Sperm cryostorage in a dry tank: An accurate alternative.

This prospective study aimed to determine the effects of dry nitrogen cryostorage on human sperm characteristics in comparison with liquid nitrogen cryostorage. For this purpose, 42 men undergoing routine semen analysis (21 normozoospermia and 21 with altered semen parameters) were analyzed. After slow freezing, half of the straws of each sample were randomly stored in liquid and dry tanks, at the top and bottom levels of the latter. After 6 months storage, thawed samples were treated by density gradient centrifugation and sperm characteristics were compared. There was no difference in sperm progressive motility (15.1% ± 14.2% vs. 15.1% ± 12.7%; p = 0.76), sperm vitality (25.5% ± 17.7% vs. 26.2% ± 19%; p = 0.71), percentages of acrosome-reacted spermatozoa (38% ± 8.5% vs. 38.5% ± 7.4%; p = 0.53) and DNA fragmentation spermatozoa (27.3% ± 12.4% vs. 28.5% ± 12.9%, p = 0.47) after cryostorage in the dry or the liquid nitrogen tank. Moreover, we did not observe differences between either cryostorage system for normal and altered sperm samples. This lack of difference was also observed whatever the floor level of cryostorage in the dry tank. The temperature measurement of the dry tank showed a stable temperature at -194 °C throughout storage whatever the storage floor level, guaranteeing the stability of the low temperatures suitable for human sperm storage. Because of its greater safety, dry storage without contact with the liquid phase should be preferred and can be a useful alternative for the cryostorage of human sperm samples.

Liver X Receptors (LXRs) Alpha and Beta Play Distinct Roles in the Mouse Epididymis.

After its production in the testis, a spermatozoon has to undergo posttesticular maturation steps to become fully motile and fertile. The first step is epididymal maturation, during which immature spermatozoa are transformed into biochemically mature cells ready to proceed to the next step, capacitation, a physiological process occurring in the female genital tract. The biochemical transformations include modification of sperm lipid composition during epididymal transit, with significant changes in fatty acids, phospholipids, and sterols between the caput and the cauda epididymal spermatozoa. Although quantitative aspects of these changes are well documented for several mammalian species, molecular mechanisms governing these steps are poorly understood. Transgenic male mice invalidated for the two liver X receptors (LXRalpha and LXRbeta, nuclear oxysterol receptors regulating cholesterol and lipid metabolism) become sterile when aging, showing an epididymal phenotype. We used single-knockout-model mice to characterize the role of each LXR isoform during sperm maturation in the epididymis. We show here that although a certain redundancy exists in the functions of the two LXR isoforms, some physiological processes are more under the influence of only one of them. In both cases, aging males showed slight subfertility, associated with dyslipidemia, emphasizing the importance of lipid metabolism in relation with male fertility.

Vitrification of human ovarian tissue: a practical and relevant alternative to slow freezing.

BACKGROUND: Cryopreservation of ovarian tissue can be used to preserve the fertility of patients who are about to receive treatment(s) that could compromise their future ovarian function. Here we evaluate the effectiveness of a vitrification protocol by carrying out a systematic comparison with a conventional slow-freezing method on human ovarian tissue. METHODS: Human ovarian samples (mean age 28.0 ± 1.1 years) were processed in parallel for each cryopreservation procedure: vitrification and slow-freezing. Following warming/thawing, histological observations and a TUNEL assay in ovarian follicles were performed and compared to unfrozen control. RESULTS: Both cryopreservation protocols gave comparable histological outcomes. Percentage of intact follicles was 83.6 % following vitrification in a 1.5 M 1,2-propanediol (PrOH), 1.5 M ethylene glycol (EG) and 0.5 M raffinose solution, 80.7 % after slow-freezing in 1.5 M PrOH and 0.025 M raffinose, and 99.6 % in fresh tissue. Follicle density was unchanged by vitrification (0.6 follicles/mm2) or slow-freezing (0.5 follicles/mm2) compared to fresh tissue (0.7 follicles/mm2). Percentage of follicles with DNA fragmentation was not statistically different in vitrified (20.8 %) or slow-frozen (31.3 %) tissues compared to the unfrozen control (35.0 %). There was no difference in proportion of stroma cells with DNA fragmentation in vitrified (6.4 %) and slow-frozen (3.7 %) tissues compared to unfrozen tissue (4.2 %). CONCLUSIONS: This vitrification protocol enables good preservation of ovarian quality post-warming. The evaluation of endocrine function after vitrification need to be perform in a higher cohort to evaluate if this protocol may offer a relevant alternative to conventional slow-freezing for the cryopreservation of human ovarian tissue.

Evaluation of atorvastatin efficacy and toxicity on spermatozoa, accessory glands and gonadal hormones of healthy men: a pilot prospective clinical trial.

BACKGROUND: Recommendations for cardiovascular disease prevention advocate lowering both cholesterol and low-density lipoprotein cholesterol systemic levels, notably by statin intake. However, statins are the subject of questions concerning their impact on male fertility. This study aimed to evaluate, by a prospective pilot assay, the efficacy and the toxicity of a decrease of cholesterol blood levels, induced by atorvastatin on semen quality and sexual hormone levels of healthy, normocholesterolaemic and normozoospermic men. METHODS: Atorvastatin (10 mg daily) was administrated orally during 5 months to 17 men with normal plasma lipid and standard semen parameters. Spermatozoa parameters, accessory gland markers, semen lipid levels and blood levels of gonadal hormones were assayed before statin intake, during the treatment, and 3 months after its withdrawal. RESULTS: Atorvastatin treatment significantly decreased circulating low-density lipoprotein cholesterol (LDL-C) and total cholesterol concentrations by 42% and 24% (p<0.0001) respectively, and reached the efficacy objective of the protocol. During atorvastatin therapy and/or 3 months after its withdrawal numerous semen parameters were significantly modified, such as total number of spermatozoa (-31%, p<0.05), vitality (-9.5%, p<0.05), total motility (+7.5%, p<0.05), morphology (head, neck and midpiece abnormalities, p<0.05), and the kinetics of acrosome reaction (p<0.05). Seminal concentrations of acid phosphatases (p<0.01), α-glucosidase (p<0.05) and L-carnitine (p<0.05) were also decreased during the therapy, indicating an alteration of prostatic and epididymal functions. Moreover, we measured at least one altered semen parameter in 35% of the subjects during atorvastatin treatment, and in 65% of the subjects after withdrawal, which led us to consider that atorvastatin is unsafe in the context of our study. CONCLUSIONS: Our results show for the first time that atorvastatin significantly affects the sperm parameters and the seminal fluid composition of healthy men.

CFTR p.Arg117His associated with CBAVD and other CFTR-related disorders.

BACKGROUND: The high frequency of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene mutation p.Arg117His in patients with congenital bilateral absence of the vas deferens (CBAVD) and in newborns screened for CF has created a dilemma. METHODS: Phenotypic and genotypic data were retrospectively collected in 179 non-newborn French individuals carrying p.Arg117His and a second CFTR mutation referred for symptoms or family history, by all French molecular genetics laboratories, referring physicians, CF care centres and infertility clinics. RESULTS: 97% of the patients had the intronic T7 normal variant in cis with p.Arg117His. 89% patients were male, with CBAVD being the reason for referral in 76%. In 166/179 patients with available detailed clinical features, final diagnoses were: four late-onset marked pulmonary disease, 83 isolated CBAVD, 67 other CFTR-related phenotypes, including 44 CBAVD with pulmonary and/or pancreatic symptoms and 12 asymptomatic cases. Respiratory symptoms were observed in 30% of the patients, but the overall phenotype was mild. No correlation was observed between sweat chloride concentrations and disease severity. Five couples at risk of CF offspring were identified and four benefited from prenatal or preimplantation genetic diagnoses (PND or PGD). Eight children were born, including four who were compound heterozygous for p.Arg117His and one with a severe CF mutation. CONCLUSIONS: Patients with CBAVD carrying p.Arg117His and a severe CF mutation should benefit from a clinical evaluation and follow-up. Depending on the CBAVD patients' genotype, a CFTR analysis should be considered in their partners in order to identify CF carrier couples and offer PND or PGD.

Quality and functionality of human ovarian tissue after cryopreservation using an original slow freezing procedure.

PURPOSE: To evaluate the efficiency of an original slow freezing protocol on the quality and function of human ovarian cortex. METHODS: Human ovarian tissues were cryopreserved using a freezing medium supplemented with propanediol and raffinose as cryoprotectants and antioxidants (L-glutamine, taurine). Samples were then frozen using a faster cooling rate than the usual one. Viability and morphology of follicles, DNA fragmentation in follicles and stroma as well as histology of the vascular endothelium were analyzed before and after freezing/thawing. Moreover, a functional analysis was performed based on the evaluation of follicular growth and development in thawed ovarian tissues that were cultured in vitro. RESULTS: Our freezing/thawing protocol allows preservation of a high proportion of viable follicles and the preservation of the different follicle developmental stages (p>0.05 versus fresh control). 70.5 ± 5.2 % of follicles retained an intact morphology after cryopreservation (p=0.04). Stroma cells but not follicles exhibited a slight increase of DNA fragmentation after thawing (p<0.05). Microvessel endothelium within thawed tissues appeared to be preserved. Granulosa cells showed signs of proliferation in follicles cultured for 12 days. Secretion of 17ß-oestradiol significantly increased during in vitro culture. CONCLUSIONS: This protocol leads to good preservation of ovarian integrity and functionality post-thawing and thus appears as a suitable technique of ovarian tissue cryopreservation in clinical settings. Further research could be extended to optimize conditions of in vitro culture.

Viability assessment of fresh and frozen/thawed isolated human follicles: reliability of two methods (Trypan blue and Calcein AM/ethidium homodimer-1).

PURPOSE: To assess the reliability of trypan blue (TB) and calcein AM/ethidium homodimer-1 (CaAM/EthD-1) staining to evaluate the viability of fresh and thawed human ovarian follicles. METHODS: Isolated follicles from fresh and thawed cortex were stained using TB versus CaAM/EthD-1 methods (n = 10 patients). Measurements were performed by two independent observers. The reliability was evaluated by intraclass correlation coefficient (ICC) and the differences between paired measurements were tested by the Wilcoxon test. RESULTS: Inter-observer reliability was excellent for each method. Nevertheless, it was even better with the TB method (ICC = 0.83) compared with CaAM/EthD-1 (ICC = 0.75). Moreover, the ICCs for viability measurements using the two methods were good for each observer (observer 1: ICC = 0.49; observer 2: ICC = 0.40). CONCLUSION: Compared with CaAM/EthD-1, TB appears to be more reliable as a staining method for follicle viability evaluation. TB staining is a quick and useful method, complementary to histological analysis for quality control in ovarian tissue cryopreservation.

In vitro alachlor effects on reactive oxygen species generation, motility patterns and apoptosis markers in human spermatozoa.

Due to its extensive production and application, the toxicity of chloracetanilide herbicide alachlor[2-chloro-2',6'-diethyl-N-(methoxymethyl)-acetanilide] should be evaluated to establish minimum effects. In this study, we have examined the in vitro effects of alachlor on human sperm motion using a computer-assisted sperm analyser (CASA). An evaluation of both reactive oxygen species (ROS) and markers of apoptosis was also performed to investigate the mechanism by which alachlor modifies the sperm movement. After exposure up to 2 h to alachlor (0, 0.18, 0.37, 0.90 and 1.85 mM), the percentage of viable, motile spermatozoa and sperm velocities were concentration and/or time dependently decreased. The most sensitive parameters were progressive motility, mean average path velocity and mean straight velocity. Alachlor (1.85 mM) induced an increase in ROS production. A decrease of mitochondrial membrane potential (DeltaPsi(m)), an increase of both phosphatidylserine (PS) externalization and DNA fragmentation, which were concentration and/or time dependent, were also observed. It is possible that toxic effects of alachlor result in an oxidative stress which could act as a mediator of apoptosis. Alachlor could also contribute to some hypofertility cases.

Oxidative DNA damage in mouse sperm chromosomes: Size matters.

Normal embryo and foetal development as well as the health of the progeny are mostly dependent on gamete nuclear integrity. In the present study, in order to characterize more precisely oxidative DNA damage in mouse sperm we used two mouse models that display high levels of sperm oxidative DNA damage, a common alteration encountered both in in vivo and in vitro reproduction. Immunoprecipitation of oxidized sperm DNA coupled to deep sequencing showed that mouse chromosomes may be largely affected by oxidative alterations. We show that the vulnerability of chromosomes to oxidative attack inversely correlated with their size and was not linked to their GC richness. It was neither correlated with the chromosome content in persisting nucleosomes nor associated with methylated sequences. A strong correlation was found between oxidized sequences and sequences rich in short interspersed repeat elements (SINEs). Chromosome position in the sperm nucleus as revealed by fluorescent in situ hybridization appears to be a confounder. These data map for the first time fragile mouse sperm chromosomal regions when facing oxidative damage that may challenge the repair mechanisms of the oocyte post-fertilization.

Levels of liver X receptors in testicular biopsies of patients with azoospermia.

OBJECTIVE: To determine whether the transcription factors liver X receptors (LXRs) and their downstream genes, which are involved in the regulation of several testicular functions in mouse models, are differentially expressed in testes of men with nonobstructive azoospermia (NOA) or obstructive azoospermia (OA). DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Patients with various types of NOA (n=22) and with OA (n=5). INTERVENTION(S): Human testicular biopsies. MAIN OUTCOME MEASURE(S): Transcript levels were measured in testicular biopsies with the use of quantitative polymerase chain reaction. Correlations of LXR mRNA levels with the number of germ cells, the expression of proliferation and apoptosis markers, and the amount of intratesticular lipids and testosterone were evaluated. The localization of LXRα was analyzed by immunofluorescence. RESULT(S): LXR mRNA levels were decreased by 49%-98% in NOA specimens and positively correlated with germ cell number. Accumulations of IDOL and SREBP1c (LXR targets involved in lipid homeostasis) were 1.8-2.1 times lower in NOA samples and mRNA levels of the SREBP1c target gene ELOVL6 were increased 1.9-2.4-fold. Interestingly, the amount of triglycerides and free fatty acids were higher in NOA testes (3.4-12.2-fold). LXRα was present in Leydig cells. Accumulations of LXR downstream genes encoding the steroidogenic proteins StAR and 3ßHSD2 were higher in NOA testes (5.9-12.8-fold). CONCLUSION(S): Knowledge of changes in the transcript levels of LXRs and some of their downstream genes during altered spermatogenesis may help us to better understand the physiopathology of testicular failure in azoospermic patients.

DNA oxidative damage in mammalian spermatozoa: where and why is the male nucleus affected?

Gamete DNA integrity is one key parameter conditioning reproductive success as well as the quality of life for the offspring. In particular, damage to the male nucleus can have profound negative effects on the outcome of fertilization. Because of the absence of repair activity of the quiescent mature spermatozoa it is easily subjected to nuclear damage, of which oxidative damage is by far the most prominent. In relation to the organization of the mammalian sperm nucleus we show here that one can correlate the nuclear regions of lower compaction with areas preferentially showing oxidative damage. More precisely, we show that oxidative DNA damage targets primarily histone-rich and nuclear matrix-attached domains located in the peripheral and basal regions of the mouse sperm nucleus. These particular sperm DNA domains were recently shown to be enriched in genes of paramount importance in postfertilization DNA replication events and in the onset of the embryonic developmental program. We propose that monitoring of sperm DNA oxidation using the type of assay presented here should be considered in clinical practice when one wants to estimate the integrity of the paternal nucleus along with more classical assays that essentially analyze DNA fragmentation and nucleus compaction.

Indoleamine 2,3-dioxygenase 1 (ido1) is involved in the control of mouse caput epididymis immune environment.

The epididymis maintains a state of immune tolerance towards spermatozoa while also protecting them and itself against infection and acute inflammation. The immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (Ido1) participates in this delicate local equilibrium. Using the mouse Ido1(-/-) model, we show here that the absence of IDO1 expression leads in the epididymis but not in serum to (1) an increase in the inflammatory state as evidenced by changes in the content of cytokines and chemokines, (2) the engagement of a Th1-driven inflammatory response as evidenced by changes in the Th17/Treg as well as Th1/Th2 equilibria, as well as (3) differences in the content of lipid intermediates classically involved in inflammation. Despite this more pronounced inflammatory state, Ido1(-/-) animals succeed in preserving the local epididymal immune situation due to the activation of compensatory mechanisms that are discussed.

Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice.

We report here that spermatozoa of mice lacking both the sperm nucleus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2)O(2)-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.

Dietary cholesterol-induced post-testicular infertility.

This work shows that an overload of dietary cholesterol causes complete infertility in dyslipidemic male mice (the Liver X Receptor-deficient mouse model). Infertility resulted from post-testicular defects affecting the fertilizing potential of spermatozoa. Spermatozoa of cholesterol-fed lxr-/- animals were found to be dramatically less viable and motile, and highly susceptible to undergo a premature acrosome reaction. We also provide evidence, that this lipid-induced infertility is associated with the accelerated appearance of a highly regionalized epididymal phenotype in segments 1 and 2 of the caput epididymidis that was otherwise only observed in aged LXR-deficient males. The epididymal epithelial phenotype is characterized by peritubular accumulation of cholesteryl ester lipid droplets in smooth muscle cells lining the epididymal duct, leading to their transdifferentiation into foam cells that eventually migrate through the duct wall, a situation that resembles the inflammatory atherosclerotic process. These findings establish the high level of susceptibility of epididymal sperm maturation to dietary cholesterol overload and could partly explain reproductive failures encountered by young dyslipidemic men as well as ageing males wishing to reproduce.

Clinical data and parenthood of 63 infertile and Y-microdeleted men.

OBJECTIVE: To collect follow-up data for infertile men with Y microdeletion. DESIGN: Retrospective, observational survey. SETTING: Multicenter IVF units associated with genetics laboratories. PATIENT(S): Sixty-three patients with Y microdeletion. INTERVENTION(S): Karyotype analysis, Y microdeletion screening, and assisted reproductive technology. MAIN OUTCOME MEASURES: Medical history, karyotype, nature of the AZF deletion, semen parameters, testis biopsy results, choice of assisted reproductive technology, and results of intracytoplasmic sperm injection (ICSI). RESULTS: Abnormal karyotypes were found in 8 men (12.7%), who were azoospermic except 1. Of these 8 men, 5 presented a combined AZFb+c deletion, and 3 had a deletion in AZFc only. Most men (39 of 63) were azoospermic, 3 were cryptoazoospermic, and 19 had extreme oligozoospermia (sperm concentration

Activated caspases in thawed epididymal and testicular spermatozoa of patients with congenital bilateral absence of the vas deferens and intracytoplasmic sperm injection outcome.

OBJECTIVE: To analyze the expression of activated caspases and membrane permeability in thawed epididymal and testicular spermatozoa of patients with congenital bilateral absence of the vas deferens (CBAVD). DESIGN: Retrospective study. SETTING: Biology and medicine of reproduction in University hospital. PATIENT(S): Eight CBAVD patients. INTERVENTION(S): Staining of activated caspases and viability (propidium iodide, PI); intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Proportion of viable (Casp-/PI-) or dead (Casp-/PI+) spermatozoa without activated caspases, viable (Casp+/PI-) or dead (Casp+/PI+) spermatozoa with activated caspases. ICSI results. RESULT(S): Higher percentage of dead (Casp+/PI+; 84.0% vs. 57.5%) and viable (Casp+/PI-; 12.0% vs. 0) spermatozoa with activated caspases were observed in testicular than in epididymal samples. No significant difference was observed between the percentage of total testicular and epididymal spermatozoa permeant for PI. The outcome of ICSI fertilization (67.5% vs. 57.4%), good morphology embryo at day 2 (75.9% vs. 61.3%), clinical pregnancy (26.7% vs. 15.4%), and implantation (15.6% vs 9.5%) rates were better when ICSI were performed with epididymal sperm samples. CONCLUSION(S): These results support the hypothesis of an abortive apoptotic process and demonstrate that combined staining of the activated caspases and membrane permeability provide complementary measurements for the evaluation of viable and functional spermatozoa to better understand ICSI outcomes with epididymal and testicular spermatozoa.

Outcome of intracytoplasmic sperm injection for a couple in which the man is carrier of CFTR p.[R74W;V201M;D1270N] and p.P841R mutations and his spouse a heterozygous carrier of p.F508del mutation of the cystic fibrosis transmembrane conductance regulator gene.

OBJECTIVE: To document the phenotype associated with the p.[R74W;V201M;D1270N] and p.P841R mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene. DESIGN: Case report. SETTING: Biology and medicine of reproduction in a university hospital. PATIENT(S): A couple in which the man is carrier of the triple mutant p.[R74W;V201M;D1270N] allele in trans to p.P841R mutation and his spouse a heterozygous carrier for the severe p.F508del mutation of the CFTR gene, who became pregnant after intracytoplasmic sperm injection (ICSI) with twins. INTERVENTION(S): Genetic counseling; CFTR gene sequencing; ICSI; children's follow-up. MAIN OUTCOME MEASURE(S): First report of a male phenotype associated with the p.P841R mutation. RESULT(S): The triple mutant p.[R74W;V201M;D1270N] allele associated with the unknown p.P841R mutations were detected in this man with congenital bilateral absence of the vas deferens, which may presume p.P841R as a severe mutation. After genetic counseling, the couple preferred prenatal diagnosis after ICSI than preimplantation genetic diagnosis, which revealed that the boys were both carriers of p.[R74W;V201M;D1270N] and p.F508del mutations. They are now 4 years old and show normal growth without nutritional deficiency. CONCLUSION(S): This case report documents for the first time a male phenotype associated with the p.P841R mutation and underlines the difficulties in counseling a man with congenital bilateral absence of the vas deferens carrying uncommon mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene before ICSI.

Annexin V binding to plasma membrane predicts the quality of human cryopreserved spermatozoa.

Cryopreservation-induced stress may result in membrane injury with consequent decrease of sperm motility and fertilizing capacity. This study has investigated the relationship between human spermatozoa tolerance to cryopreservation and the loss of plasma membrane asymmetry, especially translocation of phosphatidylserine (PS) from the inner to the outer leaflet. The prospective study was performed on semen samples from 31 men. Conventional characteristics of 20 semen were analysed, before and after cryopreservation as well as externalization of PS assessed by annexin V-staining in combination with the propidium iodide which stains dead cells. The fertilizing capacity was evaluated by a zona free hamster egg penetration test in 11 freeze/thaw spermatozoa samples. The percentages of vital annexin V-negative and annexin V-positive spermatozoa in post-thaw samples were significantly lower than in pre-freeze ones (10 +/- 3 vs. 25 +/- 5% and 22 +/- 3 vs. 34 +/- 4% respectively), while the percentages of dead spermatozoa annexin V-negative and annexin V-positive had increased (42 +/- 4 vs. 23 +/- 4% and 23 +/- 4 vs. 16 +/- 2% respectively). The percentages of progressive and total motile spermatozoa were significantly correlated with the percentage of vital annexin V-negative spermatozoa before as well as after cryopreservation. Furthermore, recovery of motile spermatozoa after freeze/thaw was strongly correlated (p < 0.002) with the proportion of vital annexin V-negative spermatozoa in fresh semen. The percentage of penetration of zona-free hamster eggs was correlated (p < 0.02) with the percentage of live annexin V-negative cryopreserved spermatozoa capacitated for 3 h. These findings provide evidence that annexinV-binding staining in combination with PI brings additional information to predict the outcome of cryopreserved ejaculated sperm and may be used as a novel and reliable marker to study the freeze/thaw process.