Quantitative Analysis of the Human Semen Phosphorometabolome by 31P-NMR.

Phosphorus-containing metabolites occupy a prominent position in cell pathways. The phosphorometabolomic approach in human sperm samples will deliver valuable information as new male fertility biomarkers could emerge. This study analyzed, by 31P-NMR, seminal plasma and whole semen from asthenozoospermic and normozoospermic samples (71% vs. 27% and 45% vs. 17%, total and progressive sperm motility, respectively), and also ejaculates from healthy donors. At least 16 phosphorus-containing metabolites involved in central energy metabolism and phospholipid, nucleotide, and nicotinamide metabolic pathways were assigned and different abundances between the samples with distinct sperm quality was detected. Specifically, higher levels of phosphocholine, glucose-1-phosphate, and to a lesser degree, acetyl phosphate were found in the asthenozoospermic seminal plasma. Notably, the phosphorometabolites implicated in lipid metabolism were highlighted in the seminal plasma, while those associated with carbohydrate metabolism were more abundant in the spermatozoa. Higher levels of phosphocholine, glucose-1-phosphate, and acetyl phosphate in the seminal plasma with poor quality suggest their crucial role in supporting sperm motility through energy metabolic pathways. In the seminal plasma, phosphorometabolites related to lipid metabolism were prominent; however, spermatozoa metabolism is more dependent on carbohydrate-related energy pathways. Understanding the presence and function of sperm phosphorylated metabolites will enhance our knowledge of the metabolic profile of healthy human sperm, improving assessment and differential diagnosis.

Effects of in vitro repaglinide supplementation on improving sperm motility parameters, viability, and DNA integrity in normozoospermic and asthenozoospermic men.

OBJECTIVE: Human sperm motility and hyperactivation (HA) are induced by different factors such as intracellular calcium concentration. Repaglinide is an antidiabetic drug that, via the blocking of ATP-sensitive potassium channels (K-ATP channels), depolarization of the ß-cell membrane, and opening of the voltage-gated calcium channels leads to an increase in intracellular calcium. The present study aimed to examine the effects of repaglinide on in vitro sperm motility parameters, viability, and DNA integrity in normozoospermic and asthenozoospermic men. METHODS: Semen samples were collected from two groups of normozoospermic donors and asthenozoospermic patients. The samples were washed free of seminal plasma and then treated with medium alone (control) or with 100 nM and 1µM concentrations of repaglinide. After 1 h of incubation, percent sperm motility and hyperactivation were assessed; after 2 h of incubation, sperm viability and DNA fragmentation rate were evaluated by the Eosin-Y and acridine orange staining, respectively. RESULTS: In both groups, repaglinide at a concentration of 100 nM and 1µM significantly improved percent sperm motility, hyperactivation, and vital sperms with normal DNA; in specimens from normozoospermic men, the 1µM concentration had a noticeable effect on progressive motility; in samples from asthenozoospermic men, the highest hyperactivation rate was seen at a concentration of 100 nM as compared with the 1µM concentration and controls (p<0.05). CONCLUSIONS: Our results suggest that repaglinide can improve sperm motility, hyperactivity, viability, and DNA integrity in both normozoospermic and asthenozoospermic men.

Scutellarein stimulates human sperm function by increasing the levels of intracellular calcium and tyrosine phosphorylation.

As a kind of flavonoid, scutellarein is widely used to protect against various human diseases. Although the protective effects of scutellarein have been well studied, its influence on human reproduction remains unknown. In this research, we evaluated the effect of scutellarein on human sperm functions in vitro. Three different concentrations of scutellarein (1, 10, 100 µM) were applied to ejaculated human sperm. Fertilisation-essential functions, as well as the intracellular calcium concentration ([Ca2+ ]i ) and protein-tyrosine phosphorylation, two factors which are vital for sperm function regulation, were evaluated. The results demonstrated that all concentrations of scutellarein utilised in this study could significantly increase sperm spontaneous capacitation and acrosome reaction through the enhancement of [Ca2+ ]i . Besides, the level of tyrosine phosphorylation of sperm could also be increased by scutellarein. Meanwhile, the sperm motility could be improved by 10 and 100 µM scutellarein, which also make a significant enhancement in sperm penetration ability and hyperactivation. This is one of the limited studies showing the regulation of scutellarein on human spermatozoa functions and is helpful to enrich its application.

The cryoprotective effect of vitamins on human spermatozoa quality: a systematic review and meta-analysis.

The Cryopreservation of spermatozoa ensures preserving fertility potential after some medical treatments such as chemotherapy and radiotherapy in cancer patients. However, many spermatozoa encounter serious damages, and their motility and viability decrease considerably after thawing. The excessive production of reactive oxygen species is one of the major causes of these damages. The supplementation of cryopreservation media with vitamins, which are well-known antioxidants, can reduce cryopreservation-induced damages. In this systematic review, we aimed to evaluate the cryoprotective effect of various vitamins on the quality of cryopreserved-thawed human spermatozoa. Two researchers searched PubMed, ISI, and Scopus databases up to March 2020. All original articles using vitamins in human spermatozoa cryopreservation media were included. We used a standardized form to extract sample size and to determine sample quality, the type and dose of vitamins, and the cryopreservation methods and their effects. We performed a meta-analysis on studies with available data (Mean + SD in cryoprotectant and cryoprotectant + cryoprotectant groups). We also performed a test of between-study heterogeneity, subgroup analysis, and meta-regression. Out of 258 studies, 16 articles were included for the analysis. Our meta-analysis revealed that using vitamins in cryopreservation media could increase motility by 4.60% (95% CI 6.16, 3.05; P = 0.0001), viability by 5.71% (95% CI 9.71, 1.72; P = 0.0001), and DNA integrity by 10.20% (95% CI 12.98, 7.42; P = 0.0001) in cryopreserved-thawed spermatozoa. We found a significant correlation between using vitamins and improved spermatozoa quality; the sperm motility and viability were improved and DNA fragmentation was reduced after thawing by vitamins. However, we could not emphasize on any type or dose of vitamins but we conclude that the anti-oxidative function of vitamins is the main reason for these benefits.

F4-Neuroprostanes: A Role in Sperm Capacitation.

F4-neuroprostanes (F4-NeuroPs), derived from the oxidative metabolization of docosahexaenoic acid (DHA), are considered biomarkers of oxidative stress in neurodegenerative diseases. Neurons and spermatozoa display a high DHA content. NeuroPs might possess biological activities. The aim of this in vitro study was to investigate the biological effects of chemically synthetized 4-F4t-NeuroP and 10-F4t-NeuroP in human sperm. Total progressive sperm motility (p < 0.05) and linearity (p = 0.016), evaluated by a computer-assisted sperm analyzer, were significantly increased in samples incubated with 7 ng F4-NeuroPs compared to non-supplemented controls. Sperm capacitation was tested in rabbit and swim-up-selected human sperm by chlortetracycline fluorescence assay. A higher percentage of capacitated sperm (p < 0.01) was observed in samples incubated in F4-NeuroPs than in the controls. However, the percentage of capacitated sperm was not different in F4-NeuroPs and calcium ionophore treatments at 2 h incubation. The phosphorylated form of AMPKα was detected by immunofluorescence analysis; after 2 h F4-NeuroP incubation, a dotted signal appeared in the entire sperm tail, and in controls, sperm were labeled in the mid-piece. A defined level of seminal F4-NeuroPs (7 ng) showed a biological activity in sperm function; its addition in sperm suspensions stimulated capacitation, increasing the number of sperm able to fertilize.

Respiratory Mitochondrial Efficiency and DNA Oxidation in Human Sperm after In Vitro Myo-Inositol Treatment.

Semen samples are known to contain abnormal amounts of reactive oxygen species (ROS) and oxygen free radicals; therefore, the identification of antioxidant molecules able to counteract the oxidative damage caused by ROS is foresight. Indeed, improving semen quality in terms of motility and reduction in DNA damage, can significantly improve the fertilization potential of sperm in vitro. To this regard, myo-inositol, based on its antioxidant properties, has been reported to be effective in improving sperm quality and motility in oligoasthenozoospermic patients undergoing assisted reproduction techniques when used as a dietary supplementation. Moreover, in vitro treatment demonstrated a direct relationship between myo-inositol, mitochondrial membrane potential and sperm motility. This experimental study aimed to evaluate the effects of myo-inositol (Andrositol-lab) in vitro treatment on sperm motility, capacitation, mitochondrial oxidative phosphorylation and DNA damage. Our results demonstrate that myo-inositol induces a significant increase in sperm motility and in oxygen consumption, the main index of oxidative phosphorylation efficiency and ATP production, both in basal and in in vitro capacitated samples. Moreover, we provide evidence for a significant protective role of myo-inositol against oxidative damage to DNA, thus supporting the in vitro use of myo-inositol in assisted reproductive techniques. Even if further studies are needed to clarify the mechanisms underlying the antioxidant properties of myo-inositol, the present findings significantly extend our knowledge on human male fertility and pave the way to the definition of evidence-based guidelines, aiming to improve the in vitro procedure currently used in ART laboratory for sperm selection.

Protective effect of the superoxide dismutase mimetic MnTBAP during sperm vitrification process.

Sperm cryopreservation is widely used in assisted reproduction and male infertility therapy; however, it induces oxidative stress affecting sperm quality. This work evaluated the effect of the antioxidant MnTBAP during vitrification steps in human spermatozoa. First, the effect of MnTBAP on viability and ROS production was evaluated. Then, the spermatozoa were vitrified in straws with the vitrification, warming and post-warming incubation media separately supplemented with MnTBAP. An untreated control was included. The sperm viability, ROS production, total and progressive motility were evaluated. The results showed that the direct exposure of spermatozoa to MnTBAP significantly decreases the ROS levels in comparison with the untreated control without affecting the viability. The supplementation of the vitrification medium with MnTBAP did not affect the parameters analysed. However, the supplementation of the warming and incubation post-warming media resulted in a decrease in ROS production and maintained viability and motility for 4 hr after warming with concentrations up to 100 µM of MnTBAP. Higher concentrations of MnTBAP caused a decrease in total motility. In conclusion, the use of MnTBAP during the warming or post-warming incubation media has beneficial effect decreasing ROS levels and maintaining the viability and motility during the vitrification procedure.

Changes in the Level of DNA Fragmentation in Sperm Cells detected by Acridine Orange Test in Men with Sub/infertility Treated with Nutritional Supplement PAPA.

BACKGROUND: When diagnosing and treating male infertility it is important to determine whether there are defects in the maturation process of sperm nuclei. Using nutritional supplements can improve the morphological and physiological condition of the spermatozoa. In recent years there has been an increase in the usage of supplements with different compositions which strives to determine the best combination and avoid side effects. AIM: To study the effect of PAPA nutritional supplement on the levels of DNA fragmentation of sperm cells tested with acridine orange test (single stranded DNA against double stranded DNA) in men with sub/infertility. MATERIALS AND METHODS: 48 men with confirmed sub/infertility underwent treatment for three months with nutritional supplement PAPA containing 9 micronutrients. The differences in levels of DNA fragmentation were determined with acridine orange test, which was conducted before and after the treatment. RESULTS: The results were statistically significant (p<0.001) showing an increase in the number of green spermatozoa (normal DNA), and a decrease of damaged ones (orange and red). After treatment the level of sperm DNA fragmentation decreased by 10.2%. CONCLUSION: Men with confirmed sub/infertility that took nutritional supplement PAPA for three moths showed a decrease in DNA fragmentation levels of 10.2% determined by AO test which implies an improvement of male fertility levels.

Antioxidant effects of penicillamine against in vitro-induced oxidative stress in human spermatozoa.

Oxidative stress contributes importantly to the aetiology of male infertility, impairing sperm function. The protective effect of antioxidants on seminal parameters has been established, and the antioxidant penicillamine has shown beneficial effects; however, its protective effect on human spermatozoa exposed to oxidative stress has not been reported. The objective of this work was to evaluate the effect of penicillamine on human spermatozoa exposed in vitro to oxidative stress. First, the effect of penicillamine on spermatozoa from normozoospermic donors was evaluated. Then, the effect of penicillamine on spermatozoa exposed to oxidative stress induced separately by ionomycin and hydrogen peroxide (H2 O2 ) was analysed. An untreated control and a control treated only with the oxidative stress inducer were included. Reactive oxygen species (ROS) levels, viability, mitochondrial membrane potential (MMP) and motility were analysed. The results showed that penicillamine, added to the incubation medium, decreased the ROS levels induced by ionomycin and H2 O2 , and this effect was associated with better preservation of MMP, motility, and ATP levels. These results highlight the potential advantages of penicillamine supplementation of sperm culture medium, especially for semen samples with high ROS levels and also in circumstances where laboratory handling can cause an increase in ROS production.

Anethole compromises human sperm function by affecting the sperm intracellular calcium concentration and tyrosine phosphorylation.

Anethole is a natural anisole derivative that has been widely used in food and daily chemical industries, agricultural applications and the traditional medicine. It is closely related to aspects of daily life, and humans can easily be exposed to it. Although the reproductive toxicity of anethole was shown in the rat, its effect on human reproduction remains unknown. In this study, we examined the effect of anethole on human sperm in vitro. Different anethole doses (0.1, 1, 10, and 100 µM) were applied to ejaculated human sperm. Fertilization-essential functions, as well as the intracellular calcium concentration ([Ca2+]i) and tyrosine phosphorylation, two vital factors for regulating sperm function, were measured. The results indicated that 10 and 100 µM anethole significantly reduced the motility, hyperactivation, and penetration ability of human sperm (P < 0.05) and inhibited the increase in human sperm functions induced by progesterone, a hormone essential for sperm function activation. Additionally, 10 and 100 µM anethole decreased both basal and progesterone-increased tyrosine phosphorylation, [Ca2+]i, and the current of CATSPER, a cation channel of sperm predominant for Ca2+ influx. These results suggest that anethole inhibits human sperm functions by reducing sperm [Ca2+]i through CATSPER and suppressing tyrosine phosphorylation in vitro, raising the fact that the caution is needed when overtaking anethole.