GM-CSF (granulocyte-macrophage colony-stimulating factor) treatment improves sperm parameters in men with oligoasthenoteratospermia via PI3K/AKT pathway.

Poor sperm quality in oligoasthenoteratospermia patients negatively affects assisted reproductive technology outcomes. Therefore, the development of sperm media is necessary to improve sperm parameters. This study investigated the effect of GM-CSF via PI3K/AKT pathway on sperm quality in OAT patients. Semen samples were collected from 20 OAT patients, and each sample was divided into two groups: Experiment and Control. In the experimental group, the samples were incubated with medium containing GM-CSF, and control samples were incubated without GM-CSF. Sperm parameters, mitochondrial membrane potential, acrosome reaction and DFI were studied; in addition, gene expression of PI3KR1, PI3KCA, GLUT1, GLUT3 and AKT1 was analysed, evaluation of PAKT/TAKT, and expression of GLUT 1, 3 was examined; subsequent fertilization rate and embryo quality were assessed. Our data showed that GM-CSF supplementation could significantly increase motility, mitochondrial activity, gene expression of PI3KCA, AKT1, the protein level of PAKT/TAKT and expression of GLUT 1, 3 while it decreases DNA fragmentation. The fertilization rate and embryo quality significantly improved in the treatment group. LY294002 had adverse effects on sperm motility and the PAKT/TAKT ratio. GM-CSF can improve in vitro sperm quality and could be a suitable supplement to sperm media for OAT patients.

Oligoasthenoteratospermia and sperm tail bending in PPP4C-deficient mice.

Protein phosphatase 4 (PPP4) is a protein phosphatase that, although highly expressed in the testis, currently has an unclear physiological role in this tissue. Here, we show that deletion of PPP4 catalytic subunit gene Ppp4c in the mouse causes male-specific infertility. Loss of PPP4C, when assessed by light microscopy, did not obviously affect many aspects of the morphology of spermatogenesis, including acrosome formation, nuclear condensation and elongation, mitochondrial sheaths arrangement and '9 + 2' flagellar structure assembly. However, the PPP4C mutant had sperm tail bending defects (head-bent-back), low sperm count, poor sperm motility and had cytoplasmic remnants attached to the middle piece of the tail. The cytoplasmic remnants were further investigated by transmission electron microscopy to reveal that a defect in cytoplasm removal appeared to play a significant role in the observed spermiogenesis failure and resulting male infertility. A lack of PPP4 during spermatogenesis causes defects that are reminiscent of oligoasthenoteratospermia (OAT), which is a common cause of male infertility in humans. Like the lack of functional PPP4 in the mouse model, OAT is characterized by abnormal sperm morphology, low sperm count and poor sperm motility. Although the causes of OAT are probably heterogeneous, including mutation of various genes and environmentally induced defects, the detailed molecular mechanism(s) has remained unclear. Our discovery that the PPP4C-deficient mouse model shares features with human OAT might offer a useful model for further studies of this currently poorly understood disorder.

Does myoinositol supplement improve sperm parameters and DNA integrity in patients with oligoasthenoteratozoospermia after the freezing-thawing process?

Sperm cryopreservation is a routine method in andrology and IVF laboratory. However, the sperm quality and its fertilizing capacity have been decreased during this process. The purpose of this experiment was to determine the role of myoinositol as a supplement in amelioration of total and progressive sperm motility, DNA fragmentation, total antioxidant capacity (TAC), reactive oxygen species (ROS), and lipid peroxidation after the freezing-thawing process on patients with oligoasthenoteratozoospermia (OAT) syndrome. Semen samples obtained from 40 patients were divided into two aliquots and freezed with simple and 2 mg/mL myoinositol (MYO) supplemented freezing media. All samples were thawed and assessed after one month. Semen parameters were analyzed in terms of the motility by CASA, the level of total ROS by fluorimetry, TAC and MDA by colorimetric assay and finally DNA fragmentation by TUNEL assay. Our results clearly showed that MYO could improve total (37.46 vs. 12.91, p < 0.001) and progressive motility (21.92 vs. 6.49, p < 0.001) in experimental group compared to control group. A higher TAC level was observed in the MYO treated group in comparison to control group (1.11 vs. 0.91, p = 0.05). While MYO supplementation could not be effective on ROS level, it reduced DNA fragmentation of sperm after freeze-thaw process (p = 0.01). Therefore, MYO could be a good supplement for sperm freezing to reduce the detrimental effects of freezing process especially on DNA integrity, which is an important factor in the success of ART, in OAT suffered patients.

Autologous sperm usage after cryopreservation-the crucial impact of patients' characteristics.

BACKGROUND: The wide implementation of sperm freezing presents a growing burden on sperm banks. OBJECTIVES: To evaluate sperm freezing and usage patterns over 30 years, according to demographic parameters of age at first cryopreservation and number of children, and indication for cryopreservation. MATERIAL AND METHODS: This retrospective, population based, cohort study included all sperm cryopreservation cases performed at a tertiary referral center from October 1993 to December 2021, among patients aged 18 years and older. First, we determined the interval between first sperm sample and use. Then, we examined sperm usage separately for: (1) age, comparing patients grouped into 5-year age cohorts; (2) paternal status according to number of children; (3) indication, comparison among seven indications. Secondary analysis included correlations between main age groups and paternal status versus the four most common indications found. RESULTS: During the study period 1490 men who cryopreserved sperm met the inclusion criteria. Average age at cryopreservation of the first sample was 33.9 ± 8.1 years. Average age at first sperm use was 37 ± 8.5 years. Cumulative sperm usage was 38.7% after 17.8 years. Increasing age was associated with progressive increase in sperm usage rate and shorter preservation period. Use significantly decreased with increasing number of children. Examination of seven reasons for sperm cryopreservation found the highest cumulative sperm usage was related to azoospermia (67.7%), followed by functional cryopreservation (39.3%), oligoasthenoteratospermia (27.3%), other (26.5%), patient's request (24%), cancer (19%), and systemic disease (7.2%). Secondary analysis defined specific usage patterns mainly related to age and indication, with less of an effect based on the number of children. DISCUSSION AND CONCLUSION: After decades of cryopreservation, the paradigm of sperm cryopreservation is mostly related to cancer patients. This should be reevaluated and evolve to include broader patient-targeted factors and perceptions.

The Effects of Varicocelectomy on Sperm DNA Fragmentation and Conventional Semen Parameters in Men with Severe Oligoasthenoteratozoospermia: A Prospective Study.

BACKGROUND: The dilation and torsion of testicular veins in the plexus pampiniformis causes Varicocele, which is a surgically repairable cause of male infertility. This study assessed the impact of varicocelectomy on semen characteristics, total motile sperm count (TMSC) and sperm DNA integrity in patients with severe oligoasthenoteratozoospermia (OAT). MATERIALS AND METHODS: In this prospective study, semen samples of 360 men with severe OAT who underwent varicocelectomy according to World Health Organization (WHO) criteria 2021 were studied (pre-operatively and at 6, 12, and 18 months post-operatively). RESULTS: The average age of our patients was 38.5 years. The mean spermatozoa concentration was found to be 1.60 ± 0.83 million/ml pre-operatively, while the mean post-operative concentration was 5.17 ± 1.23 million/ml at 6 months, 8.32 ± 0.98 million/ml at 12 months, and 13.51 ± 1.48 million/ml at 18 months (P<0.0001). The mean percentage of A+B motile spermatozoa was 2.92 ± 1.17% pre-operatively, 6.10 ± 1.51% at six months, 9.58 ± 1.49% at 12 months and 13.92 ± 1.88% at 18 months postoperatively (P<0.0001). The mean Modified David's morphology score was 3.80 ± 1.43% pre-operatively, 5.95 ± 1.23% at 6 months, 7.94 ± 1.18% at 12 months, and 10.82 ± 1.91% at 18 months post-operatively (P<0.0001). The mean of total motile sperm count (TMSC) was statistically improved after varicocelectomy (P<0.001). The mean of DNA fragmentation index (DFI) of the spermatozoa was 31.40 ± 0.52% pre-operatively, and post-operatively at 28.20 ± 0.32% at 6 months, 25.90 ± 0.31% at 12 months and 20.50 ± 0.40% at 18 months (P<0.001). CONCLUSION: Varicocelectomy was associated with significant improvement of sperm parameters and DNA fragmentation resulting in significant improvement of spermatogenesis quality. We believe that universalization in the routinely used sperm dispersion chromatin (SDC) test could be beneficial in the treatment of infertility.

Correlation between seminal plasma biochemical markers and semen parameters in idiopathic oligoasthenoteratospermia: identification of biomarkers for L-carnitine therapy.

Background: L-carnitine therapy for idiopathic sperm abnormalities exhibits variable effectiveness, and currently, there are no established criteria to predict patient response. This study investigated correlations between seminal plasma markers and semen parameters to identify biomarkers that can guide indications for L-carnitine therapy indications in patients with idiopathic sperm abnormalities. Methods: A retrospective review was conducted on 223 male patients with idiopathic oligoasthenoteratospermia, who sought medical attention at our clinic between January 2020 and October 2022. These patients underwent a pretreatment seminal plasma biochemical analysis, followed by a three-month continuous L-carnitine treatment. The correlation between seminal plasma biochemical parameters and pretreatment semen parameters was analyzed. Semen quality was compared between cases with normal and abnormal seminal plasma biochemical parameters, both pretreatment and posttreatment. The correlation between the changes in semen parameters after treatment and seminal plasma biochemical parameters were investigated. Results: Correlation analyses revealed significant associations between all pretreatment semen parameters and seminal plasma biochemical markers, except for liquefying time and the ratio of normal morphology. Subgroup analysis, stratified by seminal fructose, zinc, citric acid, and neutral glycosidase levels, demonstrated that abnormal groups exhibited significantly different levels of semen parameters compared with the normal groups. The changing difference and changing ratio in the ratio of forward motile sperm showed a negative correlation with seminal fructose levels (r=-0.165 and -0.144). The changing difference in semen volume was negatively correlated with the level of seminal neutral glycosidase (r=-0.158). The changing ratio in semen volume, sperm concentration, total sperm count, and count of forward motile sperm all exhibited negative correlations with the levels of seminal neutral glycosidase (range from -0.178 to -0.224). Conclusion: Seminal plasma biochemical markers, particularly fructose and neutral glycosidase, may serve as valuable indicators for determining the eligibility of patients with idiopathic sperm abnormalities for L-carnitine therapy.

The Association Between the Levels of Oxidative Stress Indicators (MDA, SOD, and GSH) in Seminal Plasma and the Risk of Idiopathic Oligo-asthenotera-tozoospermia: Does Cu or Se Level Alter the Association?

Epidemiological studies on the associations between the levels of oxidative stress (OS) indicators (MDA, SOD, and GSH) in seminal plasma and the risk of idiopathic oligo-asthenotera-tozoospermia (OAT) are still inconsistent. Additionally, whether the associations can be altered by the status of essential trace elements is still unknown. To investigate the relationship between MDA, SOD, and GSH levels in seminal plasma and the risk of idiopathic OAT, and further to examine whether levels of iron (Fe), copper (Cu), and selenium (Se) in seminal plasma can alter the associations. A total of 148 subjects (75 idiopathic OAT cases and 73 controls) were included in this study. Seminal plasma samples from all the participants were measured for levels of MDA, SOD, GSH, Fe, Cu, and Se. Unconditional logistic regression models were used to examine the associations between three oxidative stress indicators and the risk of idiopathic OAT. Bayesian kernel machine regression was performed to determine the joint effects of levels of three OS indicators on the risk of idiopathic OAT. Subgroup analyses were performed to explore whether the above associations can be different when Fe, Cu, and Se were in different levels. The level of MDA in seminal plasma was positively associated with the risk of idiopathic OAT, with adjusted odds ratio (OR) and 95% confidence interval (CI) of 2.38 (1.17, 4.83), and SOD and GSH levels were not associated with the risk of idiopathic OAT. In BKMR analyses, we found a significant positive association between the mixture of MDA, SOD, and GSH levels and the risk of idiopathic OAT at concentrations below the 65th percentile, while a negative association at concentrations above it. In subgroup analysis, a positive association was observed between MDA levels in seminal plasma and the risk of idiopathic OAT in the high-Cu group (adjusted OR = 3.66, 95%CI = 1.16, 11.57), while no significant association was found in the low-Cu group (adjusted OR = 1.43, 95%CI = 0.44, 4.58). Additionally, a negative association was found between GSH levels in seminal plasma and the risk of idiopathic OAT in the high-Se group (adjusted OR = 0.34, 95%CI = 0.11, 0.99), while no significant association was observed in the low-Se group (adjusted OR = 1.96, 95%CI = 0.46, 8.27). The levels of MDA, SOD, and GSH in seminal plasma were associated with the risk of idiopathic OAT, and the levels of Cu and Se in seminal plasma may alter the associations.

The Effect of Selenium Therapy on Semen Parameters, Antioxidant Capacity, and Sperm DNA Fragmentation in Men with Idiopathic Oligoasthenoteratospermia.

Idiopathic male infertility (IMI) remains challenging as the etiology of semen abnormalities is still unidentified. Sperm DNA fragmentation (SDF) has been suggested as a potential mechanism. Oral antioxidants including selenium have been tried for IMI with variable results. This study was undertaken to explore the effect of selenium therapy on semen parameters, antioxidant capacity, and SDF in infertile patients with idiopathic oligoasthenoteratospermia (OAT). Sixty-five infertile men with idiopathic OAT and fifty fertile controls were included in this prospective clinical study. Patients received selenium (200 µg/day) orally for 6 months. Seminal fluid parameters (WHO 5th criteria), total antioxidant capacity (TAC), catalase (CAT), and seminal SDF levels were assessed for all participants at the start of the study and after 6 months. Sperm concentration (P < 0.001), progressive motility (P < 0.001), and total motility (P < 0.01) significantly increased in patients after selenium therapy. Seminal TAC and CAT increased in patients post-therapy as compared to baseline values (P < 0.01). SDF levels significantly decreased (P < 0.001) in patients following selenium treatment in comparison to baseline values. SDF levels also correlated negatively with sperm progressive motility (r = - 0.44, P = 0.003) and total motility (r = - 0.48, P = 0.001). In conclusion, selenium therapy (200 µg/day) for 6 months increases sperm concentration, motility, seminal antioxidant capacity, and reduces SDF in patients with idiopathic OAT. Thus, selenium could be a promising therapy for men with IMI and may boost their fertility and fertility treatment outcomes.

The association of a probiotic with a prebiotic (Flortec, Bracco) to improve the quality/quantity of spermatozoa in infertile patients with idiopathic oligoasthenoteratospermia: a pilot study.

The hypothesis that the assumption of a probiotic associated with a prebiotic (Flortec, Bracco; one sachet contains: Lactobacillus paracasei B21060 5 × 109 cells + arabinogalctan 1243 mg + oligo-fructosaccharides 700 mg + l-glutamine 500 mg) could improve the quality and quantity of spermatozoa in idiopathic oligoasthenoteratospermia (iAOT) patients to a larger extent than a control substance (starch) was tested. Forty-one patients with no chromosomal aberrations were randomized into two groups: 20 received Flortec, one sachet per day for 6 months, whereas 21 received the control substance. The following data were collected: clinical history, scrotal Doppler scans, Y microdeletion, karyotype and cystic fibrosis screens, follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), testosterone (T), and prolactin (PRL) levels, and two semen analyses. Both the Flortec and the starch groups underwent two semen analyses and one FSH, LH, T, E2, and PRL measurement in the blood, at the beginning of the study, and after 6 months. The comparisons were carried out using a non-parametric (Wilcoxon signed rank) test. The side effects were assessed and compared using the chi-squared test. Group 1 (Flortec) had their sperm count improved: volume of the ejaculate (median from 2.4 to 3.1 mL; p < 0.01), sperm concentration (median: from 15.2 × 106 /mL to 28.3 × 106 /39 mL; p < 0.01), progressive motility (median: from 16.2 to 42.0%; p < 0.01), and the percentage of typical forms (median: from 7 to 16.3%; p < 0.01); in addition, their FSH, LH, and T levels improved (p < 0.01), while those of E2 and PRL did not. Group 2 (control substance) had no modification in 42 of the parameters studied. There were no side effects in either group. These data showed that Flortec constitutes a safe therapy for improving the volume of the ejaculate and the quality/quantity of spermatozoa in iOAT patients.