Frequency of sex chromosomal disomy in spermatozoa of normal and oligozoospermic Iranian patients and its effects on fertilisation and implantation rates after ICSI

Chromosomal aneuploidy is a well‐known phenomenon in human gametes including spermatozoa. Success rate of fertilisation and implantation in subfertile patients with male factor has always been shown to be very low. We tried to relate the possible impact of sex chromosomal aneuploidy in spermatozoa used for intracytoplasmic sperm injection (ICSI) on fertilisation and implantation rate. To evaluate the frequency of disomy for X and Y chromosomes in sperm samples retrieved from normal and oligozoospermic individuals, primed in situ labelling (PRINS) technique was used. Following ICSI, the rate of eight‐cell embryos for each category was determined and followed up for successful implantation. Results showed a statistically significant higher frequency of disomy for all chromosomes under study in spermatozoa of oligozoospermic patients compared with normal men (P < 0.01). The rate of eight‐cells embryo formation was significantly lower than in normal group (P < 0.01). The number of embryos transferred for both groups were nearly similar. Implantation rate for oligozoospermic patients was much lower than that of the normal group but was not significantly different (P > 0.05). These results demonstrate that men especially with severe oligozoospermia have an elevated risk for chromosome abnormalities in their spermatozoa. These abnormalities might affect fertilisation and pre‐embryo formation with less impact on implantation.     

N‐acetyl‐L‐cysteine pre‐treatment protects cryopreserved bovine spermatozoa from reactive oxygen species without compromising the in vitro developmental potential of intracytoplasmic sperm injection embryos

Excess of reactive oxygen species (ROS) on in vitro embryo production systems negatively affects the quality and developmental potential of embryos, as result of a decreased sperm quality and increased DNA fragmentation. This issue is of major importance in assisted fertilisation procedures such as intracytoplasmic sperm injection (ICSI), because this technique does not allow the natural selection of competent spermatozoa, and therefore, DNA‐damaged spermatozoa might be used to fertilise an egg. The aim of this study was to investigate a new strategy to prevent the potential deleterious effect of ROS on cryopreserved bovine spermatozoa. We evaluated the effect of a sperm pre‐treatment with different concentrations of N‐acetyl‐L‐cysteine (NAC) on ROS production, viability and DNA fragmentation and assessed the effect of this treatment on the in vitro developmental potential and quality of embryos generated by ICSI. The results show a strong scavenging effect of 1 and 10 mm NAC after exposure of spermatozoa to a ROS inducer, without compromising the viability and DNA integrity. Importantly, in vitro developmental potential and quality of embryos generated by ICSI with spermatozoa treated with NAC were not affected, confirming the feasibility of using this treatment before an ICSI cycle.     

Effect of brain‐derived neurotrophic factor on sperm function,oxidative stress and membrane integrity in human

Oxidative stress has negative impacts on the clinical outcomes of assisted reproduction techniques. The brain‐derived neurotrophic factor (BDNF) promotes the viability of nerve cells and is known to decrease oxidative stress and apoptosis in different cells. The aim of this study was to evaluate the effect of BDNF treatment on human sperm functions that are known to be essential for fertilisation. Our findings showed that treatment of human spermatozoa with 0.133 nM BDNF significantly increased the percentages of both total (P = 0.001) and progressive (P < 0.01) motile sperm cells compared to those observed in the nontreated (control) group. We also showed that the mean fluorescence intensity of DCFH‐DA, as an indicator of intracellular reactive oxygen species, was significantly lower (P < 0.05) in spermatozoa treated with BDNF compared to the control group. Treatment of spermatozoa with BDNF significantly decreased the percentages of both dead (P = 0.001) and apoptotic‐like sperm cells (P < 0.05) compared to the control group. On the other hand, BDNF treatment significantly increased the percentage of viable sperm cells compared to the control (P = 0.001). In conclusion, BDNF has protective effects against oxidative stress in spermatozoa and could improve sperm functions that are essential for sperm–egg fusion and subsequent fertilisation.     

Relationship between DYNLT1 and Beclin1 expression and the fertilising potential of human spermatozoa

This study aimed to evaluate dynein light chain type 1 (DYNLT1) mRNA expression in mature spermatozoa and to investigate its association with Beclin1 expression to help in understanding of pathogenesis of male infertility. It included 60 infertile men divided into idiopathic (n = 20), accessory gland inflammation (n = 20), and varicocele (n = 20) groups, and 20 healthy fertile men as a control group. Semen parameters were evaluated according to the 2010 World Health Organization criteria. Mature spermatozoa were isolated by Sil‐select gradient. Relative quantification of DYNLT1 and Beclin1 mRNA expression in whole sperm pellet and mature spermatozoa was done using real‐time PCR. Beclin1 protein was assessed in whole sperm pellet and mature spermatozoa by ELISA. Beclin1 mRNA and protein were significantly increased in spermatozoa from infertile patients of different aetiologies in comparison to healthy controls (p < .05). However, DYNLT1 mRNA expression was significantly decreased in infertile groups than controls (p < .05). Mature spermatozoa extracted from all studied subjects showed increased DYNLT1 mRNA and decreased Beclin1 mRNA and protein expression compared with the whole sample. It is concluded that decreased Beclin1 and increased DYNLT1 mRNA expression in mature spermatozoa may provide an insight into the biological processes that are activated or suppressed during sperm maturation.     

Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos

Gamete co‐incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co‐incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1 mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000 nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18 h. In Experiment 1, only 1 mmol melatonin showed lesser blastocyst rates than control (C: 23.2 ± 6.7% versus 1 mmol: 2.0 ± 1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000 nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls (P < 0.05). IVF media with 1 mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production.     

Human sperm handling in intracytoplasmic sperm injection processes: In vitro studies on mouse oocyte activation,embryo development competence and sperm oxidation–reduction potential

Polyvinylpyrrolidone (PVP) and hyaluronic acid (HA) are routinely used in handling spermatozoa for intracytoplasmic sperm injection (ICSI). As there are still concerns about possible adverse effects on the embryo, this study investigated sperm handling in a mouse ICSI model to (i) evaluate oocyte activation after injection of spermatozoa selected for rotational or linear motion in PVP; (ii) assess the effect of sperm selection in PVP, HA and medium on oocyte activation; (iii) examine the effects of PVP and HA on parthenogenetic oocyte activation and embryo development; and (iv) assess the oxidation–reduction potential (ORP) of spermatozoa exposed to PVP, HA or medium. Oocyte activation was higher when spermatozoa exhibited rotational motion rather than linear motion (79% vs. 52%; p = .05). There was no difference in oocyte activation and embryo development after parthenogenetic oocyte activation after sperm injection using PVP, HA or medium‐incubated spermatozoa. PVP‐selected spermatozoa exhibited lower (p < .0001) ORP levels than using HA. Thus, results indicate that the sperm handling method and the type of medium used impact ICSI outcomes. Overall, sperm incubation in PVP, HA and medium yields similar outcomes with regard to oocyte activation and embryo development. However, PVP provides more antioxidative protection than HA and should therefore be preferred for sperm manipulation.     

Antioxidant activity of Nigella sativa Seeds Aqueous Extract and its use for cryopreservation of buffalo spermatozoa

The free radical scavenging activity (RSA) of Nigella sativa extract and its efficiency for cryopreservation of buffalo spermatozoa was investigated. In experiment 1, Nigella sativa extract was prepared and evaluated for RSA using 2,2‐diphenyl‐1‐picrylhydrazyl assay. The results showed increased pattern of RSA at 1%–5% of Nigella sativa extract. In experiment 2, buffalo semen from three bulls (24 ejaculates) was incubated at 0%, 0.1%, 0.3%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5% and 6% extract to assess in vitro tolerability to Nigella sativa in terms of progressive motility (PM). Buffalo spermatozoa showed tolerance to all levels; rather, sperm PM was increased at 1%–4% extract. In experiment 3, semen from three bulls (24 ejaculates) was cryopreserved with 0%, 1%, 2%, 3%, 4% and 5% of Nigella sativa extract. Sperm PM and plasma membrane integrity (PMI) were evaluated after dilution and cooling, while PM, PMI, viability and DNA integrity were evaluated after thawing. Nigella sativa extract at 4% in extender improved (p < .05) post‐dilution, post‐cooling and post‐thaw sperm quality. In conclusion, Nigella sativa extract at all concentrations (1%–6%) showed antioxidant activity and its supplementation at 4% in extender improved buffalo sperm quality at all stages of cryopreservation.     

Comparison of sperm morphology and nuclear sperm quality in SPATA16‐ and DPY19L2‐mutated globozoospermic patients

The aim of this study was to compare the sperm morphology and nuclear sperm quality (sperm aneuploidy and DNA fragmentation) in two groups of globozoospermic patients: DPY19L2‐mutated patients (n = 6) and SPATA16‐mutated patients (n = 2). Results for these two groups were also compared to a group of fertile men (n = 25). Fluorescence in situ hybridisation was performed for chromosomes X, Y and 18. Sperm DNA fragmentation was evaluated by TUNEL assay. Sanger sequencing was performed for mutations screening of DPY19L2 and SPATA16 genes. Sperm analysis revealed a classic phenotype of total globozoospermia in DPY19L2‐mutated group and a particular phenotype characterised by a predominance of double/multiple round‐headed (39.00 ± 4.2%) and multi‐tailed spermatozoa (26.00 ± 16.97%) in SPATA16‐mutated group. FISH analysis showed a significantly higher aneuploidy rate in globozoospermic patients compared to controls (p < 0.05), and a higher rate was observed in SPATA16‐mutated group compared to DPY19L2‐mutated group (p < 0.05). DNA fragmentation index was significantly higher in globozoospermic men compared to controls (p < 0.001), and there is no statistically significant difference between the two globozoospermic groups. We showed that SPATA16 defects could be associated with an abnormal meiosis leading to a particular morphological sperm defect of double/multiple round‐headed and multi‐flagella and a higher sperm aneuploidy rate than in case of DPY19L2‐defects in classic globozoospermia.     

Effect of You Gui Wan on mouse sperm fertilising ability in vivo and in vitro

This study is to explore whether YGW has an impact on sperm fertilising ability in mice. Twenty male mice were randomly divided into two groups. In vivo experiments, one group of animals were orally administrated with YGW decoction and another group administered with saline for 14 days. Afterwards, the animals were mated with their female partners. Percentages of retrieved zygotes were then compared. In vitro experiments, in vitro fertilisation (IVF) assay, sperm acrosome reaction and acrosin activity were used to compare sperm fertilising ability between the two groups. The YGW‐treated group had a significantly higher percentage of zygotes than the saline controls (P = 0.005). The IVF rates induced by spermatozoa from the herb‐treated mice were also significantly higher than those from the control animals (P = 0.015). The sperm acrosin activity of the herb‐treated group was significantly higher than that of the saline‐treated group (P = 0.048), although there was no significant difference in testicular weight, sperm count and sperm motility. These data suggest that YGW decoction has a significant effect on normal sperm fertilising ability both in vivo and in vitro, which may be due to, at least in part, increments in the sperm acrosin activity.     

Rat sperm immobilisation effects of a protein from Ricinus communis (Linn.): an in vitro comparative study with nonoxynol‐9

Previous study conducted in our department showed that 50% ethanolic extract of the root of Ricinus communis possess reversible antifertility effect and a 62‐kDa protein (Rp) from this extract is responsible for the antifertility effects. In this study, we compared the spermicidal effect of this Rp with nonoxynol‐9 (N‐9) in vitro. The sperm immobilisation studies showed that 100 μg ml^?1 of Rp was able to immobilise the sperms completely within 30 s. Sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo‐osmotic swelling in Rp and N‐9 treated groups in comparison with the control. In Rp and N‐9 treated groups, the number of acrosome‐reacted cells was found to be high and also caused agglutination of the spermatozoa, indicating the loss of intactness of the plasma membrane, which was further supported by the significant reduction in the activity of membrane bound 5′‐nucleotidase, acrosomal acrosin. In short, the protein Rp possesses spermicidal activity in vitro and its effects are similar to that of nonoxynol 9.     

In vitro effects of nicotine on human spermatozoa

Washed human spermatozoa from 12 normozoospermic donors were treated with different concentrations of nicotine 0.1, 1.0, 5.0 and 10.0 mm and were compared to spermatozoa suspended in nutrient medium only (control). Computer‐aided sperm analysis was used to assess sperm kinematic properties after 30, 60, 120 and 180 min of incubation. Viability was assessed by means of a dye exclusion staining technique (eosin/nigrosin), while acrosome‐reacted cells were identified under a fluorescent microscope using fluorescein isothiocyanate–Pisum sativum agglutinin as a probe. Nicotine significantly reduced total motility, progressive motility, curvilinear velocity, amplitude of lateral head displacement, beat cross‐frequency, viability and caused spontaneous acrosome reaction at concentrations of ≥5.0 mm after 2 and 3 h of exposure. Nicotine concentrations of 0.1 and 1.0 mm had no significant effect (P < 0.05) on spermatozoa except that 1.0 mm significantly decreased (P < 0.05) sperm progressive motility at 2 and 3 h of incubation as well as viability after 3 h of incubation. This study concludes that the occurrence of high levels of nicotine in the body and seminal fluid might adversely affect fertilisation capacity of human spermatozoa through a mechanism that involves decreased motility, viability and premature induction of the acrosome reaction.     

Spermicidal activity of Indian seaweeds: an in vitro study

Contraceptive properties of seaweeds are still stands as lacuna; in this context, the screening of in vitro male contraceptive properties of crude ethanolic extract of Indian seaweeds against normal human sperm is carried out. In total, twelve seaweeds were screened for in vitro spermicidal activity. Among these twelve seaweeds, Halimeda gracilis showed 100% inhibition of human spermatozoa at 10 mg ml^?1 concentration in 20 s and its EC₅₀ value was 2.05 mg ml^?1 in 20 s. Further, dose‐ and time‐dependent spermicidal assay revealed that the sperm was completely immobilised for 20 s. Plasma membrane of sperm was damaged due to the exposure of H. gracilis extract. MTT assay with H. gracilis extract showed 88.5% of cytotoxic incidence. H. gracilis extract tested for cytotoxicity against Artemia salina recorded LC₅₀ value of 34.8 μg ml^?1. Phytochemical analysis of H. gracilis extract evidenced the presence of alkaloids, flavonoids, proteins and sugars. Results of this study clearly inferred that the synergistic effect of active principles reside within the H. gracilis extract had shown better contraceptive activity.     

Evaluation of the morphokinetic parameters and development of pre‐implantation embryos obtained by testicular,epididymal and ejaculate spermatozoa using time‐lapse imaging system

Low sperm quality has negative effects on fertilisation and embryo development. The males with azoospermia apply for testicular sperm extraction (TESE) or microsurgical epididymal sperm aspiration (MESA) in order to retrieve sperm. To date, there have not been any reports investigating morphokinetic parameters of pre‐implantation embryos using testicular and epididymal spermatozoa. Therefore, we aimed to correlate embryo development and assess morphogenetic parameters in embryos obtained by TESE and MESA using time‐lapse imaging. A total of 60 patients undergoing IVF treatments were included in this study. Twenty men with normal semen parameters were selected as control group. Twenty men undergoing TESE and 20 men undergoing MESA were also included in this study. The morphokinetic parameters of time intervals between the second polar body (PB2) extrusion, pronuclei formation and disappearance and cleavage divisions showed significant variations in TESE, MESA and control groups. Furthermore, the pregnancy rates (positive beta‐hCG) were shown to be similar in both TESE and the control group (55% in each group), whereas for the MESA group, this rate was significantly lower (39%, p = 0.049). Further extrapolation of these results may implicate that the obstructive azoospermia patients should undergo TESE instead of MESA for better blastocyst development and higher pregnancy rates.     

Oxidative stress‐induced alterations in seminal plasma antioxidants: Is there any association with keap1 gene methylation in human spermatozoa?

Kelch‐like ECH‐associated protein 1 (keap1)‐nuclear factor‐erythroid 2‐related factor 2 (Nrf2) pathway is one of the master regulators of cellular defence against oxidative stress. Epigenetic alterations like hypermethylation of keap1 gene impair keap1‐Nrf2 system in several oxidative stress–associated diseases. The objective of this study was to evaluate the epigenetic status of keap1 in sperm DNA of normozoospermic subjects, having different levels of reactive oxygen species (ROS) in seminal plasma. Semen samples were obtained from 151 apparently healthy male partners of couples who attended the Avicenna infertility clinic. Samples were categorised into four groups according to their ROS levels: group A (n = 39, ROS < 20 RLU/s per 10⁶ spermatozoa), group B (n = 38, 20 ≤ ROS < 40 RLU/s per 10⁶ spermatozoa), group C (n = 31, 40 ≤ ROS < 60 RLU/s per 10⁶ spermatozoa) and group D; (n = 43, ROS ≥ 60 RLU/s per 10⁶ spermatozoa). Keap1 methylation status was assessed using methylation‐specific PCR along with seminal total antioxidant capacity. The results showed no significant alterations in keap1 methylation in any groups, whereas the total antioxidant capacity enhanced with increasing levels of ROS exposure. These results indicate that keap1 was not methylated during ROS elevation and oxidative stress, suggesting that the cells have adopted other mechanisms to elevate antioxidant level.     

Selection of normal spermatozoa with a vacuole‐free head (x6300) improves selection of spermatozoa with intact DNA in patients with high sperm DNA fragmentation rates

Intracytoplasmic morphologically selected sperm injection (IMSI, 6300× magnification with Nomarski contrast) of a normal spermatozoon with a vacuole‐free head could improve the embryo's ability to grow to the blastocyst stage and then implant. However, the most relevant indications for IMSI remain to be determined. To evaluate the potential value of IMSI for patients with a high degree of sperm DNA fragmentation (n = 8), different types of spermatozoa were analysed in terms of DNA fragmentation. Motile normal spermatozoa with a vacuole‐free head selected at 6300× magnification had a significantly lower mean DNA fragmentation rate (4.1 ± 1.1%, n = 191) than all other types of spermatozoa: non‐selected spermatozoa (n = 8000; 26.1 ± 1.5% versus 4.1 ± 1.1%; P < 0.005), motile spermatozoa (n = 444; 20.8 ± 2.7% versus 4.1 ± 1.1%; P < 0.001) and motile, normal spermatozoa selected at 200× magnification (n = 370; 18.7 ± 2.7% versus 4.1 ± 1.1%; P < 0.001) and then motile, morphometrically normal spermatozoa with anterior vacuoles (n = 368; 15.9 ± 2.9% versus 4.1 ± 1.1%; P < 0.05) or posterior vacuoles (n = 402; 22.5 ± 3.6% versus 4.1 ± 1.1%; P < 0.001) selected at 6300× magnification. For patients with high sperm DNA fragmentation rates, selection of normal spermatozoa with a vacuole‐free head (6300×) yields the greatest likelihood of obtaining spermatozoa with non‐fragmented DNA.     

Aphrodisiac properties of hydro‐alcoholic extract of Cassia auriculata flower in male rats

Cassia auriculata is a commonly found plant in Asia, widely used in Ayurveda and Siddha medicines as a tonic, astringent and in general for diabetes. Herbal tea made from this plant has been marketed as a product for restoring sexual vitality, to increase sperm count and counteract ejaculatory disorders. However, the scientific evidences are scarce to prove this concept. Here, we examined the effect of hydro‐alcoholic extract obtained from C. auriculata flower upon the expression of male Wistar albino rat’s sexual behaviour. Sildenafil was used as a positive control. Penile erection index (PEI), mount latency (ML), intromission latency (IL), ejaculation latency (EL), mounting frequency (MF), intromission frequency (IF), ejaculation frequency (EF) and post‐ejaculatory interval (PEjI) were recorded for days 0, 7, 14 and 28 and also after the withdrawal of the treatment on days 7 and 15. Significant reduction in ML, IL and PEjI, and increment in EL, PEI, MF, IF and EF were observed (p < 0.05, <0.01). However, neither extract nor sildenafil sustains the effect after withdrawal of the treatment. The present finding demonstrates the aphrodisiac potential of hydro‐alcoholic extract of C. auriculata flower in vivo and lends support to the traditional utilisation as a sexual stimulating agent.     

Immobilising effect of Ruta graveolens L. on human spermatozoa: coumarin compounds are involved

This study was designed to find out Ruta graveolens L. functional components, which have immobilisation effect on human spermatozoa for contraceptive use. A five‐step fractionation method was used to derive different components from rue aqueous extract by using hexane, chloroform, ethanol, acetone and ultrapure water. Gas chromatography–mass spectrophotometery (GC–MS) of all fractions and the aqueous extract were performed to determine the chemical components. The immobilisation assay and membrane integrity test were also performed with four different coumarins, which were found in GC–MS in a concentration of 10 μm . Hexane, chloroform, acetone and ethanol fractions could significantly decrease motility of sperms within the first and the second hours. Hexane fraction had also significant immediate effect. The aqueous fraction had no effect on sperm motility. Meanwhile, GC–MS revealed that aqueous extract and effective fractions had similar coumarin compounds. We performed the immobilisation assay on four different coumarins, which were found in GC–MS in a concentration of 10 μm . Reduction of sperm motility was only significant for xanthotoxin. In the sperm viability and membrane integrity tests, hexane and ethanolic fractions could impair sperm vitality significantly, in contrast to coumarins. These results indicated that a part of immobilising effect of rue could be due to its coumarins. The possible mechanism could be blocking of spermatozoa potassium channels.     

In vitro effect of gold and silver nanoparticles on human spermatozoa

The cytotoxicity of Au/Ag nanoparticles (NPs) on human spermatozoa was investigated in vitro. Semen from donors were incubated (37 °C, 60′–120′) with 30, 60, 125, 250 and 500 μM Au/Ag‐NPs. Sperm motility was evaluated following WHO guidelines; sperm viability was assessed with eosin Y test. Au‐NPs were characterised and localised with field emission gun‐based scanning transmission electron microscope/energy dispersive spectroscopy and transmission electron microscopy. Both tested NPs exerted a significant dose‐dependent effect on motility and viability of human spermatozoa (P < 0.001). Ag‐NPs seem to show a slightly elevated toxicity although not significant (P > 0.05). Au‐NPs were localised in spermatozoa, whereas Ag‐NPs were undetectable. In conclusion, Au‐NPs and Ag‐NPs do not appear to be harmful for human spermatozoa up to high concentrations (250–500 μM) that are probably difficult to reach in vivo. It is mandatory to explore the genotoxic effect of NPs in germ cells.     

Male immunity to the chlamydial 60 kDa heat shock protein (HSP 60) – associated with semen quality?

The role of Chlamydia trachomatis for male infertility is a matter of constant debate. It is assumed that in its persistent form this pathogen may produce high levels of 60 kD heat shock protein (Chlam HSP60). Cross‐reactivity between epitopes of the bacterial and human HSPs, involved in many steps of the reproductive process, might induce an autoimmune response with potential impairment of semen quality and sperm fertilising capacity. This prospective study included asymptomatic males of a total of 128 unselected subfertile couples (median duration of infertility 3 years) to determine the clinical relevance of male immunity to Chlam HSP60 during infertility investigation. After medical history and clinical examination of both partners, serum antibodies (Ab) to Chlam HSP60 were determined. Same day semen quality evaluation included microscopical standard sperm analysis, determination of seminal white blood cells (WBC) and of antisperm Ab (ASA) of the Ig G‐ and Ig‐A class (mixed antiglobulin reaction, MAR), microbial screening and examination of sperm functional capacity. Sperm/mucus interaction was tested in vitro and in vivo. Simultaneously, patients′ female partners were tested for Chlam HSP60 Ab and results were compared with a standard serology evaluation for antichlamydial IgG Ab. The presence of ChlamHSP60 Ab (positive in 24% of males) was not significantly associated with semen quality, seminal WBC and antisperm AB of the IgG‐ or Ig A‐class, the outcome of the microbial screening nor with sperm functional capacity and results of sperm/mucus interaction testing in vitro and in vivo. Chlam HSP60 Ab were significantly more frequent in female partners of Chlam HSP60 Ab‐positive men, and results correlated with the outcome of standard chlamydial serology evaluation. In conclusion, when serum Chlam HSP60 Ab are used as marker, male immunity to the chlamydial 60 kD heat shock protein is not associated with semen quality, sperm functional capacity and other clinically relevant parameters of male fertility.     

Effect of short‐term semen storage in salmon (Oncorhynchus mykiss) on sperm functional parameters evaluated by flow cytometry

The short‐term storage of salmonid semen is a viable method for in vitro fertilisation. Previous studies have found that short‐term storage affects sperm motility, compromising quality and fertilising capacity. However, the functional characteristics of the spermatozoa of O. mykiss during storage time and its relation to the spawning period are little known. This study was designed to evaluate the effects of in vitro short‐term storage on sperm functional parameters in O. mykiss, determined by flow cytometry. Semen samples of the first spawning – undiluted (SSD) and diluted (SD) (Storfish^® 1 : 2v/v; IMV AI solutions, France) – were stored at 4 °C for 14 days. Motility, viability (PMI: plasma membrane integrity) and mitochondrial membrane potential (ΔΨM) were assessed. On the fifth day of storage, spermatozoa showed a motility >70% (SSD: 78.3% versus SD 85.0%), PMI (81.5% SSD/87.2% SD) and ΔΨM (72.5% SSD/SD 80.0%) (P < 0.05). However, a significant decline in the percentage of all functional parameters (P < 0.05) was observed after 5 days of storage for all samples of both undiluted (SSD) and diluted semen. In conclusion, the results here provide new data on O. mykiss sperm quality with respect to in vitro short‐term storage evaluated by flow cytometry.