The amount of 4'-hydroxy praziquantel was also affected by both liquid and freeze-dried grapefruit juice administration: the AUC and C(max) increased four- and three-fold, respectively and the T(max) was significantly enhanced. These findings demonstrate that both freeze-dried grapefruit juice and commercial liquid grapefruit juice significantly increase plasma concentrations and T(1/2) of praziquantel in dogs.

Animals↗
PubMed2004

Yeast adapt to near-freezing temperatures by STRE/Msn2,4-dependent induction of trehalose synthesis and certain molecular chaperones.

Virtually nothing is known about the biochemical adaptations in eukaryotic cells that may enhance survival at low temperatures or upon freezing. Here we demonstrate an adaptive response in yeast that is activated below 10 degrees C and increases tolerance to low temperatures and freezing. This response involves a dramatic accumulation of the chemical chaperone trehalose and induction of trehalose-synthesizing enzymes (Tps1, Tps2) and certain heat shock proteins (Hsp104, Hsp42, Hsp12, Ssa4). mRNAs for these proteins increase dramatically below 10 degrees C and even at 0 degrees C. Their expression requires Msn2,4 transcription factors but also involves marked mRNA stabilization. Upon return to 30 degrees C, TPS1, TPS2, and HSP104 mRNAs, trehalose levels and tolerance to freezing fall dramatically within minutes. Mutants lacking trehalose or Msn2,4 die more rapidly at 0 degrees C and upon freezing. Thus, below 10 degrees C, yeast show an adaptive response that sustains viability at low or freezing temperatures, which are commonly encountered in natural environments and laboratory refrigerators.

Adaptation, Physiological↗
PubMed2000

Method of quantifying the loss of acidification activity of lactic acid starters during freezing and frozen storage.

We have developed a method to quantify the resistance to freezing and frozen storage of lactic acid starters, based on measuring the time necessary to reach the maximum acidification rate in milk (tm) using the Cinac system. Depending on the operating conditions, tm increased during the freezing step and storage. The loss of acidification activity during freezing was quantified by the difference (delta tm) between the tm values of the concentrated cell suspension before and after freezing. During storage at -20 degrees C, linear relationships between tm and the storage time were established. Their slope, k, allowed the quantitation of the decrease in acidification activity during 9-14 weeks of frozen storage. The method was applied to determine the resistance to freezing and frozen storage of four strains of lactic acid bacteria and to quantify the cryoprotective effect of glycerol.

Cryoprotective Agents↗
PubMed1996

Unprotected freezing of human spermatozoa exerts a detrimental effect on their oocyte activating capacity and chromosome integrity.

The influence of unprotected freezing of mammalian spermatozoa on their oocyte activating capacity and chromosome integrity is unknown. However, this type of sperm treatment has been used in assisted reproduction by intracytoplasmic sperm injection in cattle and humans. The mouse oocyte injection test was used to analyse the influence of unprotected freezing of human spermatozoa on their reproductive characteristics. Mouse oocytes were microinjected with intact human spermatozoa or spermatozoa treated with two cycles of unprotected freeze-thawing. Oocytes surviving the injection were either cultured without further treatment or exposed to ethanol solution to induce parthenogenetic activation. Both injected and activated oocytes were used for sperm chromosome analysis. The results revealed a significant reduction in oocyte activating capacity and a tenfold increase in the incidence of structural chromosomal abnormalities in human spermatozoa treated by unprotected freezing. We conclude that unprotected freezing of human spermatozoa has a detrimental effect on their reproductive characteristics. Our data also provide a new perspective on the stability of mammalian spermatozoa to physical factors and demonstrate the importance of detailed analysis of the stability of sperm structures for successful development of new approaches in assisted reproduction.

Animals↗
PubMed2000

Nitrogenase reduction of carbon disulfide: freeze-quench EPR and ENDOR evidence for three sequential intermediates with cluster-bound carbon moieties.

Freeze-quenching of nitrogenase during reduction of carbon disulfide (CS(2)) was previously shown to result in the appearance of a novel EPR signal (g = 2.21, 1.99, and 1.97) not previously associated with any of the oxidation states of the nitrogenase metal clusters. In the present work, freeze-quench X- and Q-band EPR and Q-band (13)C electron nuclear double resonance (ENDOR) spectroscopic studies of nitrogenase during CS(2) reduction disclose the sequential formation of three distinct intermediates with a carbon-containing fragment of CS(2) bound to a metal cluster inferred to be the molybdenum-iron cofactor. Modeling of the Q-band (35 GHz) EPR spectrum of freeze-trapped samples of nitrogenase during turnover with CS(2) allowed assignment of three signals designated "a" (g = 2.035, 1.982, 1.973), "b" (g = 2.111, 2.002, and 1.956), and "c" (g = 2.211, 1. 996, and 1.978). Freezing samples at varying times after initiation of the reaction reveals that signals "a", "b", and "c" appear and disappear in sequential order. Signal "a" reaches a maximal intensity at 25 s; signal "b" achieves maximal intensity at 60 s; and signal "c" shows maximal intensity at 100 s. To characterize the intermediates, (13)CS(2) was used as a substrate, and freeze-trapped turnover samples were examined by Q-band (13)C ENDOR spectroscopy. Each EPR signal ("a", "b", and "c") gave rise to a distinct (13)C signal, with hyperfine coupling constants of 4.9 MHz for (13)C(a), 1. 8 MHz for (13)C(b), and 2.7 MHz for (13)C(c). Models for the sequential formation of intermediates during nitrogenase reduction of CS(2) are discussed.

Azotobacter vinelandii↗
PubMedDate not supplied

Collapse temperature of freeze-dried Lactobacillus bulgaricus suspensions and protective media.

Optimization of the freeze-drying process needs to characterize the physical state of frozen and dried products. A protocol to measure the collapse temperature of complex biological media such as concentrated lactic acid bacteria using freeze-drying microscopy was first elaborated. Afterward, aqueous solutions of one or several components as well as concentrated lactic acid bacterial suspensions were analyzed in order to study how the structure of these materials is degraded during freeze-drying. A similar behavior toward collapse was observed for all aqueous solutions, which was characterized by two temperatures: the "microcollapse" temperature (T(microc), beginning of a local loss of structure) and the "collapse" temperature (T(c), beginning of an overall loss of structure). For aqueous solutions, these two temperatures were close, differing by less than 3 degrees C. Nevertheless, when lactic acid bacteria were added to aqueous solutions, the collapse temperatures increased. Moreover, the interval between microcollapse and collapse temperatures became larger. Lactic acid bacterial cells gave a kind of "robustness" to the freeze-dried product. Finally, comparing glass transition, measured by differential scanning calorimetry (DSC) and collapse temperature for aqueous solutions with noncrystallizable solutes, showed that these values belonged to the same temperature range (differing by less than 5 degrees C). As suggested in the literature, the glass transition temperature can thus be used as a first approximation of the collapse temperature of these media. However, for lactic acid bacterial suspensions, because the difference between collapse and glass transition temperatures was about 10 degrees C, this approximation was not justified. An elegant physical appearance of the dried cakes and an acceptable acidification activity recovery were obtained, when applying operating conditions during freeze-drying in vials that allowed the product temperature to be maintained during primary drying at a level lower than the collapse temperature of lactic acid bacterial suspensions. Consequently, the collapse temperature T(c) was proposed as the maximal product temperature preserving the structure from macroscopic collapse and an acceptable biological activity of cells.

Cell Culture Techniques↗
PubMed2000

Effect of freezing on the activity of catalase in apple flesh tissue.

Catalase (CAT, EC 1.11.1.6) activity was measured in flesh tissue of six apple cultivars (Malus domestica Borkh. cvs. Braeburn, Gala, Jonagold, McIntosh, Red Delicious, and Spartan). Activity of CAT was determined for fresh and frozen tissue of the same fruit. Freezing resulted in reductions of 50 to 90% in CAT activity compared with the activity measured in crude extracts from fresh tissues. The rate of freezing had an impact on the level of reduction of CAT activity, with slower freezing procedures leading to greater losses in activity. Six additives to the extraction buffer were tested to evaluate their potential to reduce the inactivation of CAT from frozen tissue, but only EDTA and Tween 20 showed any benefit. However, EDTA and Tween 20 provided only partial recovery in CAT activity. In contrast, crude extracts prepared from fresh tissue showed no appreciable loss in CAT activity after frozen storage for two weeks at -80 degrees C. Gel electrophoresis and immunological analysis indicated that the loss in CAT activity from tissue freezing could be attributed to loss of both the tetrameric CAT enzyme structure and total CAT protein. The implications of using freezing to preserve apple tissue samples prior to catalase activity analysis is discussed.

Catalase↗
PubMed1998

Stability of lipid/DNA complexes during agitation and freeze-thawing.

It is well established that cationic liposomes facilitate the delivery of DNA and offer substantial advantages over viral-based delivery systems. However, these synthetic vectors readily aggregate in liquid formulations which in clinical trials requires preparation of lipid/DNA complexes at the bedside immediately before injection. This temporal requirement could be eliminated if complexes were formulated as stable preparations that could be shipped, stored, and administered as needed. To this end, our study investigates the stability of lipid/DNA complexes during physical stresses that might be encountered during shipping and storage, i.e., agitation and freeze-thawing. Our data show that agitation significantly reduces transfection rates in complexes prepared with three different commercially available lipid formulations. Additional experiments indicate that slow freezing is more damaging than rapid freezing, and that sucrose is able to preserve transfection and complex size during freeze-thawing. These results are consistent with previous reports and demonstrate that frozen formulations may be suitable for maintaining transfection rates of lipid/DNA complexes. Under certain conditions, we observe a reproducible 3-fold increase in transfection after freeze-thawing that is prevented by high concentrations of sucrose. Together, these data suggest that physical stresses can alter structural characteristics of lipid/DNA complexes that can markedly affect rates of DNA delivery.

Animals↗
PubMed1997

Dependence of the molecular mobility and protein stability of freeze-dried gamma-globulin formulations on the molecular weight of dextran.

PURPOSE: The effect of the molecular weight of dextran on the molecular mobility and protein stability of freeze-dried serum gamma-globulin (BGG) formulations was studied. The stabilizing effect of higher molecular weight dextran is discussed in relation to the molecular mobility of the formulations. METHODS: The molecular mobility of freeze-dried BGG formulations containing dextrans of various molecular weights was determined based on the free induction decay of dextran and water protons measured by proton NMR. The protein stability of the formulations was determined at temperatures ranging from 20 to 70 degrees C by size exclusion chromatography. RESULTS: Changes in the molecular mobility of freeze-dried formulations that occurred at temperatures below the glass transition temperature could be detected as the molecular mobility-changing temperature (Tmc), at which dextran protons started to exhibit a Lorentzian relaxation decay due to higher mobility in addition to a Gaussian relaxation decay. Tmc increased as the molecular weight of dextran increased. The proportion of dextran protons which exhibited the higher mobility relaxation process (Phm) at temperatures above Tmc decreased as the molecular weight of dextran increased. Protein stability was closely related to molecular mobility. The temperature dependence of the denaturation rate changed at around Tmc, and denaturation in the microscopically liquidized state decreased as Phm decreased with increasing molecular weight of dextran. CONCLUSIONS: The effect of the molecular weight of dextran on the protein stability of freeze-dried BGG formulations could be explained in terms of the parameters obtained by 1H-NMR such as Tmc and Phm. These parameters appear to be useful in preformulation and stability prediction of freeze-dried formulations.

Calorimetry, Differential Scanning↗
PubMed1996

Preparation and characterization of freeze-dried chitosan-poly(ethylene oxide) hydrogels for site-specific antibiotic delivery in the stomach.

PURPOSE: The purpose of this study was to develop novel drug delivery systems with pH-sensitive swelling and drug release properties for localized antibiotic delivery in the stomach. METHODS: The drug delivery systems were synthesized by crosslinking chitosan and poly(ethylene oxide) (PEO) in a blend to form semi-interpenetrating polymer network (semi-IPN). Scanning electron microscopy was used to compare the surface and bulk morphology of the freeze-dried and air-dried chitosan-PEO semi-IPN. The hydrogels were allowed to swell and release the antibiotics--amoxicillin and metronidazole--in enzyme-free simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.2) at 37 degrees C. RESULTS: Freeze-dried chitosan-PEO semi-IPN with a porous matrix had swollen extensively as compared to the air-dried hydrogel. The swelling ratio of freeze-dried and air-dried chitosan-PEO semi-IPN after 1 h in SGF was 16.1 and 2.30, respectively. More than 65% of the entrapped amoxicillin and 59% of metronidazole were released from the freeze-dried chitosan-PEO semi-IPN after 2 h in SGF. CONCLUSIONS: The results of this study suggest that freeze-dried chitosan-PEO semi-IPN could be useful for localized delivery of antibiotics in the acidic environment of the gastric fluid.

Amoxicillin↗
PubMed2002

Crystallization of mannitol below Tg' during freeze-drying in binary and ternary aqueous systems.

PURPOSE: To characterize the phase transitions in a multicomponent system during the various stages of the freeze-drying process and to evaluate the crystallization behavior below Tg' (glass transition temperature of maximally freeze-concentrated amorphous phase) in frozen aqueous solutions and during freeze-drying. METHODS: X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to study frozen aqueous solutions of mannitol with or without trehalose. By attaching a vacuum pump to the low-temperature stage of the diffractometer, it was possible to simulate the freeze-drying process in situ in the sample chamber of the XRD. This enabled real-time monitoring of the solid state of the solutes during the process. RESULTS: In rapidly cooled aqueous solutions containing only mannitol (10% w/w), the solute was retained amorphous. Annealing of frozen solutions or primary drying. both below Tg', resulted in crystallization of mannitol hydrate. Similar effects were observed in the presence of trehalose (2% w/w). At higher concentrations (> or =5% w/w) of this noncrystallizing sugar. annealing below Tg' led to nucleation but not crystallization. However, during primary drying, crystallization of mannitol hydrate was observed. CONCLUSIONS: The combination of in situ XRD and DSC has given a unique insight into phase transitions during freeze-drying as a function of processing conditions and formulation variables. In the presence of trehalose, mannitol crystallization was inhibited in frozen solutions but not during primary drying.

Cold Temperature↗
PubMed2003

The effect of additives on the crystallization of cefazolin sodium during freeze-drying.

PURPOSE: To monitor the phase transitions during freeze-drying of cefazolin sodium (I) as a function of process and formulation variables. METHODS: Aqueous solutions of I were frozen under controlled conditions in the sample chamber of a variable temperature X-ray powder diffractometer (XRD). The instrument was modified so that the chamber could be evacuated and the samples dried under reduced pressures. Thus, the entire freeze-drying process was carried out in the XRD holder with real time monitoring of the phase transitions during the different stages of freeze-drying. RESULTS: When aqueous solutions of cefazolin sodium (10% w/w) were cooled to -40 degrees C, the XRD pattern revealed only the crystallization of ice. Annealing the frozen sample led to the crystallization of I as the pentahydrate. Differential scanning calorimetry revealed that the presence of isopropyl alcohol (IPA) (5% w/w) led to a decrease in the Tg', the glass transition temperature of the system, and lowered the temperature of crystallization. The crystallization was studied at -8 and at -15 degrees C in the XRD, and, as expected, more rapid crystallization was observed at the higher temperature. Primary drying at -8 degrees C led to the dehydration of the pentahydrate, resulting in a poorly crystalline product. Again, XRD permitted real time monitoring of the decrease in intensities of some characteristic peaks of the pentahydrate. The in situ XRD technique also enabled us to study the effects of processing conditions (different primary and secondary drying temperatures) and crystalline bulking agents on the solid-state of I in the lyophile. When I was lyophilized using mannitol or glycine as an additive, without an annealing step, the drug was X-ray amorphous although the additive crystallized. When annealed and freeze-dried, I remained crystalline in the presence of glycine but not in the presence of mannitol. CONCLUSIONS: The in situ XRD technique has enabled us to characterize the phase transitions during freeze-drying of cefazolin sodium in multicomponent systems.

Cefazolin↗
PubMed2004

Poly(lactide-co-glycolide) microsphere formulations of darbepoetin alfa: spray drying is an alternative to encapsulation by spray-freeze drying.

PURPOSE: The purpose of this work was to evaluate spray-freeze drying and spray drying processes for encapsulation of darbepoetin alfa (NESP, Aranesp). METHODS: Darbepoetin alfa was encapsulated in poly(lactide-co-glycolide) by spray-freeze drying and by spray drying. Integrity was evaluated by size-exclusion chromatography and Western blot. Physical properties and in vitro release kinetics were characterized. Pharmacokinetics and pharmacodynamics were evaluated in nude rats. RESULTS: Microspheres produced by spray drying were larger than those produced by spray-freeze drying (69 microm vs. 29 microm). Postencapsulation integrity was excellent for both processes, with < 2% dimer by size-exclusion chromatography. In vitro release profiles were similar, with low burst (< 25%) and low cumulative protein recovery at 4 weeks (< or = 30%), after which time covalent dimer (< or = 6.5%) and high molecular weight aggregates (< or = 2.3%) were recovered by denaturing extraction. After a single injection, darbepoetin alfa was detected in serum through 4 weeks for all microsphere formulations tested in vivo, although relative bioavailability was higher for spray-freeze drying (28%) compared with spray drying (21%; p = 0.08) as were yields (73-82% vs. 34-57%, respectively). For both processes hemoglobin was elevated for 7 weeks, over twice as long as unencapsulated drug. CONCLUSIONS: Spray drying, conducted at pilot scale with commercial equipment, is comparable to spray-freeze drying for encapsulation of darbepoetin alfa.

Animals↗
PubMed1999

Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men.

Cryopreservation is known to impair sperm motility and decrease the fertilization rate by detrimental effects on acrosomal structure and acrosin activity. However, the consequences of cryopreservation on the integrity of the sperm nucleus, chromatin stability and centrosome are less clear. The present study was designed to determine the effect of the freeze-thawing procedure on chromatin condensation (aniline blue staining) and the morphology (strict criteria) and membrane integrity of human spermatozoa. The structural and functional characteristics of the sperm plasma membrane were measured by the eosin-test and hypo-osmotic swelling test which were done separately. Sperm cryopreservation was performed on semen samples from two groups of men classified as fertile (n = 20) and subfertile (n = 72), based on their reproductive history and semen analysis according to WHO guidelines. The mean percentage of condensed chromatin, morphologically normal spermatozoa and membrane integrity in all semen samples investigated (n = 92) decreased significantly (p = 0.0001) after freeze-thawing, in comparison to the value observed prior to freezing. By comparing the semen samples between fertile and subfertile patients, significantly (p = 0.0009) greater damage was demonstrated in the subfertile than in the fertile group. Furthermore, no significant difference was observed between the two groups with regard to the morphological alteration and structural as well as functional damage of the sperm membrane. In conclusion, the freeze-thawing procedure significantly affects chromatin structure and sperm morphology, especially in the head and the tail regions, and this may explain the lower fertilization rate and IVF/ICSI outcome when frozen-thawed spermatozoa are used. In addition, this study demonstrates that chromatin condensation is a sensitive parameter for the evaluation of cryodamage of semen samples from fertile and subfertile patients, though subfertile patients with very poor semen characteristics have yet to be studied. It is therefore recommended that chromatin condensation be used as an additional parameter for the assessment of sperm quality after freeze-thawing.

Cell Membrane↗