Normozoospermic Semen Samples Research Articles

Effects of astaxanthin supplementation during vitrification and liquid nitrogen vapor freezing on motility, morphology, survival, reactive oxygen species (ROS), and DNA fragmentation of post-cryopreserved human sperm.

To investigate the effect of astaxanthin supplementation in cryopreservation media on post-thawed sperm motility, viability, morphology, reactive oxygen species (ROS), and DNA fragmentation in two cryopreservation techniques using vitrification and liquid nitrogen vapor freezing. Thirty normozoospermic semen samples were used in the study. Post-prepared semen samples were divided into 1) non-cryopreserved control, 2) and 3) vitrified without (V) and with astaxanthin 0.5 µM (V+ATX), 4) and 5) frozen in liquid nitrogen vapor without (L) and with astaxanthin 0.5 µM (L+ATX). Cryopreservation using vitrification and liquid nitrogen vapor freezing significantly decreased sperm motility and viability and increased ROS levels. However, no changes were seen in sperm morphology or DNA fragmentation. The addition of astaxanthin in cryopreservation media significantly increased post-thawed motility in both vitrification (77.6±8.9% vs. 69.0±9.5% in V+ATX and V) and vapor freezing (57.0±13.3% vs. 47.7±14.6% in L+ATX and L); it significantly increased sperm viability in vitrification (75.0±11.9% vs. 65.9±11.1% in V+ATX and V), and significantly decreased ROS level in both vitrification (4.7 (2.6-8.3) RLU/sec/106 vs. 10.6 (9.4-16.0) RLU/sec/106 in V+ATX and V) and vapor freezing (4.6 (3.3-10.5) RLU/sec/106 vs. 10.3 (7.9-18.6) RLU/ sec/106 in L+ATX and L). Astaxanthin supplementation in cryopreservation media did not affect sperm morphology or DNA fragmentation. Astaxanthin supplementation improved post-cryopreserved sperm motility, decreased ROS levels in both vitrification and liquid nitrogen vapor freezing and improved sperm viability only in the vitrification technique.

P-271 Deep sequencing of spermatozoal accessible chromatin reveals complex arrangements underlying spermatozoal chromatin packaging and fertilization

Abstract Study question Could structural aspects of sperm DNA packaging contribute to fertilization and early embryo development? Summary answer Structural aspects of sperm DNA include complex patterns of repetitive-DNA and developmental gene packaging that could delineate a paternal impact on fertilization and early development. What is known already Genetics, epigenetics and transcriptomics of ejaculate human spermatozoa could have important effects on the establishment of embryonic chromatin architecture that may impact early fetal development. Spermatozoa are transcriptionally silent; however, a small fraction (∼5%-15%) of their chromatin escapes repackaging by protamines and remains in a more open nucleosomal conformation that may be poised for transcription. The sensitivity of this fraction to progressive salt/detergent disruption is likely assayed in sperm chromatin dispersion (SCD) or halo tests that are commonly used in assessments of sperm DNA fragmentation. Study design, size, duration Eleven healthy male donors and 9 women undergoing IVF treatment agreed to participate in this study. Eleven normozoospermic semen samples were donated by male donors, aged 25-39 years, who abstained from sexual activity for 3 days. Seven MII oocytes and 5 pronuclear zygotes were donated from 5 and 4 women, respectively, undergoing IVF treatment. The samples were collected within a time frame of 6 months. Female donors provided either MII oocytes or pronuclear zygotes. Participants/materials, setting, methods Sperm chromatin was processed by differential salt extraction tailored with endonuclease or micrococcal nuclease treatment and RNA isolated using established protocols. Oocytes and pronuclear zygotes were analyzed at the single cell level. DNA library construction employed the NEBNext Ultra approach (NEB) and RNA libraries the Ovation Single Cell (NuGEN) for Illumina. Bioinformatics analysis included FastQC, HISAT2, StringTie, GREAT, edgeR and other source-software. Public datasets were employed to further increase the statistical power. Main results and the role of chance The aim of our approach was to dissect the accessible sperm chromatin fraction, identifying regions of great importance that could contribute towards the formation of the zygote and/or early embryonic development. Differing extraction methods revealed complementary accessible and resistant fractions in sperm, with accessible regions being significantly enriched for chromatin important in very early embryonic development. These chromatin fractions contained various genomic features including promoter sequences, CpG-islands and CTCF-binding sites that could attract transcription binding factors in the paternally-derived chromatin. Accessible sperm chromatin also contains high levels of specific DNA repeat classes of retroviral origin, known to impact early gene expression and gene expression programs. Accessible sperm chromatin was strongly associated with developmental and regulatory signatures with Bonferroni P values ranging between 2.07x10-4 and 1.03x10-20, potentially revealing a role for paternal chromatin in the initial start-up of early zygotic development. We further investigated paternally- and maternally- derived transcripts and identified mono-allelically transcribed genes present in the zygotes, that were entirely absent from all seven oocyte transcriptomes, suggesting a paternal origin, and potentially important roles following oocyte fertilization. Limitations, reasons for caution Due to the restraints of obtaining human material, the study relied on a relatively small sample size. However, sequencing results showed quite homogeneous clustering in both DNA/RNA samples, with at least 5 biological replicates from at least 4 donors per group, potentially reducing the impact of low sample size. Wider implications of the findings A better understanding of differentially accessible sperm chromatin and residual RNA, could help support the discovery of biomarkers that can be used to select more competent spermatozoa for IVF cycles, resulting in higher success rates in couples undergoing ART. Trial registration number not applicable

Investigation of the effects of thymoquinone (TQ) and polyvinylpyrrolidone (PVP) on the motility, viability, and chromatin status of sperm in normozoospermic semen samples

Objective: Adverse effects of polyvinylpyrrolidone (PVP) on sperm membrane and chromatin have been proven in many studies. Among the natural products proposed as an alternative for PVP, thymoquinone (TQ)-a major constituent of Nigella sativa plant- has been suggested as a potential natural spermostasis. Therefore, this study aimed to compare the effects of TQ with PVP for sperms motility, survival, DNA denaturation, and DNA fragmentation in normozoospermic men (men with a normal or healthy sperm profile). Methods: An experimental trial was carried out on 30 normozoospermic men of the Andrology Unit of (Shahid Beheshti Hospital, Isfahan, Iran). Each washed semen samples were divided into four fractions and was randomly treated with TQ (50 μg/ml), %5 PVP, and %10 PVP (M/V) which was compared to untreated fraction (control). Results: There was a significant difference between the four groups in terms of motility, viability, DNA denaturation, and fragmentation (P <0.05). TQ caused sperm immobility, while 5% PVP and 10% PVP decreased (98 and 99%, respectively) sperm motility compared to control. TQ did not affect sperm viability compared to the control group, but PVP decreased it. Besides, TQ did not affect DNA denaturation and fragmentation, but PVP increased it. Conclusion: TQ could be used as an alternative natural spermostasis with less adverse effects rather than PVP which causes more efficient immobilization and isolation of individual sperm cells.

P-067 Characterization of seminal microbiota in patients with asthenozoospermic and normozoospermic using next-generation sequencing

Abstract Study question How are the semen microbiota compositions of patients with asthenozoospermic and normozoospermic different, According to data from 16S rRNA gene-specific Next-generation sequencing (NGS)? Summary answer Large numbers of microbes can be present in seminal fluid, and there are differences in the semen microbiota between asthenozoospermic and normozoospermic semen samples. What is known already Male factor is attributable in up to 50% of cases of infertility. Most studies on normal and abnormal sperm microbiota are based on 16S rRNA gene-specific next-generation sequencing (NGS). Microbiome analysis based on subunit 16S rRNA sequencing is a fast tool that can enable the identification of all the pathogenic microorganisms associated with semen in clinical pathology. Studies on the impact of semen micro-biomes in asthenozoospermic and normozoospermic samples could improve the results of assisted reproductive technologies. The major bacterial diversity in asthenozoospermic and normozoospermic samples belongs to the genera Lactobacillus, Prevotella, Staphylococcus, and Anaerococcus. Study design, size, duration Two eighty patients with their own semen samples were included in the study. The study population consists of patients attending to the “Pacific IVF Center” Pacific Medical University and Hospital (Udaipur, India) from November 2020 to October 2022. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 127) was asthenozoospermic, and Group 2 (n = 153) had normozoospermic semen samples, Patients were aged between (20-45 years). Ethical approval was obtained from the institute. Participants/materials, setting, methods Genomic DNA was extracted from samples using commercially available (Qiagen, DNeasy Power Soil kit). The amount of extracted DNA was measured using a Nanodrop spectrophotometer (Thermo Scientific). The microbiota of semen was analyzed using 16S ribosomal RNA (rRNA) gene amplification (MinION) Oxford Nanopore Ltd. Bioinformatics analysis was performed using QIIME2 and Microbiome Analyst packages. Alpha, beta diversity, and taxonomic characterization were compared for the seminal microbiome in asthenozoospermic and normozoospermic semen samples. Main results and the role of chance Different bacterial communities were detected when asthenozoospermic and normozoospermic semen samples were analyzed. In patients with asthenozoospermic parameters, a higher alpha diversity index tendency was found. In normozoospermic semen samples, a p-value of bacterial diversity was non-significant (p = 0.14 for the Shannon index), and significant differences in asthenozoospermic semen samples (p = 0.01 for the Shannon index). In the beta diversity analysis, no significant differences were observed between both groups Relative abundance analysis identified the bacterial communities, Lactobacillus, Streptococcus, and Ureaplasma in normozoospermic semen samples, and Anaerococcus and Gardnerella in asthenozoospermic samples, Comparison of asthenozoospermic and normozoospermic samples with alpha diversity (p = 0.06 for the Shannon index and p = 0.08 for the Simpson index) and beta diversity (p < 0.001) showed significant differences. Relative abundance in normozoospermic semen samples identified the bacterial communities, Lactobacillus (p = 3.70E-4), Prevotella (p = 8.11E-4), and in asthenozoospermic semen samples Anaerococcus (p = 0.004) and Gardnerella (p = 0.004). Limitations, reasons for caution The limited sample size is the primary constraint of this study. Larger studies with a larger sample size are needed to confirm the distinct microbiome patterns identified in asthenozoospermic and normozoospermic samples in connection to male infertility. Wider implications of the findings Further research could determine the detection rate of the described bacterial diversity in semen with other pathologies. Determining the relationship between sperm microbiota characteristics and infertility could enable the development of new algorithms for treating patients with reproductive disorders, An abnormal seminal microbiome appears to be strongly correlated with infertility. Trial registration number N/A

P-096 Prediction of fertilization success by voltage-dependent intracellular Ca2+ dynamics in non-normozoospermic human semen samples undergoing in vitro fertilization: preliminary results

Abstract Study question Does the change in intracellular calcium levels evoked by artificial membrane depolarizations predict fertilization success in a non-normozoospermic semen sample undergoing assisted reproductive techniques? Summary answer The increase in the intracellular calcium after artificial plasma membrane depolarization correlates negatively with fertilization rates in conventional IVF but not with ICSI What is known already Sperm capacitation-related features, like the regulation of the intracellular calcium concentration ([Ca2+]i) and the hyperpolarization of the plasma membrane potential (Vm), have been proposed as predictors of success during artificial reproduction techniques (ART). Intriguingly, upon Vm depolarization, sperm exhibit a rapid and transitory increase in the [Ca2+]i controlled, likely, by the activation of the sperm-specific calcium channel CatSper. The regulation of CatSper activity is essential for sperm function and is crucial for fertilization. Therefore we wondered if the Vm depolarization-dependent rise in the [Ca2+]i can be used to predict fertilization success rates in non-normozoospermic semen samples of men undergoing ART. Study design, size, duration This prospective study employed unidentified semen samples from 29 non-normozoospermic men (37.8 ± 5.6 years old) undergoing fertility treatment from March 2022 to the present at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and procedures with less than 3 oocytes were excluded. Ten normozoospermic semen samples were analyzed as a control. The depolarization-dependent [Ca2+]i changes were evaluated by flow cytometry under four different Vm conditions: resting (variable among samples), –64, –46, and –32 mV. Participants/materials, setting, methods Semen samples were obtained on the same day as oocyte retrieval. Sperm cells were washed and incubated in capacitating HTF media for further use in conventional IVF or ICSI procedures. When available, the surplus cells were stained with vitality, Ca2+, and Vm-sensitive fluorescent dyes. Fluorescence changes were evaluated by AccuriC6 flow cytometer. The kinetical analysis of the transitory [Ca2+]i increase as well as the statistical comparisons were performed using custom-made R programming language code. Main results and the role of chance Analysis of resting Vm showed that normozoospermic semen samples presented more depolarized Vm than non-normozoospermic controls (–75.7 ± 6.9 vs –67.3 ± 10.5 mV, respectively, p = 0.02). The former group of semen samples was either used for conventional IVF (n = 11) or ICSI (n = 14). The semen samples in these last categories showed a tendency to present a more depolarized Vm than non-normozoospermic samples (–69.9 ± 8.8 and –66.6 ± 11.3 mV, respectively). No differences were seen in the resting Ca2+ levels. We then induced a Vm hyperpolarization by adding the K+ ionophore, valinomycin followed by increasing concentrations of extracellular K+. Each depolarizing stimulus evoked a fast and transitory increase in the [Ca2+]i. We then calculated seven different kinetic parameters of such responses: basal Ca2+ level, increase and decrease velocity, maximum Ca2+ reached, response durability, and area under the curve (AUC). The basal Ca2+ level reached after depolarizing stimuli correlated negatively (R = –0.64, p = 0.03) with the conventional IVF fertilization ratio (number of zygotes/total number of mature oocytes) when the Vm was clamped either to –64 and –46 mV. A negative and significant correlation (R = –0.76, p = 0.006) was seen for the AUC analysis at –64 mV. No relevant correlations were found in the ICSI samples. Limitations, reasons for caution This is an in vitro study, and caution must be taken when extrapolating these results in vivo. The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples (in progress). Wider implications of the findings Further research into the molecular regulation of the CatSper channel could offer promising alternatives for clinicians to appropriately choose between IVF and ICSI for each couple. This protocol is fast and simple to perform in a laboratory equipped with a flow cytometer or any fluorescence-based reader. Trial registration number NA

P-046 Sperm mitochondrial DNA copy number and mitochondrial function in normozoospermic men may predict fertilization rates after intracytoplasmic sperm injection

Abstract Study question Do sperm mitochondrial DNA copy number (mtDNAcn) and mitochondrial function correlate with fertilization rates after intracytoplasmic sperm injection (ICSI)? Summary answer We reveal a negative correlation between sperm mtDNAcn and fertilization rates after ICSI, while higher mitochondrial function corresponded to an increase in sperm fertilization capacity. What is known already Over 40% of male-factor infertility is of unknown origin (unexplained and idiopathic in nature). As current diagnoses remain largely descriptive, the identification of markers predicting the fertilization capacity of sperm will hold the key towards improved treatment. Mitochondria are fundamental for supporting spermatogenesis and fertilization. A recent study showed that altered levels of sperm mitochondrial proteins led to oocyte-activation deficiency, resulting in fertilization failure after ICSI. Poor semen parameters have also been linked to high mtDNAcn, however the relationship between mtDNAcn in sperm and oocyte-activation capacity remains unexplored. Further characterization may uncover prognostic markers associated with sperm-related fertilization failure. Study design, size, duration Twenty normozoospermic semen samples were included in the study. These were obtained from oocyte donation ICSI cycles, performed between March 2021 and November 2022. Both fresh and cryopreserved sperm samples were analysed. Five million sperm were collected from the fresh ejaculate for genomic DNA (gDNA) isolation and mtDNAcn quantification, while the remainder of the sample was used for mitochondrial functional analysis. These analyses were also performed on washed samples, cryopreserved after swim-up. Participants/materials, setting, methods Mean paternal age and BMI were 40 ± 5.8 years and 25 ± 2.3 kg/m2, respectively. We evaluated mtDNAcn using qPCR, by assessing the ratio of mitochondrial-to-nuclear genes (NT-ND4/B2M). For our functional studies, we quantified mitochondrial membrane potential (MMP) in individual spermatozoa by JC-1 staining and flow cytometry. Flow cytometry analysis was performed on ≥ 10 million sperm treated with a mitochondrial uncoupler for 15 minutes (1 µM trifluoromethoxy-carbonylcyanide-phenylhydrazone, FCCP) and controls. Spearman correlation was applied. Main results and the role of chance We observed variable fertilization rates in our cohort (69.0 ± 24.9%). One sperm sample resulted in total fertilization failure (TFF) after ICSI and was analyzed independently. In fresh samples, mtDNAcn (5.8 AU ± 2.61) correlated negatively with progressive motility (rs = -0.65) and fertilization rate (rs = -0.32). Notably, the sample resulting in TFF presented with the highest mtDNAcn value (13.9 AU ± 2.77). Flow cytometry analysis revealed two distinct sperm populations, corresponding to motile and immotile sperm. While immotile sperm presented with low or undetectable JC-1 aggregates (red signal), motile sperm showed different levels of MMP (ratio INTred/INTgreen) (2.3 AU ± 1.83). In motile sperm, our results revealed a positive correlation between MMP and fertilization rate (rs = 0.59). As with raw semen, we observed a negative association between MMP and mtDNAcn in motile sperm (rs = -0.53). Limitations, reasons for caution Cryopreservation is known to affect sperm motility and active mitochondria in sperm, however its effect on MMP intensity remains to be established. Our limited sample size warrants careful interpretation. Wider implications of the findings Our findings suggest a negative correlation between sperm mtDNAcn and fertilization rates after ICSI, while fertilization capacity improved with increasing MMP. Sperm mtDNAcn and MMP may have prognostic value for male fertility and ICSI outcomes. Further validation may deliver clinically useful markers, aiding infertility diagnosis, treatment and counselling. Trial registration number NA

P-094 Dynamics of progesterone-induced intracellular Ca2+ changes in sperm predicts zygote formation rate by IVF: Preliminary results

Abstract Study question Can the overall intracellular Ca2+ dynamics evoked by progesterone stimulation predict the fertilizing potential of a non-normozoospermic semen sample under fertility treatment? Summary answer Some kinetic parameters of the transient intracellular Ca2+ increase evoked by progesterone stimulation correlate with fertilization rates in conventional IVF but not with ICSI. What is known already Stimulation with female hormone progesterone induces the activation of the sperm-specific ion channel CatSper which in turn produces a fast increase in the intracellular Ca2+ concentration ([Ca2+]i). After reaching a maximum [Ca2+]i, different Ca2+ clearance mechanisms become active producing a transient followed by a plateau. We investigated whether the [Ca2+]i dynamics upon progesterone stimulus may be used to assess the fertilizing potential of sperm samples from men undergoing assisted reproduction. Study design, size, duration This prospective study employed unidentified semen samples from 39 non-normozoospermic men (age 38 ± 6 years old) undergoing fertility treatment from March 2022 to the present at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and ART procedures with less than 3 oocytes were excluded. Ten normozoospermic semen samples were also analyzed. The progesterone-dependent [Ca2+]i changes were evaluated using time-lapse flow cytometry. Participants/materials, setting, methods Semen samples were obtained on the same day as oocyte retrieval. Sperm cells were washed and incubated in capacitating HTF media for further use in conventional IVF or ICSI procedures. When available, the surplus cells were stained with vitality and Ca2+-sensitive fluorescent dyes. Fluorescence changes were evaluated by AccuriC6 flow cytometer. Progesterone-induced [Ca2+]i transient as well as the correlations with the fertilization rates were calculated using custom-made R programming language code. Main results and the role of chance The progesterone (1 µM) stimulation evoked a fast and transitory increase in the [Ca2+]i. We then calculated six different kinetics parameters of such responses: basal Ca2+ level, increase and decrease in velocity, maximum Ca2+ level, total response duration, and the area under the curve (AUC). There were no significant differences in any of these parameters between normozoospermic donors (and non-normozoospermic patients. The former group of semen samples were divided into either conventional IVF (n = 17) or ICSI (n = 21). In IVF but not ICSI samples, the duration, and the area under the curve of the progesterone response tended to be higher than in normozoospermic (2.39±0.7 min, p = 0.03, and 113±30 AUF*min, p = 0.18, respectively). Furthermore, both parameters showed a tendency to correlate negatively with the conventional IVF (R= –0.45, p = 0.07 and R= –0. 5, p = 0.04, respectively) but not with ICSI fertilization ratio (number of zygotes/total number of mature oocytes). These results suggest that cells with a better Ca2+ recovery mechanism are well suited for IVF. Interestingly, we observed no difference between the magnitude of the [Ca2+]i increase after progesterone stimulus among donors, IVF, or ICSI samples, as was previously reported. Differences in the concentration of progesterone could explain such results. Limitations, reasons for caution The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples (in progress). It is well known that progesterone induces variable responses even among the same donor cells. Our population-based analysis may be underestimating such single cells variations. Wider implications of the findings Current protocols for sperm analysis fail to successfully predict the fertilizing capability of semen samples from men seeking reproductive treatment. Further research into the molecular regulation of Ca2+ dynamics could offer promising alternatives for clinicians to appropriately choose between IVF and ICSI for each couple. Trial registration number .

Effects of cryopreservation in the presence of Natural Deep Eutectic Solvents (NADESs) on sperm parameters

Natural Deep Eutectic Solvents (NADESs) are being considered as a potential alternative to traditional cryoprotective agents (CPAs) in sperm freezing. The study aimed to assess the effects of NADESs as a CPA on human sperm parameters. A total of 32 normozoospermic semen samples were collected from the Alzahra infertility treatment center (Iran) between July 2021 and September 2022. The samples were categorized into eight different groups: 1) a control (nonfrozen), and groups frozen with 2) SpermFreeze Solution, 3) ChX (Choline chloride and Xylitol), 4) ChS (Choline chloride and D-sorbitol), 5) ChG (Choline chloride and Glucose), 6) ChU (Choline chloride and Urea), 7) EtP (Ethylene glycol and l-proline), and 8) GlyP (Glycerol and l-proline). The study also analyzed the quality of sperm parameters, such as chromatin condensation and integrity, acrosome integrity, and survival, along with the expression of some genes that affect sperm fertility (TRPV1, TRPV4, SPACA3, and OGG1). The study found there were notable variations in sperm parameters (such as viability, chromatin condensation and integrity, and acrosome integrity) among frozen groups with some NADESs compared to the SpermFreeze Solution and control groups (P < 0.05). Analysis of gene expression demonstrated that the levels of TRPV1, TRPV4, SPACA3, and OGG1 genes were superior in the GlyP group compared to the other groups (P < 0.05). Additionally, the ChS and ChU groups exhibited preserved expression of these genes compared with the SpermFreeze Solution group. The use of NADESs led to the discovery of a more appropriate CPA that has low toxicity and is highly effective in maintaining the fertility potential of sperm.

Oligoasthenozoospermic and Normozoospermic Indian Males Yielded Similar Fertilization Capability by Intracytoplasmic Sperm Injection

Background: The present study evaluated the fertilization outcomes by intra-cytoplasmic sperm injection (ICSI) technique with oligoastheno-zoospermic (OAZ) compared to normozoospermic (NZ) semen. Purpose: To find the fertilizing capability of human sperm in oligo-asthenozoospermic and normozoospermic semen samples. Methods: Fresh semen ejaculates belonging to NZ and OAZ groups (n=50 in each group) patients attending the infertility clinics were evalu-ated for semen volume, sperm motility, and sperm concentration. Ferti-lization rates with ICSI along with post-capacitation motility and con-centration were studied in both groups. Results: The motility and concentration in fresh semen samples patients of the NZ group (32.44±1.50%, 71.96±6.45 M/ml, respectively) were significantly (P&lt;0.05) higher compared to the OAZ group (26.76±1.38, 10.31±0.48 M/ml, respectively).However, the results of our study re-vealed similar fertilization rates in-vitro by ICSI in the NZ (98%) and OAZ (98%) groups. Conclusion:The in-vitro processing by the ICSI technique is an effective approach to achieving good fertilization rates in sub-fertile males.

Prolonged semen incubation alters the biological characteristics of human spermatozoa

The present study assessed the biological characteristics of human spermatozoa at different time intervals (0, 1, 1.5, and 2 hours) after incubation at 37°C. Twenty-five normozoospermic semen samples were incubated at 37°C. Incubation was performed at four time intervals of 0 (after liquefaction), 1, 1.5, and 2 hours. The samples were evaluated for sperm parameters at each time interval. The rate of sperm progressive motility decreased at 1.5 hours compared to 0 hours as well as 2 hours compared to 1 hour and 0 hours. The rate of non-motile spermatozoa also decreased after 2 hours compared to after 0 hours. No significant changes were observed in sperm viability (p=0.98) and non- progressive motility (p=0.48) at any time intervals. Abnormal sperm morphology increased at 1.5 hours of incubation time (p<0.001). No significant changes were observed in DNA fragmentation at 1 hour compared to 0 hours (median [interquartile range]: 19.5 [4] vs. 19 [4]), as well as at 1.5 hours compared to 1 hour (20 [5]). However, a significant increase in DNA fragmentation was observed at 1.5 hours compared to 0 hours. The mitochondrial membrane potential decreased remarkably after 1 hour of incubation time. No significant differences were observed in the acrosome reaction or malonaldehyde levels at any time point (p=0.34 and p=0.98, respectively). The incubation of normozoospermic samples before use in assisted reproductive technology should be less than 1.5 hours to minimize the destructive effects of prolonged incubation time on general and specific sperm parameters.

O-185 Cryopreservation of Human Spermatozoa: Utilization of L-Proline as a Novel Additive to Improve Sperm Quality Following Freezing-Thawing Process

Abstract Study question Does supplementation of human sperm freezing media with L-Proline improve sperm quality and DNA Integrity following the freezing-thawing process? Summary answer The inclusion of L-proline as a novel additive to human sperm cryopreservation media improves sperm parameters and DNA integrity via mitigating oxidative stress. What is known already Sperm cryopreservation is an essential aspect of assisted reproductive technique (ART) and male fertility preservation. Although frozen-thawed semen has great practical benefits for reproduction, it is widely reported that the cryopreservation process induces physical and chemical detrimental changes in sperm functions. Indeed, freezing induces nitro-oxidative stress, lipid peroxidation, DNA fragmentation, and apoptosis. L-proline plays versatile roles in osmotic protection and oxidative stress, cell signaling, programmed cell death, and nutrient adaptation. This multifunctional amino acid is a natural osmoprotectant and the protective effect of L-proline against freezing-thawing-induced damages in stallion and donkey spermatozoa have been reported. Study design, size, duration Thirty normozoospermic semen samples were collected by masturbation after 3–5 days of sexual abstinence from men who were referred to the IVF clinic of Kermanshah Motazedi Hospital from December 2020 to June 2021. Each prepared semen sample was aliquoted to 4. In aliquots 1 to 4, experimental concentrations of L-proline (0, 1, 2, and 4 mmol/L), were included in the freezing medium. Participants/materials, setting, methods 30 normozoospermic, healthy, non-smoker men were enrolled. Sperm parameters (progressive motility, viability, and morphology) were assessed. Sperm chromatin quality was measured by Aniline blue (AB), Toluidine blue (TB), and Chromomycin A3 (CMA3) staining. DNA integrity was evaluated by the Sperm chromatin dispersion (SCD) test. Furthermore, the levels of reactive oxygen species (ROS), lipid peroxidation (LPO), and total antioxidant capacity (TAC) were determined in sperm freezing media. All assessments were conducted before cryopreservation and after thawing. Main results and the role of chance Our findings showed that sperm progressive motility and viability were significantly higher in the 4 mmol/L L-proline treated groups compared to the control groups (p = 0.003, p = 0.0200) respectively. The percentage of normal morphology was improved in L-Proline-treated groups. However, this improvement was not considerable (p &amp;gt; 0.5). Moreover, the level of ROS production significantly diminished in the 4 mmol/L L-proline group compared to the control group (p &amp;lt; 0.001). Besides, the level of TAC was significantly enhanced in the 4 mmol/L L-proline group by comparison to the control group (p &amp;lt; 0.001). Furthermore, the results of AB and TB tests demonstrated that chromatin packaging after supplementation with 4 mmol/L L-proline significantly improved compared to the control group (p &amp;lt; .0001). In this regard, there was a significant improvement between the 4 mmol/L L-proline group and the control group in terms of CMA3 evaluation (p &amp;lt; .0001). Also, there was a difference between the 4 mmol/L L-proline group and the control group in reducing the level of fragmented DNA. Although 4 mmol/L of Praline could diminish the SCD level compared to the control group, this amelioration was not statically significant (p = 0.053). Limitations, reasons for caution We were not able to perform further studies to verify the effects of L-proline on mitochondrial activity and membrane potential, and sperm apoptosis as well as to confirm the efficiency and safety of L-proline in terms of more indicators such as the in vitro embryonic development and live birth rate. Wider implications of the findings These findings can draw attention to the potential role of L-proline as a novel additive and antioxidant to human freezing medium in preserving sperm quality and protecting spermatozoa against ROS attack during sperm cryopreservation in infertility clinics. Trial registration number N/A

Impact of calcium and reactive oxygen species on human sperm function: Role of NOX5

NOX5 is introduced as a new therapeutic target for infertility treatment. This study aimed to compare the basal and stimulated reactive oxygen species (ROS) production and sperm function in human teratozoospermic (n= 15) and normozoospermic (n= 17) semen samples following calcium overload and NOX5 activation. Washed spermatozoa incubated for 1h under five various conditions: control group, adding a calcium ionophore A23187, phorbol myristate acetate (PMA), A23187 + PMA, and diphenylene iodonium (DPI) + A23187 + PMA. ROS generation was measured immediately after treatment for 30 min. Motility, viability, acrosome reaction, and apoptosis were evaluated after 1-h incubation. ROS production significantly increased when A23187 or PMA was added to the sperm medium. DPI had suppressive effects on ROS generation. Progressive and total motility significantly decreased following calcium elevation and NOX5 activation, which was somewhat returned by DPI. Necrotic and live cells in teratozoospermia was, respectively, higher and lower than normozoospermia samples. Incubation with A23187 significantly increased the percentage of early and late apoptosis. Teratozoosperm are more vulnerable than normal spermatozoa, and produce more basal and stimulated ROS. It seems that calcium overload induces apoptosis in spermatozoa and loss of viability through MPT pore opening and increased intracellular ROS.

Protective roles of seminal plasma exosomes and microvesicles during human sperm cryopreservation

Research questionDo seminal plasma microvesicles and exosomes, as two subtypes of extracellular vesicles, exert cryoprotective properties in sperm cryopreservation? DesignMicrovesicles and exosomes isolated from normozoospermic semen samples (n = 10) by serial ultracentrifugation were determined using scanning electron microscopy, dynamic light scattering and western blot analysis. The interactions between extracellular vesicles and spermatozoa were detected using Dil labelling. Purified spermatozoa from different normozoospermic samples (n = 25) were then treated individually with exosomes or microvesicles for 1 h and subsequently cryopreserved. The effects of extracellular vesicles during cryopreservation were investigated by determining post-thaw sperm motility, morphology, viability, reactive oxygen species (ROS) generation, lipid peroxidation, total antioxidant capacity (TAC), mitochondrial membrane potential (MMP), DNA integrity, and apoptosis rate. ResultsMicrovesicles and exosomes displayed a round-shape morphology, with about 70% of exosomes ranging from 43–144 nm, microvesicles ranging from 144.5–486 nm and both expressed tetraspanin markers. Fluorescence microscopy showed that exosomes and microvesicles absorbed mainly in the sperm head and less frequently in the neck and tail. The post-thawing results indicated that the diluent with exosomes or microvesicles had improved sperm motility (P = 0.007), morphology (P < 0.001) and viability (P < 0.001) compared with untreated samples. The ROS levels decreased significantly (P = 0.001), with a consequent decrease in DNA damage (P = 0.001). The TAC activity (P = 0.001) and MMP levels (P = 0.001) were also significantly improved; levels of malondialdehyde (MDA) (P = 0.62) and apoptosis rate (P = 1.000) remained unchanged. ConclusionSeminal plasma microvesicles and exosomes could protect spermatozoa from cryopreservation chilling injuries.

Sperm-friendly lubricant: Fact or fiction.

To assess the effects of "sperm-friendly" coital lubricants on sperm motility. This study compared the effects of five lubricants (Optilube®, Pre-Seed®, Yes Baby®, olive oil, and egg white) on sperm motility in 60 normozoospermic semen samples obtained from men attending a private fertility clinic. Samples were exposed to each of the lubricants, with untreated samples serving as controls, and were examined microscopically at four defined time-points from 2 to 72h after liquefaction. Sperm motility was graded according to World Health Organization criteria. With the exception of egg white, all lubricants caused significant (P<0.001) reductions in sperm forward progression compared with untreated controls until 24h after liquefaction. Furthermore, between-group comparisons of the commercially available lubricants revealed statistically significant differences in forward progression motility: Pre-Seed® was superior to Optilube® (P<0.001), which in turn was superior to Yes Baby® (P<0.001) at 2-4h after exposure. Significance (P<0.001) between Pre-Seed® and Yes Baby® was maintained until 24h. Although spermatozoa exposed to Pre-Seed® demonstrated greater motility than spermatozoa exposed to Yes Baby®, claims that these lubricants are sperm-friendly were refuted. Conversely, egg white was shown to be a sperm-friendly lubricant for couples who are trying to conceive.

L-proline as a novel additive to cryopreservation media improved post-thaw quality of human spermatozoon via reducing oxidative stress.

Sperm cryopreservation as a routine technique in assisted reproductive technique (ART) laboratories has detrimental effects on spermatozoa. Various methods have been introduced to improve it. The aim of this research was to evaluate the effects of L-proline supplementation in cryopreservation medium on normozoospermic semen samples. A total of 30semen samples were collected from normozoospermic men. Cryopreservation media were supplemented with different concentrations of L-proline (0, 1, 2 and 4mmol/L). The semen samples were cryopreserved. After thawing, sperm parameters and chromatin integrity (aniline blue (AB), toluidine blue (TB), sperm chromatin dispersion test (SCD) and chromomycin A3 (CMA3)), reactive oxygen species (ROS), and total antioxidant capacity (TAC) and malondialdehyde (MDA) levels were evaluated. A total of 4mmol/L L-proline significantly improved progressive motility and viability (p<0.05). MDA and ROS levels significantly diminished in samples were cryopreserved by 4mmol/L L-proline supplemented cryopreservation media (p<0.001). Also, it significantly increased TAC level. Also, chromatin damages (AB, TB and CMA3) significantly improved in samples were cryopreserved by 4mmol/L L-proline supplemented cryopreservation media (p<0.05). The results support that the usage of L-proline supplemented cryopreservation media to improve sperm quality after cryopreservation.

Protamine Characterization by Top-Down Proteomics: Boosting Proteoform Identification with DBSCAN.

Protamines replace histones as the main nuclear protein in the sperm cells of many species and play a crucial role in compacting the paternal genome. Human spermatozoa contain protamine 1 (P1) and the family of protamine 2 (P2) proteins. Alterations in protamine PTMs or the P1/P2 ratio may be associated with male infertility. Top-down proteomics enables large-scale analysis of intact proteoforms derived from alternative splicing, missense or nonsense genetic variants or PTMs. In contrast to current gold standard techniques, top-down proteomics permits a more in-depth analysis of protamine PTMs and proteoforms, thereby opening up new perspectives to unravel their impact on male fertility. We report on the analysis of two normozoospermic semen samples by top-down proteomics. We discuss the difficulties encountered with the data analysis and propose solutions as this step is one of the current bottlenecks in top-down proteomics with the bioinformatics tools currently available. Our strategy for the data analysis combines two software packages, ProSight PD (PS) and TopPIC suite (TP), with a clustering algorithm to decipher protamine proteoforms. We identified up to 32 protamine proteoforms at different levels of characterization. This in-depth analysis of the protamine proteoform landscape of normozoospermic individuals represents the first step towards the future study of sperm pathological conditions opening up the potential personalized diagnosis of male infertility.

Effect of autologous platelet-rich plasma on human sperm quality during cryopreservation

As a source of growth factors, platelet-rich plasma (PRP) has been widely used in the repair of various injuries due to its cytoprotective properties in regenerative medicine. The objective of the present study was to investigate the effect of autologous PRP supplementation on the quality of frozen-thawed human sperm. Twelve normozoospermic semen samples were collected, and each sample was divided into 4 aliquots and added with different proportions of PRP (0%, 2%, 5%, and 10%) separately, followed by cryopreservation. Sperm motility, viability, membrane integrity, DNA fragmentation index (DFI), reactive oxygen species (ROS) levels and mitochondrial membrane potential were measured and analyzed. The results showed that the addition of 5% PRP improved sperm progressive motility (30.3 ± 2.7 VS. 28.1 ± 2.6), viability (65.5 ± 4.2 VS. 59.6 ± 3.9), and membrane integrity (52.4 ± 3.6 VS. 49.2 ± 3.4) after cryopreservation (P < 0.05). Slight attenuation of ROS generation, restoration of mitochondrial membrane potential and reduction of DNA fragments were also observed in post-thawed sperm supplemented with PRP, but there was no significant (P > 0.05). In conclusion, autologous PRP has a partial protective effect on cryopreservation of human spermatozoa, and the combined application with other high-efficiency cryoprotectant is worthy of further study.

Gene transcription profiling of astheno- and normo-zoospermic sperm subpopulations.

Spermatozoa contain a repertoire of RNAs considered to be potential functional fertility biomarkers. In this study, the gene expression of human sperm subpopulations with high (F1) and low (F2) motility from healthy normozoospermic (N) and asthenozoospermic (A) individuals was evaluated using RNA microarray followed by functional genomic analysis of differentially expressed genes. Results from A–F1 versus N–F1, A–F2 versus N–F2, N–F1 versus N–F2, and A–F1 versus A–F2 comparisons showed a considerably larger set of downregulated genes in tests versus controls. Gene ontology (GO) analysis of A–F1 versus N–F1 identified 507 overrepresented biological processes (BPs), several of which are associated with sperm physiology. In addition, gene set enrichment analysis of the same contrast showed 110 BPs, 36 cellular components, and 31 molecular functions, several of which are involved in sperm motility. A leading-edge analysis of selected GO terms resulted in several downregulated genes encoding to dyneins and kinesins, both related to sperm physiology. Furthermore, the predicted activation state of asthenozoospermia was increased, while fertility, cell movement of sperm, and gametogenesis were decreased. Interestingly, several downregulated genes characteristic of the canonical pathway protein ubiquitination were involved in asthenozoospermia activation. Conversely, GO analysis of A–F2 versus N–F2 did not identify overrepresented BPs, although the gene set enrichment analysis detected six enriched BPs, one cellular component, and two molecular functions. Overall, the results show differences in gene transcription between sperm subpopulations from asthenozoospermic and normozoospermic semen samples and allowed the identification of gene sets relevant to sperm physiology and reproduction.

Effects of temperature and storage time on the motility, viability, DNA integrity and apoptosis of processed human spermatozoa.

The aim of this study was to evaluate motility, viability, DNA integrity and apoptosis of spermatozoa when washed semen samples were kept for up to 12days at 4-6°C and 25°C. In this experimental study, 26 normozoospermic semen samples were washed twice in Modified Ham's F10 and resuspended in IVF fertilisation medium. Half of the specimens were stored at 4-6°C, and the other half was kept at 25°C for 12days. The proportions of viable, motile, spermatozoa with double-stranded DNA and apoptotic spermatozoa were examined during storage time. Apoptosis was measured using annexin V-PI staining followed by flow cytometry. Results showed that sperm motility and viability decreased during 12days of sample storage (p<.001). There was no significant difference between the two temperatures in terms of motility and viability for up to 2days (p<.05). The percentage of spermatozoa with double-stranded DNA remained unchanged during the 12days of storage at both temperatures (p>.05). Although there was no difference between the two temperatures in terms of motility, viability and apoptosis during the first two days of storage, storage of spermatozoa at 4-6°C is better than storage for a longer period than storage at 25°C. Sperm DNA resisted against denaturation during storage.

Canthaxanthin protects human sperm parameters during cryopreservation.

Different antioxidants have been introduced to reduce oxidative stress during the cryopreservation. The main goal of this study was to evaluate the effects of canthaxanthin on human sperm parameters during the freeze-thaw process. This study was performed on 25 normozoospermic semen samples dividing into five groups including 0, 0.1, 1, 10, and 25µM of canthaxanthin. The prepared spermatozoa were cryopreserved by rapid freezing technique. Sperm motility, viability (eosin-nigrosin), morphology (Papanicolaou), acrosome reaction (double staining), DNA denaturation (acridine orange), chromatin packaging (aniline blue and toluidine blue), and DNA fragmentation (sperm chromatin dispersion test) were evaluated before freezing and after thawing. All sperm parameters after thawing significantly were decreased compared to before freezing. Twenty-five micromolar canthaxanthin could significantly improve the progressive and total motility, viability, normal morphology, chromatin packaging, acrosome integrity and DNA denaturation and fragmentation. Ten micromolar canthaxanthin significantly improved total motility, viability, normal morphology, chromatin packaging, acrosome integrity and DNA denaturation and fragmentation. Whereas, in 1µM group, there were significant differences only in improvement of acrosome integrity, chromatin packaging (toluidine blue) and DNA denaturation and fragmentation. But, in 0.1µM group, there were no significant differences in any of measured parameters. It seems that canthaxanthin ameliorates detrimental effects of cryopreservation on human sperm parameters.