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Background: Formalin is commonly applied as an antiseptic and tissue fixative. It has reactive molecules that lead to its cytotoxic effects. According to recent studies, formalin causes a change in the testicular and sperm structure and L-carnitine (LC) acts as an antioxidant to counteract its effects. Objectives: This study aimed to investigate the protective effects of LC on the parameters, chromatin condensation and apoptosis of mice sperm exposed to formalin. Materials and Methods: In this experimental study, 24 balb/c mice (25-40 gr ,10-12 wk) were divided into three groups (n = 8/each): group I without any injections or gavage; group II, received 10 mg/ kg formalin intraperitoneally (I.P); and group III was exposed to formalin and LC, where a dose of 10 mg/kg formalin was injected I.P daily and LC the dose of 100 mg/kg was kept in a solvent solution. After 31 days, the sperm examination was performed as follows: to evaluate chromatin and DNA quality of the sperm, we applied aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3), and terminal transferase-mediated deoxy uridine triphosphate biotin end labeling (TUNEL) tests. Results: Sperm parameters such as count, motility, morphology, and viability displayed a significant decrease in the formalin group. While the data exhibited a considerable augment in sperm parameters in the formalin + LC than the formalin and control groups (p < 0.001), significant differences were detected between groups with respect to TB staining, TUNEL test, CMA3 test and AB staining in the formalin and formalin + LC groups. Conclusion: LC can reduce the negative effects of formalin on sperm parameters, chromatin stability, and percentage of apoptosis in an animal model. Key words: Formalin, L-carnitine, Mice, Sperm chromatin, Apoptosis.

Assisted reproductive technologies involving the use of spermatozoa and eggs for in vitro fertilization (IVF) have come as the solution for many infertile couples to become parents. However, in some cases, the use of ejaculated spermatozoa delivers poor IVF performance. Some studies have suggested the use of testicular spermatozoa in severe male infertility cases, but no guidelines regarding their utilization are currently available. In the present study, we found the mRNA protamine 1/protamine 2 (P1/P2) ratio to be a valuable biomarker of poor sperm function that could be used as a diagnostic key for the identification of cases that would benefit from the use of testicular spermatozoa. A total of 23 couples undergoing egg donation cycles with at least one previous cycle failure were studied. All couples underwent two consecutive intracytoplasmic sperm injection (ICSI) cycles with either ejaculated or testicular spermatozoa (TESA). The sperm mRNA P1/P2 ratio, fertilization rate, blastocyst rate, and pregnancy and live birth rate were compared. Results showed improved ICSI and clinical outcomes in cycles with testicular spermatozoa in men with altered mRNA P1/P2 ratios. TESA cycles presented significantly higher rates of fertilization (mean ± standard deviation: 76.1% ± 15.1% vs 65.5% ± 18.8%), blastocyst formation (55.0% ± 20.3% vs 30.8% ± 23.8%), and good morphological quality blastocyst (28.9% ± 22.9% vs 13.5% ± 17.9%) and also improvements on pregnancy (60.9% vs 0%) and healthy birth rates (56.5% vs 0%) than EJACULATE cycles. The results described here suggest that in patients with previous IVF/ICSI failures and aberrant mRNA protamine ratios, the use of testicular spermatozoa may be a good alternative to improve clinical outcomes.

Background Spermatogenesis is the process by which germ cells develop into spermatozoa in the testis. Sperm protamines are small, arginine-rich nuclear proteins which replace somatic histones during spermatogenesis, allowing a hypercondensed DNA state that leads to a smaller nucleus and facilitating sperm head formation. In eutherian mammals, the protamine-DNA complex is achieved through a combination of intra- and intermolecular cysteine cross-linking and possibly histidine-cysteine zinc ion binding. Most metatherian sperm protamines lack cysteine but perform the same function. This lack of dicysteine cross-linking has made the mechanism behind metatherian protamines folding unclear. Results Protamine sequences from UniProt’s databases were pulled down and sorted into homologous groups. Multiple sequence alignments were then generated and a gap weighted relative entropy score calculated for each position. For the eutherian alignments, the cysteine containing positions were the most highly conserved. For the metatherian alignment, the tyrosine containing positions were the most highly conserved and corresponded to the cysteine positions in the eutherian alignment. Conclusions High conservation indicates likely functionally/structurally important residues at these positions in the metatherian protamines and the correspondence with cysteine positions within the eutherian alignment implies a similarity in function. One possible explanation is that the metatherian protamine structure relies upon dityrosine cross-linking between these highly conserved tyrosines. Also, the human protamine P1 sequence has a tyrosine substitution in a position expecting eutherian dicysteine cross-linking. Similarly, some members of the metatherian Planigales genus contain cysteine substitutions in positions expecting plausible metatherian dityrosine cross-linking. Rare cysteine-tyrosine cross-linking could explain both observations.

Background: Prescribing antidepressant drugs is becoming common. These drugs are known to affect sexual functions. Objective: The study is aimed to assess the effects of amitriptyline and venlafaxine on sperm parameters and evaluate Malondialdehyde (MDA) and 1, 1-Diphenyl-2-picryl-hydrazyl values in BALB/ mice spermatozoa. Materials and Methods: Forty adult male BALB/c mice were separated into five groups. Group Ι (control) received distilled water; group ΙΙ amitriptyline (4 mg/kg); group ΙΙΙ amitriptyline (4 mg/kg) +vitamin C (10 mg/kg); group ΙV venlafaxine (2 mg/kg); and group V received vitamin C (10 mg/kg) + venlafaxine (2 mg/kg). All drugs were administered by oral gavage for 35 days. After excision of caudal epididymis, it was located in 1 mL Ham's F10 medium at 37oC for 15 min and then analysis of sperm parameters was performed. To examine lipid peroxidation and total antioxidant capacity, the MDA and 1, 1-Diphenyl-2-picryl-hydrazyl were measured, respectively. Results: The mean sperm parameters in the group treated with amitriptyline were significantly lower than in the other groups. MDA tests showed a significant difference between amitriptyline and control groups (p=0.007). Conclusion: The results of this study showed that amitriptyline consumption can weaken sperm parameters, which can be attributed to the increased production of ROS and toxicity resulting from amitriptyline consumption. Moreover, venlafaxine improved sperm parameters in mice and the lipid peroxidation in this group did not change compared to the control group.

F2-isoprostanes (F2-IsoPs), stereoisomers of prostaglandin F2α generated by the free radical-induced oxidation of arachidonic acid, have been associated with different male infertility conditions. This study aimed to evaluate the role of seminal isoprostane levels and sperm characteristics in the reproductive outcome and embryo quality of 49 infertile couples. Semen analysis was performed following WHO guidelines. Sperm chromatin maturity was detected using an aniline blue (AB) assay, and DNA integrity was assessed using the acridine orange (AO) test. Seminal F2-IsoP levels were quantified by gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI–MS/MS) analysis. Correlations among variables and their impact on in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) outcome were investigated. F2-IsoP levels are positively correlated with double-stranded DNA sperm (p &lt, 0.001) and negatively correlated with mature sperm chromatin (p &lt, 0.001). Patients with positive outcomes had an increased percentage of sperm with double-stranded DNA, as did patients producing high-quality embryo, who showed higher F2-IsoP levels compared to those detected in the low-quality embryo group. An intriguing relationship between a mild increase in F2-IsoP levels, DNA integrity, and embryo quality seems to indicate that the non-enzymatic oxidation of arachidonic acid can be also a marker of metabolic activity in human semen.

Background: Teratoasthenozoospermia (TA) is a severe form of male infertility with no clear etiology. Objective: To compare the level of intracellular anion superoxide (O2–), heat shock protein A2 (HSPA2) and protamine deficiencies in ejaculated spermatozoa between teratoasthenozoospermic and normozoospermic men. Materials and Methods: In this case- control study, semen samples of 20 infertile men, with TA (with normal morphology lower than 4%_ and total motility lower than 40% ) as the case group and 20 normozoospermic fertile men as the control group were evaluated for intracellular O2 – and HSPA2 by flow cytometry and protamine deficiency by Chromomycin A3 (CMA3) test. Results: The rate of CMA3+ spermatozoa in the case group was higher than controls (p=0.001). The percentages of HSPA2+ spermatozoa in the cases were significantly lower than controls (p=0.001). Also, intracellular O2 – levels in the case group were significantly higher than controls (p=0.001) and had positive correlations with sperm apoptosis (r=0.79, p=0.01) and CMA3 positive sperm (r=0.76, p=0.01), but negative correlations with normal morphology (r=-0.81, p=0.01) and motility (r=-0.81, p=0.01). There was no significant correlation between intracellular O2 – and HSPA2 in the case group (r=0.041, p=0.79). Conclusion: We suggest that the increase in intracellular O2 –, decrease in spermatozoa HSPA2+, and high percentages of spermatozoa with immature chromatin might be considered as etiologies of infertility in TA patients

Background: Methamphetamine (MA) was shown to have harmful effects on male reproductive system. Objective: To investigate probable effects of daily administration of MA on sperm parameters and chromatin/DNA integrity in mouse. Material and Methods: Thirty-five NMRI male mice were divided into five groups including low, medium, and high dosage groups which were injected intraperitoneally with 4, 8 and 15 mg/kg/day for 35 days, respectively. Normal saline was injected in sham group and no medications were used in control group. Then, the mice were killed and caudal epididymis of each animal was cut and placed in Ham’s F10 medium for sperm retrieval. To evaluate sperm chromatin abnormalities, the aniline blue, toluidine blue and chromomycine A3 were used. For sperm DNA integrity and apoptosis, the acridine orange, sperm chromatin dispersion, and TUNEL assay were applied. For sperm morphology, Papanicolaou staining was done Results: Normal morphology and progressive motility of spermatozoa decreased in medium and high dosage groups in comparison with the control group (p=0.035). There was a significant increase in rate of aniline blue, toluidine blue, and chromomycine A3 positive spermatozoa in high dosage group. In a similar manner, there was an increase in rates of acridine orange, TUNEL and sperm chromatin dispersion positive sperm cells in high dosage group with respect to others. Conclusion: MA abuse in a dose-dependent manner could have detrimental effects on male reproductive indices including sperm parameters and sperm chromatin/DNA integrity in mice.

Objective: To evaluate the effects of low level ultraviolet (UV) radiation on sperm chromatin structure.Methods: The target study was divided into three groups: (i) males with proven fertility (n=40 ) was taken as Group I (control); (ii) Oligoasthenozoospermic (OAT) cases as Group II (n=36); (iii) males with unexplained infertility (MUI) cases (n=42) as Group III. Specific techniques were used to study the impact of UV radiation (Pre and Post UV exposure) on the sperm nuclear DNA viz. Aniline blue staining was for detection of immature chromatin. Chromomycin A3 fluorescence staining was used to determine protamine-DNA dissociation by intense fluorescence of protamine deficient sperm cells and neutral comet assay was for evaluation of DNA fragmentation. Statistical analysis was carried out using Student's t-test (GraphPad Prism Version-6). Level of significance was considered at P

Salinity represents a critical environmental and an ecological factor in the reproduction of marine species. As global climate changes and anthropogenic factors affect salinity, in this study, we have analyzed the responses of Mytilus galloprovincialis spermatozoa to hyposaline stress. We exposed mussels, in laboratory tanks, for 24 hr at 18°C to control (35.9 psu) and three hyposaline (17.1, 22.6, and 26.2 psu) conditions, and evaluated the expression of sperm hsp70 and protamine-like proteins genes. Further we analyzed the electrophoretic pattern, the DNA binding and the release from sperm nuclei of protamine-like proteins. For all experimental approaches used, the results obtained at 17.1 psu condition were very similar to those obtained in the control condition, while alterations were always recorded at 22.6 and 26.2 psu conditions. Particularly, at 22.6 and 26.2 psu, was observed: 42.5- and 17.1-fold increase in hsp70 expression, respectively, and hypoexpression of PL-II/PLIV protamine-like proteins genes. Further, electrophoretic mobility shift assays and salt-induced release of nuclear proteins from sperm nuclei, revealed alterations in the PL proteins/DNA binding, in these two hyposaline conditions. The similarity between the results obtained in control and in the more severe hyposaline condition (17.1 psu) could indicate a phenomenon of fertility preservation strategy due to gamete plasticity.

This study aims to assess the effects induced by 24hr exposure to a subtoxic copper concentration on the reproductive system (gonads, spermatozoa, and protamine-like [PL] proteins) of Mytilus galloprovincialis. Inductively coupled plasma-mass spectrometry indicated accumulation of this metal in gonads, spermatozoa, and PL proteins of exposed mussels. Further, real-time polymerase chain reaction analyses showed altered expression levels of mt10 and PL proteins genes in spermatozoa and gonads, respectively, of exposed mussels. Protamine-like proteins, which represent the main basic component of sperm chromatin of this organism, showed a higher DNA binding affinity and a different DNA binding mode in exposed mussels. Moreover, an increased amount of NaCl was required for the release from sperm nuclei of PL-III, the main PL protein component. Finally, PL proteins extracted from exposed mussels promoted DNA oxidative damage in the presence of H O-2 (2.) These results demonstrate that the tolerable copper amount could also affect the properties of PL proteins and determine the negative effects on the reproductive system of this organism. These analyses could be useful to develop quick and efficient chromatin-based genotoxicity tests for pollution biomonitoring programs.

International audience; Fertility is a growing healthcare issue for a rising number of cancer survivors. In men, cancer itself and its treatment can negatively affect spermatogenesis by targeting the dividing spermatogonia and their cellular environment, ultimately leading to a reduction of testicular germ cells and sperm count. Experimental data and prospective longitudinal studies have shown that sperm production can recover after cancer treatment. But despite this, yet unpredictable, recovery in sperm production, cancer survivors are more at risk to produce sperm with aneuploidy, DNA damage, abnormal chromatin structure, and epigenetic defects even 2 years post-treatment. Sperm DNA alteration is of clinical concern, as these patients may father children or seek assisted reproduction technologies (ART) using gametes with damaged genome that could result in adverse progeny outcomes. Interestingly, large cohort studies revealed lower birth rate but no significant impact on the health of the children born from male cancer survivors (naturally or using ART). Nevertheless, a better understanding of how cocktail of chemotherapy and new anticancer agents affect spermatogenesis and sperm quality is needed to reduce side effects. Moreover, developing new fertility preservation strategies is essential as sperm cryopreservation before treatment is currently the only option but does not apply for prepubertal/young postpubertal patients.

Protamine-like proteins (PL-II, PL-III and PL-IV) represent the major basic nuclear component of Mytilus galloprovincialis L sperm chromatin. The present study investigates the effects induced on the properties of PL-II protein after exposure of Mytilus galloprovincialis L for 24 h to 1.5 and 5 µM CdCl2. We found cadmium accumulation in protamine-like proteins with a linear grow up with the exposition dose. In particular, after 5 µM CdCl2 mussels exposure, the mobility of PL-II band changed in SDS-PAGE, suggesting structural rearrangement in presence of cadmium. Structural analysis using fluorescent probes, indicated that at 5 µM CdCl2 the complete conformational change of PL-II protein was reached. In the same condition of mussels exposure of 5 µM CdCl2, PL-II protein changed its DNA binding mode, which determined a closer DNA binding, because higher amount of NaCl were required for PL-II protein release by sperm nuclei. These results supported the hypothesis that mussel exposure to this CdCl2 dose, although lower to toxic ones, affects the properties of this protein and as a consequence chromatin organization of spermatozoa that is essential for the success of fertilization.

The sperm nucleus is prone to sustain DNA damage before and after ejaculation. Distribution of the damage is not homogeneous, and the factors determining differential sensitivity among nuclear regions have not yet been characterized. Human sperm chromatin contains three structural domains, two of which are considered the most susceptible to DNA damage: the histone bound domain, harboring developmental related genes, and the domain associated with nuclear matrix proteins. Using a quantitative polymerase chain reaction (qPCR) approach, we analyzed the number of lesions in genes homeobox A3 (HOXA3), homeobox B5 (HOXB5), sex-determining region Y (SRY)-box 2 (SOX2), β-GLOBIN, rDNA 18S, and rDNA 28S in human sperm after ultraviolet irradiation (400 μW cm−2, 10 min), H2O2 treatment (250 mmol l−1, 20 min), and cryopreservation, which showed differential susceptibility to genetic damage. Differential vulnerability is dependent on the genotoxic agent and independent of the sperm nuclear proteins to which the chromatin is bound and of accessibility to the transcription machinery. Immunodetection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that the highest level of oxidation was observed after H2O2 treatment. The distribution of oxidative lesions also differed depending on the genotoxic agent. 8-OHdG did not colocalize either with histone 3 (H3) or with type IIα + β topoisomerase (TOPO IIα + β) after H2O2 treatment but matched perfectly with peroxiredoxin 6 (PRDX6), which is involved in H2O2 metabolism. Our study reveals that the characteristics of the sperm head domains are responsible for access of the genotoxicants and cause differential degree of damage to nuclear areas, whereas chromatin packaging has a very limited relevance. The histone-enriched genes analyzed cannot be used as biomarkers of oxidative DNA damage.

International audience; BACKGROUND: Cryopreserved human sperm are used in assisted reproductive technology. However, the effect of cryopreservation on sperm DNA integrity is unclear.OBJECTIVES: The objectives of this study were to: (i) determine the impact of semen cryopreservation on human sperm DNA integrity and chromatin structure; (ii) test if parameters obtained from TUNEL and SCSA® correlate; and (iii) verify correlation between sperm motility, morphology and viability with TUNEL and SCSA® parameters.MATERIALS AND METHODS: Men attending a fertility clinic were recruited and grouped according to their sperm parameters (n = 9/group): normozoospermia, oligoasthenoteratozoospermia and teratozoospermia. Each semen sample was processed as follow: (i) directly frozen at -80 °C; (ii) diluted in Sperm Maintenance Medium, cooled for 30 min at 4 °C and frozen at -80 °C; (iii) diluted in Sperm Maintenance Medium; or (iv) in SpermFreeze. Each mixture from method (iii) and (iv) was then suspended for 30 min in liquid nitrogen vapor and plunged into liquid nitrogen. After at least two months of storage, samples were thawed at room temperature and analyzed for motility and viability, TUNEL and SCSA® assays.RESULTS: Progressive motility and viability decreased after freeze-thawing. TUNEL scores increased significantly in all samples after freezing-thawing while no significant change in the DNA fragmentation index (DFI) from SCSA® was observed. No change in the percentage high DNA stainability (HDS) was observed in normozoospermic samples; however it was significantly increased in all the methods in oligoasthenoteratozoospermic and in the methods (ii)-(iv) in teratozoospermic samples. The DFI and TUNEL scores correlated significantly with each other and inversely with sperm motility, viability and morphology.DISCUSSION AND CONCLUSION: Cryopreservation seems to be deleterious for the integrity of human sperm DNA and compaction. However, the sperm DFI was not affected during cryopreservation under the various methods of storage tested. Clinicians and investigators should take this information into consideration when using cryopreserved sperm for assisted reproduction.

Due to socioeconomic factors, more couples are choosing to delay conception than ever. Increasing average maternal and paternal age in developed countries over the past 40 years has raised the question of how aging affects reproductive success of males and females. Since oxidative stress in the male reproductive tract increases with age, we investigated the impact of advanced paternal age on the integrity of sperm nucleus and reproductive success of males by using a Prdx6−/− mouse model. We compared sperm motility, cytoplasmic droplet retention sperm chromatin quality and reproductive outcomes of young (2-month-old), adult (8-month-old), and old (20-month-old) Prdx6−/− males with their age-matched wild type (WT) controls. Absence of PRDX6 caused age-dependent impairment of sperm motility and sperm maturation and increased sperm DNA fragmentation and oxidation as well as decreased sperm DNA compaction and protamination. Litter size, total number of litters and total number of pups per male were significantly lower in Prdx6−/− males compared to WT controls. These abnormal reproductive outcomes were severely affected by age in Prdx6−/− males. In conclusion, the advanced paternal age affects sperm chromatin integrity and fertility more severely in the absence of PRDX6, suggesting a protective role of PRDX6 in age-associated decline in the sperm quality and fertility in mice., Graphical abstract, Highlights • Absence of PRDX6 promotes increased levels of 8-OHdG in mouse spermatozoa. • Sperm DNA damage and impairment of sperm maturation and motility is exacerbated by the absence of PRDX6 in aging males. • Prdx6−/− fathers are subfertile producing smaller litters and pups with low birth weight compared to wild-type fathers. • PRDX6 is important to protect paternal DNA and sperm functions during aging.

Testicular varicocele, a dilation of the veins of the pampiniform plexus thought to increase testicular temperature via venous congestion, is commonly associated with male infertility. Significant study has clarified the negative impact of varicocele on semen parameters and more recent work has shed light on its detrimental effects on the molecular and ultrastructural features of sperm and the testicular microenvironment, as well as more clearly defined the positive impacts of treatment on couples’ fertility. The relationship between varicocele and testicular endocrine function, while known for some time based on histologic evaluation, has become more apparent in the clinical setting with a growing link between varicocele and hypogonadism. Finally, in the pediatric setting, while future study will clarify the impact of varicocele on fertility and testicular function, recent work supports a parallel effect of varicocele in adolescents and adults, suggesting a re-evaluation of current treatment approaches in light of the progressive nature of the condition and potential increased risk of future disease.

Ezrin, radixin, moesin and merlin (ERM) proteins are highly homologous actin-binding proteins that share extensive sequence similarity with each other. These proteins tether integral membrane proteins and their cytoplasmic peripheral proteins (e.g., adaptors, nonreceptor protein kinases and phosphatases) to the microfilaments of actin-based cytoskeleton. Thus, these proteins are crucial to confer integrity of the apical membrane domain and its associated junctional complex, namely the tight junction and the adherens junction. Since ectoplasmic specialization (ES) is an F-actin-rich testis-specific anchoring junction-a highly dynamic ultrastructure in the seminiferous epithelium due to continuous transport of germ cells, in particular spermatids, across the epithelium during the epithelial cycle-it is conceivable that ERM proteins are playing an active role in these events. Although these proteins were first reported almost 25 years and have since been extensively studied in multiple epithelia/endothelia, few reports are found in the literature to examine their role in the actin filament bundles at the ES. Studies have shown that ezrin is also a constituent protein of the actin-based tunneling nanotubes (TNT) also known as intercellular bridges, which are transient cytoplasmic tubular ultrastructures that transport signals, molecules and even organelles between adjacent and distant cells in an epithelium to coordinate cell events that occur across an epithelium. Herein, we critically evaluate recent data on ERM in light of recent findings in the field in particular ezrin regarding its role in actin dynamics at the ES in the testis, illustrating additional studies are warranted to examine its physiological significance in spermatogenesis.

In mammals, sperm guidance in the oviduct appears essential for successful sperm arrival at the oocyte. Hitherto, three different potential sperm guidance mechanisms have been recognized: thermotaxis, rheotaxis, and chemotaxis, each of them using specific stimuli - a temperature gradient, fluid flow, and a chemoattractant gradient, respectively. Here, we review sperm behavioral in these mechanisms and indicate commonalities and differences between them.

In this study annual fluctuations of DNA fragmentation and quality of cold-stored equine sperm were evaluated. Ejaculates were collected weekly during one year from 15 stallions. Ejaculate volume, sperm concentration and total sperm count were determined and semen was then extended and cold-stored for 48 h. Sperm motility was evaluated by CASA before and after 24 as well as 48 h of cold storage. In addition, the percentages of sperm with intact plasma membrane and acrosome (PMAI %) and with low intracellular Ca2+ level were determined in cold-stored semen (24 h, 48 h). SCSA™ was performed to assess mean DFI, SD of DFI and % DFI in raw frozen-thawed as well as in extended sperm after 24 and 48 h of storage. The month of semen collection affected (P < 0.05) all parameters evaluated in raw semen and all criteria except progressive motility as well as rapid cells in semen stored for 24 and 48 h, respectively. Ejaculate volume was higher and sperm concentration lower in summer compared to winter and motility lower in July than in any other month of the year (P < 0.05). In semen processed in April and stored for 24 h the percentage of rapid cells was improved compared to January and after 48 h of storage progressive motility (%) was higher in January and October than in July (P < 0.05). After 24 h of cold storage PMAI % was higher in October than in January and after 48 h values were higher in September compared to January and February as well as from April to July (P < 0.05). Regarding sperm with low intracellular Ca+2 level (%) after storage for 24 and 48 h, higher values were measured in winter and in October compared to April, June and July (P < 0.01). Seasonal changes in DNA fragmentation were most evident with respect to mean DFI. In raw frozen-thawed semen mean DFI was lower from August to November than in June and July (P < 0.001). Values were lower during winter compared to spring and early summer (P < 0.05) and lower in December than from April to September (P < 0.001). After 24 h of cold storage mean DFI was lower in September and October when compared to January, February, May, July and November (P < 0.05) and after 48 h storage mean DFI was reduced in spring and autumn compared to February, June and July (P < 0.05). In conclusion, a seasonal effect was evident on semen characteristics of raw and cold-stored sperm. Semen quality was impaired in midsummer when low sperm motility and viability were combined with an elevated DNA fragmentation and Ca2+ level of sperm.

The study of the sperm proteins is crucial for understanding its normal function and alterations in infertile patients. The sperm is a highly specialized cell with a very large flagella, with little cytoplasm and a highly condensed nucleus. The most abundant proteins in the nucleus of mammalian sperm are the protamines. The main functions of the protamines are the condensation of the DNA, possibly contributing to the generation of a more hydrodynamic sperm head and to the protection of the genetic message. However, in addition to protamines, about 5.0-15.0% of the paternal genome is also complexed with histones and histone variants. It has also demonstrated a differential distribution of genes in regions associated with histone and protamine-associated regions, suggesting a potential epigenetic relevance in embryonic development. More recently, detailed lists of proteins have been described corresponding to the different compartments of the sperm cell thanks to the application of recent proteomic techniques based on mass spectrometry (MS). Differential proteomics is also being applied to identify the presence of protein abnormalities found in infertile patients

A strategy to circumvent the poor polyphenols bioavailability is to load these compounds into liposomes. We evaluated the in vitro effects of quercetin (Q) and Q-loaded liposomes (QLL, 30, 50, 100 ?M) on motility, viability and chromatin integrity of swim-up selected human sperm. Antioxidant power was assayed against tert-butylhydroperoxide induced lipid peroxidation (LPO) using C11-BODIPY581/591 fluorescent probe and transmission electron microscopy. QLL showed decreased toxicity for sperm motility and viability and increased DNA damage compared to Q. The percentage of sperm with fluorescence, marker of LPO, was decreased in samples incubated with Q vs QLL (P < 0.001). The ultrastructure of acrosomes and membranes was preserved with Q 30/100 ?M, whereas QLL did not prevent membrane injury.Q alone appeared more effective than Q incorporated into liposomes; however liposomes could be considered as carriers that may convey different compounds inside sperm; they may therefore represent a field of research rich of many applications.

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 °C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 μm Fe2+/1 mm ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA®) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 ± 2.9%) and OXI (11.6 ± 7.6%) (p, This study has been supported by the Spanish Ministry of Education and Science (AGL2004-05904GAN) and by the Council for Education and Science of Junta de Castilla-La Mancha (PAC-06-047). Felipe Martinez-Pastor was supported by the ‘Juan de la Cierva’ program (Spanish Ministry of Education and Science).

Background Sperm DNA integrity is essential for the accurate transmission of genetic information. The clinical significance of this assessment lies in its association with not only natural conception rates, but also the success of assisted reproduction technology (ART). It has been reported that sperm chromatin structure assay (SCSA) identified thresholds for negative pregnancy outcome after ART when the DNA fragmentation index (DFI), previously known as COMPalphat, was >30%. Methods In a prospective clinical study, we examined 34 male infertile patients, the husbands of women undergoing conventional IVF or ICSI. SCSA and ART were carried out on semen aliquots taken from the same ejaculate. Fertilization rate, embryo quality and pregnancy rates were correlated to SCSA parameters, DFI and highly DNA stainable (HDS) cells. Results No differences were seen in SCSA parameter values between patients initiating pregnancies and not doing so in either ICSI or conventional IVF. Pregnancies and normal delivery were obtained even with high levels of DFI. Conclusions There is still controversy over whether analytical techniques currently in use are able to identify the level of damage to spermatozoa. Large-scale studies should be conducted in different clinical settings to determine the effects of sperm DNA damage on the outcome of ART.

Purpose : We have carried out experiments to determine if human cervical mucus can act as an in vitro selective barrier against spermatozoa morphologically normal that carry genetic structural abnormalities. Methods : Sperm chromatin abnormalities have been evaluated by Chromomycin A3 and “endogenous” nick translation. Results : The data obtained have shown that spermatozoa possessing higher levels of DNA protamination are more proficient in crossing the cervical mucus barrier. Moreover, the levels of positivity to endogenous nick translation treatment was practically zero in such spermatozoa. Conclusions : We suggest that sperm penetration of cervical mucus could be used to select sperm preparations free of fragmented DNA or chromatin structural abnormalities for assisted reproduction.

Introduced in 2001, intracytoplasmic morphologically selected sperm injection (IMSI) represents a more sophisticated way of ICSI whereby, prior to injection, the spermatozoon is selected at higher magnification. Doing so, the spermatozoon can be evaluated for fine integrity of its nucleus and the injection of a normal spermatozoon with a vacuole-free head can be assured. Additional research is needed to unravel the underlying mechanisms responsible for the presence of vacuoles in sperm heads. Associations with acrosome status, chromatin condensation, DNA fragmentation and sperm aneuploidy have been documented, however, controversy on their nature exists. Spermatozoon shape and large vacuoles are detected and deselected in conventional ICSI as well. However, the detection of subtle small vacuoles depends on the resolving power of the optical system and may impact oocyte fertilization, embryo development and implantation. Several comparative studies have indicated that the use of high-magnification sperm selection was associated with both higher pregnancy and delivery rates, whereas also lower miscarriage rates were observed. However, still to date randomized, well-powered studies to confirm these findings are scarce and show conflicting results. Hence, the most relevant indications for IMSI still remain to be determined. Two groups of patients have been put forward i.e. severe male-factor infertility patients and patients with a history of repeated ICSI failures. However, for both groups limited to no proof of any benefit does exist. IMSI is a time-consuming procedure at the expense of oocyte ageing. The lack of proof and understanding of its benefit does not justify its routine clinical application at present.

In this study we investigated whether morphology and chromatin anomalies in human spermatozoa can influence fertilization after intracytoplasmic sperm injection (ICSI). We examined unfertilized oocytes, using the fluorochrome Hoechst 33342, to determine whether a relationship exists between failure of fertilization and sperm chromatin quality. Sperm chromatin packaging quality was assessed using the chromomycin A3 (CMA3) fluorochrome, and the presence of DNA damage in spermatozoa, using in-situ nick translation, Normal males present sperm parameters with a normal morphology of > 20%, CMA3 fluorescence of < 30% and exhibit endogenous nicks in < 10% of their spermatozoa. When patients were separated according to these values no difference was observed in their fertilization rates after ICSI. When the unfertilized ICSI oocytes were examined, we found that patients with CMA3 fluorescence of

This study aimed to investigate the association between anomalies in sperm chromatin packaging, morphology and fertilization in patients undergoing routine in-vitro fertilization (IVF) or subzonal insemination (SUZI). Sperm chromatin packaging was assessed using chromomycin A3 (CMA3), a fluorochrome specific for guanine-cytosine rich sequences of DNA. One hundred to 150 sperm cells were assessed in 55 patients to compare sperm chromatin packaging and morphology to fertilization after IVF or SUZI. When the morphology and CMA3 fluorescence of individual spermatozoa was assessed, > 75% of the macrocephalic sperm fluoresced in all patients. In contrast, a mean of 37% of the spermatozoa with normal morphology fluoresced in IVF patients compared with 58% of the normal spermatozoa in male factor patients treated by SUZI. SUZI patients displaying a high fluorescence (> 70%) in their spermatozoa also had a significantly lower fertilization rate. Lower packaging quality in morphologically normal spermatozoa may represent a major limiting factor in the fertilizing ability of male factor patients. This study confirms that a high percentage of CMA3 positivity is present in certain forms of male factor infertility and that such a test may be used to distinguish separate populations in morphologically normal spermatozoa.

It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin, which acquires fully condensed status at the caput epididymidis. The purpose of this study was to compare the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay, the comet assay, the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test by analysing spermatozoa from the caput and cauda epididymidis in order to demonstrate the ability of each technique to discriminate between different degrees of sperm maturity related to chromatin compaction and DNA fragmentation. Our results suggest that some populations of DNA-fragmented spermatozoa associated with immature sperm can only be identified using the comet assay and the SCSA but not with the SCD test or the TUNEL assay. © 2012 AJA, SIMM & SJTU. All rights reserved.

Semen quality is conventionally determined according to the number, motility, and morphology of spermatozoa in an ejaculate. In turn, it is generally accepted that an association exists between these semen parameters and fertilizing ability. With the advent of in vitro fertilization (IVF) and related techniques such as intracytoplasmic sperm injection (ICSI), it has become increasingly apparent that the number, motility, and morphology of spermatozoa are not always indicative of a male’s fertility status. Methods exploring sperm DNA stability and integrity have been applied during the last decade to evaluate fertility disorders and to increase the predictive value of sperm analysis for procreation in vivo and in vitro. It has been shown that infertile men have an increased sperm histone–protamine ratio compared to fertile counterparts. This alteration of histone–protamine ratio, also called abnormal packing, increases susceptibility of sperm DNA to external stresses due to poorer chromatin compaction. Recent studies have also underlined the link between protamine deficiency and sperm DNA damage that resulted in poor fertilizing capacity.

Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with mate infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization. (C) 2009 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

As a number of children born by assisted reproductive technology (ART) are increasing each year across the developed world, the health of such offspring is a matter of public concern. Does the integrity of the paternal genome impact on offspring health? In societal terms, as birth rates fall, and the Western population become unsustainable, do the benefits outweigh the costs of creating and providing for this ART conceived sub population? There are little data to date to answer these questions. The long‑term health of such children has largely been ignored, and success measured only by early (pre-birth) outcomes such as embryo quality or pregnancy. However, there are powerful paradigms such as ageing and smoking that give vital clues as to the potential impact of unhealthy spermatozoa on disease risk, mental and physical health, fertility and mortality of these offspring.

The classical idea about the function of the mammalian sperm chromatin is that it serves to transmit a highly protected and transcriptionally inactive paternal genome, largely condensed by protamines, to the next generation. In addition, recent sperm chromatin genome-wide dissection studies indicate the presence of a differential distribution of the genes and repetitive sequences in the protamine-condensed and histone-condensed sperm chromatin domains, which could be potentially involved in regulatory roles after fertilization. Interestingly, recent proteomic studies have shown that sperm chromatin contains many additional proteins, in addition to the abundant histones and protamines, with specific modifications and chromatin affinity features which are also delivered to the oocyte. Both gene and protein signatures seem to be altered in infertile patients and, as such, are consistent with the potential involvement of the sperm chromatin landscape in early embryo development. This present work reviews the available information on the composition of the human sperm chromatin and its epigenetic potential, with a particular focus on recent results derived from high-throughput genomic and proteomic studies. As a complement, we provide experimental evidence for the detection of phosphorylations and acetylations in human protamine 1 using a mass spectrometry approach. The available data indicate that the sperm chromatin is much more complex than what it was previously thought, raising the possibility that it could also serve to transmit crucial paternal epigenetic information to the embryo.

This chapter explores the possibility that capacitation and apoptosis are linked processes joined by their common dependence on the continued generation of reactive oxygen species (ROS). According to this model capacitation is initiated in spematozoa following their release into the female reproductive tract as a consequence of intracellular ROS generation, which stimulates intracellular cAMP generation, inhibits tyrosine phosphatase activity and enhances the formation of oxysterols prior to their removal from the sperm surface by albumin. The continued generation of ROS by capacitating populations of spermatozoa eventually overwhelms the limited capacity of these cells to protect themselves from oxidative stress. As a result the over-capacitation of spermatozoa leads to a state of senescence and the activation of a truncated intrinsic apoptotic cascade characterized by enhanced mitochondrial ROS generation, lipid peroxidation, motility loss, caspase activation and phosphatidylserine externalization. The latter may be particularly important in instructing phagocytic leukocytes that the removal of senescent, moribund spermatozoa should be a silent process unaccompanied by the generation of proinflammatory cytokines. These observations reveal the central role played by redox chemistry in defining the life and death of spermatozoa. A knowledge of these mechanisms may help us to engineer novel solutions to both support and preserve the functionality of these highly specialized cells.

The semen quality of seven young adult boars was assessed for percentages of sperm motility, normal acrosomes, abnormal sperm, cells positive to sHOST (short Hipoosmotic Swelling Test), HPNA cells (sHOST Positive with Normal Acrosome cells) and the percentage of sperm heads, which exhibited DNA fragmentation using the Sperm Chromatin Dispersion test (SCD). These parameters were analysed in sperm samples both undiluted and diluted using a commercial extender and stored at 15 °C for 21 days. Results showed that semen quality decreases faster in the undiluted semen samples from day 0 to day 7 compared to diluted semen samples that remained with a high quality up to day 11. The undiluted semen exhibited a low DNA fragmentation index (DFI) during the first days and then a significant increase from day 7 up to day 21. This increase in the DFI coincided with the lowest levels of the other semen quality parameters. On the contrary, the samples diluted in the commercial extender showed very low levels of DNA fragmentation in all boars during the preservation period. When the evolution of DNA fragmentation was analysed in the undiluted samples, differences were found among boars. These differences were not shown in the samples diluted in the extender where the basal DFI remained stable during the 21 days. The main conclusion of this study was that some sperm extenders delay or partially prevent sperm DNA fragmentation. © 2006 Elsevier Inc. All rights reserved.