Sperm Fractions Research Articles

Advancements in differentiation between sperm cells and epithelial cells for efficient forensic DNA analysis in sexual assault cases.

Most of the sexual assault casework samples are of mixed sources. Forensic DNA laboratories are always in the requirement of a precise technique for the efficient separation of sperm and non-sperm DNA from mixed samples. Since the introduction of the differential extraction technique in 1985, it has seen significant advancements in the form of either chemicals used or modification of incubation times. Several automated and semi-automated techniques have also adopted the fundamentals of conventional differential extraction techniques. However, lengthy incubation, several manual steps, and carryover over non-sperm material in sperm fraction are some of the major limitations of this technique. Advanced cell separation techniques have shown huge promise in separating sperm cells from a mixture based on their size, shape, composition, and membrane structure and antigens present on sperm membranes. Such advanced techniques such as DEParray, ADE, FACS, LCM, HOT and their respective pros and cons have been discussed in this article. As current-day forensic techniques should be as per the line of Olympic slogan i.e., faster, higher, stronger, the advanced cell separation techniques show a huge potential to be implemented in the casework samples.

P-523 Single molecule chromatin footprinting reveals only low occurrence of nucleosomes at multiple loci throughout chromatin of human and mouse spermatozoa

Abstract Study question What fraction of spermatozoa from mouse and human semen contain nucleosomes at specific loci? What chromatin features affect nucleosome retention in spermatozoa? Summary answer On average, 1-2% mouse spermatozoa and 5-9% human spermatozoa contain nucleosomes at sampled genomic loci, with moderately higher frequencies at unmethylated regions of DNA. What is known already Nucleosomes constitute the basic packaging and gene regulatory unit of DNA in eukaryotic cells. During final stages of spermatogenesis in mammals, however, most nucleosomes are replaced by protamines. Previous global chromatin immunoprecipitation-sequencing studies reported the presence of nucleosomes at gene regulatory and other genomic regions. Such nucleosomes were proposed to convey intergenerational inheritance of epigenetic information. Additional studies revealed that infertile men with decreased success of assisted reproductive therapy have protamine deficiency or displayed more widely distributed nucleosomes in their sperm genome. Direct links between altered levels of nuclear proteins and fertilizing potential of sperm have, however, not been established. Study design, size, duration We conducted a case study on nucleosome retention levels in sperm from four selected fertile human samples. This study was complemented in mice, in which we characterized the dynamic changes in nucleosome frequencies at genomic loci during differentiation of haploid spermatids into spermatozoa, and their responses in mutant spermatozoa lacking DNA methylation at certain loci. Participants/materials, setting, methods We used the single-molecule sequencing technique called Nucleosome Occupancy and Methylome Sequencing to quantify nucleosome occupancy rates at selected loci in spermatozoa from ejaculated semen of four fertile semen donors. We also profiled round spermatids, early and late elongating spermatids and spermatozoa of C57BL/6J wild-type mice as well as spermatozoa of mice conditionally deficient for the methyltransferases Dnmt3a and Dnmt3b, lacking DNA methylation at certain genomic regions. Main results and the role of chance To our knowledge, this is the first study to quantify nucleosome occupancy in mouse male gametes and human spermatozoa at single-molecule resolution. In mice, we observed that chromatin remodelling during spermatid elongation resulted in a progressive loss of nucleosomes and their positioning, a transient gain in global chromatin accessibility which was succeeded by increasing chromatin inaccessibility and nuclear compaction. In both human and mouse sperm, we profiled nucleosome occupancy levels at different genomic loci and measured only low percentages, ranging from ∼1-2% in mouse and 5-9% in human. In both organisms we observed a near two-fold higher nucleosome occupancy at loci being unmethylated at their DNA over loci being highly methylated. Mice partially deficient in genomic DNA methylation manifested a similar fold increase in nucleosome occupancy in absence of DNA methylation. These data reveal a moderate modulatory role for DNA methylation established in spermatogonial stages in regulating nucleosome eviction during spermiogenesis. The overall low nucleosome occupancy frequencies in mammalian spermatozoa suggest a stochastic or random rather than a programmed role of nucleosomes in regulating paternal gene expression during ensuing embryonic development. Limitations, reasons for caution Amplicon-based sequencing provides deep sequencing coverage for selected genomic loci. We lack, however, information on the distribution of nucleosomes among loci within the genome of single spermatozoa. In human, we studied a limited number of human donors. Wider implications of the findings This study resolves a long-standing question on the frequency and local distribution of nucleosomes in murine and human spermatozoa. The variability in nucleosome occupancy between spermatozoa within and between samples of human individuals inspires further investigations into the relationship between chromatin states and quality of individual spermatozoa for embryonic development. Trial registration number SNCTP000003654

P-053 Comparative analysis between microfluidic sperm sorting and Swim-up technique to select spermatozoa with better characteristics in hyper viscous samples

Abstract Study question What is the best technique to select spermatozoa in hyper viscous semen samples? Summary answer Microfluidics is the best technique that allows to select spermatozoa with better seminal characteristics and hyperactivated motility, and less sperm DNA fragmentation. What is known already Density gradient centrifugation and Swim-up (SU) are the two traditional selection methods used during assisted reproduction techniques (ART) to collect the highly motile sperm fraction. Many studies reported that centrifugation increase sperm DNA fragmentation and oxidative status which could affect reproductive outcomes. To overcome the limitations of centrifugation, an alternative approach by using microfluidic-based devices was recently designed. In microfluidic protocol, sample is applied to the device’s inlet port, connected to a lower chamber. This chamber consists of a microporous filter, where the most motile spermatozoa migrate upward through the filter. Study design, size, duration An observational study was conducted on 29 semen samples from patients undergoing routine semen analysis for couple infertility in the Andrology Laboratory of Careggi University Hospital of Florence from January 2023 to January 2024. In these samples we compare two different methods for sperm selection: indirect SU technique and microfluidic devices (MSS). Participants/materials, setting, methods We evaluated seminal parameters (sperm progressive and total motility, viability, and concentration), sperm kinematic parameters by Computer-Assisted Sperm Analysis (C.A.S.A. system), sperm DNA fragmentation by TUNEL/PI assay and sperm oxidative status by CellROX® Orange (which we have recently shown to identify a fraction of viable oxidized spermatozoa with better characteristics (Traini G., et al. 2022)) in 29 normozoospermic and 18 hyper viscous semen samples selected by SU and MSS. Main results and the role of chance In normozoospermic samples, sperm selection with both indirect SU technique and microfluidic devices, ameliorated sperm characteristics compared to not selected samples. Spermatozoa selected by MSS showed statistically significant higher progressive and total motility, viability and concentration compared to those selected by indirect SU. Moreover, an increase in most of kinematic semen parameters (like VAP, VCL and VSL) and hyperactivated motility was observed in MSS selected samples. Also, sperm DNA fragmentation was significantly reduced after MSS selection compared to indirect SU. We evaluated the percentage of viable oxidized spermatozoa by using CellROX® Orange. A significant increase of the percentage of CellROX® Orange positive spermatozoa was observed in most of MSS selected samples. In all 18 hyper viscous samples, the results obtained in normozoospermic samples were confirmed. Indeed, an overall improvement of sperm progressive and total motility, viability and concentration together with an increase in most of kinematic semen parameters and in hyperactivated motility were evidenced in MSS selected spermatozoa respect to those selected by indirect SU. Again, a significant decrease of sperm DNA fragmentation and an increase of the percentage of CellROX® Orange positive spermatozoa, were observed after MSS selection. Limitations, reasons for caution The number of semen samples included in the study is not very elevated. Results obtained in hyper viscous semen samples should be confirmed in a greater number of samples. Wider implications of the findings Our data indicate that MSS selects the sperm fraction with better characteristics and less DNA fragmentation. These results suggest that MSS could improve ART outcomes, particularly for specific categories of patients, including hyper viscous semen samples, for which the selection by SU does not always guarantee good yields. Trial registration number not applicable

P-103 Sperm selection for ICSI by rheotaxis and thermotaxis in In-Situ handmade microfluidics of fluidic walls: preliminary results of a Randomized Controlled Trial

Abstract Study question Does sperm selection by rheotaxis and thermotaxis in In-Situ handmade microfluidics of fluidic walls optimize ICSI outcomes and workflow? Summary answer In-Situ handmade rheo/thermotaxis microfluidics of fluidic walls is as effective and more time-efficient than centrifugation density gradients to support pre-implantation embryonic development to blastocyst. What is known already Microfluidics technologies stand as the latest sperm selection methodology for ICSI. To outline their application in ART we conceived a lab-on-a-chip approach for sperm selection by In-Situ handmade microfluidics of fluidic walls (isM) in the same ICSI plate which integrates two operative zones: one to allow seminal plasma dispersion and sperm thermotaxis and another one to allow sperm rheotaxis. Our microfluidics system can be prepared with any media suitable for ICSI, disregarding centrifugation, washing, plasticware and other cells. The isM protocol proved non-inferior to centrifugation density gradients (CDG) to support embryonic development and implantation in a previous sibling-oocyte study. Study design, size, duration The present RCT was approved by our ethical committee for research with medical products (CEIm approval number 2023/394). This preliminary analysis aims to detect deviations in the pre-defined primary outcomes: Normal Fertilization Rate, Day 5/6 usable blastocyst rate per MII, and per zygote. This analysis includes data from 21 couples undergoing an ICSI treatment in our center during December 2023-January 2024. Only patients without clinical or analytical suspicion of sperm DNA fragmentation were included. Participants/materials, setting, methods Patients were randomly allocated to isM (n 9) or to CGD (n 12) groups. All participants signed an informed consent. Sperm samples were processed directly after liquefaction. For CGD, samples were prepared in 80-40% PureSperm® layers by isopycnic centrifugation followed by washing and dilution steps. The isM system was prepared by displaying three microdroplets of different volumes following a L-shape interconnection and allowing a temperature gradient of 2ºC between the first and the second microdroplet. Main results and the role of chance Sperm characteristics showed the following total mean values for the isM and CGD groups, respectively: concentration (58,2±38,0 × 106/ml and 52,7±29,1 × 106/ml) and progressive sperm concentration (16,6±17,0 × 106/ml and 16,8±13,4 × 106/ml). Women average age was 36,7±2,9 isM and 35,8±2,5 CDG. This analysis includes a total of 168 MII oocytes (n = 69 isM and n = 99 CDG), with a mean number of MII oocytes per patient of 7,4±2,1 isM and 8,3±1,9 CDG. Comparisons between the two groups showed that isM protocol optimizes ICSI outcomes by means of Normal Fertilization Rate: 88,4% vs 76,8%; p < 0,05, Day 5 A/B grade blastocyst rate: 36,1 vs 31,6%; ns, Day 5/6 usable blastocyst rate per MII: 58,0% vs 44,4%; ns and Day 5/6 usable blastocyst rate per diploid zygote: 65,6% vs 57,9%; ns. The isM protocol also optimizes the IVF workflow compared to CDG, as the time to obtain sperm for ICSI after liquefaction was 26 vs 77 minutes, respectively. Limitations, reasons for caution The study aims for a total size of 50 ICSI cycles to validate statistically our data at a power of 80%. The isM protocol depends on a free hand preparation. A ready-to-use surface would benefit the operability, albeit the use of templates and the training reduces inter and intra-operator variability. Wider implications of the findings The potential benefits of the isM protocol on fertilization could be significant for patients with a limited number of available oocytes for ICSI. According to the literature, microfluidics reduce the DNA fragmentation index in the selected sperm fraction. The isM method could be especially beneficial in patients with this pathology. Trial registration number NCT number assignment pending

Variation of sperm quality and circular RNA content in men exposed to environmental contamination with heavy metals in 'Land of Fires', Italy.

Can illegal discharge of toxic waste into the environment induce a new condition of morpho-epigenetic pathozoospermia in normozoospermic young men? Toxic environmental contaminants promote the onset of a new pathozoospermic condition in young normozoospermic men, consisting of morpho-functional defects and a sperm increase of low-quality circular RNA (circRNA) cargo, tightly linked to contaminant bioaccumulation in seminal plasma. Epidemiological findings have reported several reproductive anomalies depending on exposure to contaminants discharged into the environment, such as germ cell apoptosis, steroidogenesis defects, oxidative stress induction, blood-testis barrier dysfunctions, and poor sperm quality onset. In this scenario, a vast geographical area located in Campania, Italy, called the 'Land of Fires', has been associated with an excessive illegal discharge of toxic waste into the environment, negatively impacting human health, including male reproductive functions. Semen samples were obtained from healthy normozoospermic men divided into two experimental groups, consisting of men living in the 'Land of Fires' (LF; n = 80) or not (CTRL; n = 80), with age ranging from 25 to 40 years. The study was carried out following World Health Organization guidelines. Quality parameters of semen from CTRL- and LF-normozoospermic men were evaluated by computer-assisted semen analysis; high-quality spermatozoa from CTRL and LF groups (n = 80 for each experimental group) were obtained using a 80-40% discontinuous centrifugation gradient. Seminal plasma was collected following centrifugation and used for the dosage of chemical elements, dioxins and steroid hormones by liquid chromatography with tandem mass spectrometry. Sperm morpho-functional investigations (cellular morphology, acrosome maturation, IZUMO1 fertility marker analysis, plasma membrane lipid state, oxidative stress) were assessed on the purified high-quality spermatozoa fraction by immunochemistry/immunofluorescence and western blot analyses. Sperm circRNA cargo was evaluated by quantitative RT-PCR, and the physical interaction among circRNAs and fused in sarcoma (FUS) protein was detected using an RNA-binding protein immunoprecipitation assay. Protein immunoprecipitation experiments were carried out to demonstrate FUS/p-300 protein interaction in sperm cells. Lastly, in vitro lead (Pb) treatment of high-quality spermatozoa collected from normozoospermic controls was used to investigate a correlation between Pb accumulation and onset of the morpho-epigenetic pathozoospermic phenotype. Several morphological defects were identified in LF-spermatozoa, including: a significant increase (P < 0.05 versus CTRL) in the percentage of spermatozoa characterized by structural defects in sperm head and tail; and a high percentage (P < 0.01) of peanut agglutinin and IZUMO1 null signal cells. In agreement with these data, abnormal steroid hormone levels in LF seminal plasma suggest a premature acrosome reaction onset in LF-spermatozoa. The abnormal immunofluorescence signals of plasma membrane cholesterol complexes/lipid rafts organization (Filipin III and Flotillin-1) and of oxidative stress markers [3-nitrotyrosine and 3-nitrotyrosine and 4-hydroxy-2-nonenal] observed in LF-spermatozoa and associated with a sperm motility reduction (P < 0.01), demonstrated an affected membrane fluidity, potentially impacting sperm motility. Bioaccumulation of heavy metals and dioxins occurring in LF seminal plasma and a direct correlation between Pb and deregulated circRNAs related to high- and low-sperm quality was also revealed. In molecular terms, we demonstrated that Pb bioaccumulation promoted FUS hyperacetylation via physical interaction with p-300 and, in turn, its shuttling from sperm head to tail, significantly enhancing (P < 0.01 versus CTRL) the endogenous backsplicing of sperm low-quality circRNAs in LF-spermatozoa. Participants were interviewed to better understand their area of origin, their eating habits as well as their lifestyles, however any information incorrectly communicated or voluntarily omitted that could potentially compromise experimental group determination cannot be excluded. A possible association between seminal Pb content and other heavy metals in modulating sperm quality should be explored further. Future investigations will be performed in order to identify potential synergistic or anti-synergistic effects of heavy metals on male reproduction. Our study provides new findings regarding the effects of environmental contaminants on male reproduction, highlighting how a sperm phenotype classified as normozoospermic may potentially not match with a healthy morpho-functional and epigenetic one. Overall, our results improve the knowledge to allow a proper assessment of sperm quality through circRNAs as biomarkers to select spermatozoa with high morpho-epigenetic quality to use for ART. This study was supported by 'Convenzione Azienda Sanitaria Locale (ASL) Caserta, Regione Campania' (ASL CE Prot. N. 1217885/DIR. GE). The authors have no conflict of interest to declare. N/A.

A new effective method for isolating single spermatozoa from surgically obtained material in azoospermia

A new effective method for isolating single spermatozoa from surgically obtained material in azoospermia

Selection of reference genes for normalizing gene expression data across seasons in spermatozoa of water buffalo (Bubalus bubalis).

Selection of the most stably expressed reference genes is key to monitoring accurate target gene expression across any tissue or cell type. The mRNA in spermatozoa stores valuable information related to changes in spermatogenesis due to variations in environmental conditions, especially during heat stress, which affects various sperm functions. Semen quality in buffalo bulls is significantly influenced by the seasons. In the study, a panel of nine genes was evaluated to identify the most stably expressed internal control gene (ICG) for the normalization of real-time gene expression data generated across various seasons for Murrah buffalo bulls' spermatozoa. Sperm cells were purified from the semen samples collected during different seasons, with temperature-humidity index (THI) ranging from 80.80 ± 1.47 (hot summer) to 55.88 ± 1.98 (winter), using the BoviPure™ gradient purification method. The RNA isolated from the purified spermatozoa fraction was quality checked prior to reverse transcription and subjected to qPCR (quantitative real-time PCR) based expression analysis. An automated 'endoGene' pipeline was employed to apply the geNorm, NormFinder, and BestKeeper algorithms for data analysis. The result indicated that GAPDH and PP1A were the most stably expressed among the gene panel, whereas ATPSF1 and ACTB were the two least stable expressed reference genes. Further, the most suitable ICGs identified were validated by normalization of real time expression data of heat stress and sperm quality genes, HSFY2 and AKAP4, respectively. The genes identified would help in generating the most reliable results for the expression profiling of the genes dictating sperm quality and heat stress cope-up mechanism in buffalo spermatozoa, collected during different seasons.

Modified differential lysis for sexual assault samples using a combined enzymatic and alkaline approach.

Traditional sexual assault sample processing methods are time-consuming and inefficient.This modified differential lysis method produces lysates with sufficient DNA yield and quality.A combined technique using enzymatic and alkaline lysis can accomplish fractional separation.Lysis with prepGEM and NaOH absent purification is compatible with downstream processes.

Utilizing differential extraction thresholds to deduce the existence of spermatozoa in forensic casework samples

Testing of evidence in an alleged sexual assault case not only seeks to address the question of who was involved, but also looks to answer the question of what biological source provided the DNA. It can be difficult to obtain positive serological data on challenging samples such as laundered items, low-level DNA samples, or sexual assault kit swabs obtained after a prolonged interval from the time of assault. In the absence of confirmatory serological results, an expert witness often cannot speak to the biological source of the DNA. In order to determine quantitation thresholds which could be used to deduce the presence of spermatozoa (sperm) within a sample, we evaluated the fractionation of male DNA utilizing our laboratory’s differential extraction method. Study samples included serial dilutions of semen and semen/saliva mixtures, post-coital and laundered samples as well as casework data from 1,729 samples that were processed using a differential extraction. Based on this data, it was determined that a sample which had at least 200 picograms of male DNA and at least 10% of the total male DNA in fraction 2 (F2, also known as the sperm-enriched or sperm fraction) could be reported as positive for the presence of sperm. No false positive results were obtained from the study-generated samples when using these thresholds to infer the presence of sperm. Additionally, samples that contained sperm, but were negative using traditional serological methods, could be detected. However, not all sperm-containing samples fractionated above both thresholds; therefore, serological testing may still be necessary to minimize false negative results. The thresholds developed here, proved reliable to deduce the presence of sperm in real casework samples.

USING MICROFLUIDIC SPERM SORTING CHIPS IN PATIENTS WITH INFERTILITY

The purpose of the work is to analyze the effectiveness of the FERTILE microfluidic sperm sorting chip.&#x0D; Materials and Methods. The technique under consideration is currently experimental. It was used in patients after obtaining voluntary informed consent. A retrospective analysis of medical records of 4 groups of patients was carried out. The patients were divided into 2 experimental and 2 control groups. In total the authors analyzed 54 medical records. The microfluidic sperm sorting chip (FERTILE/FERTILE PLUS CHIP) are intended for the selection of normal mobile spermatozoids, which can be used for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). The indicators of preimplantation embryo development in vitro were assessed.&#x0D; Results. Embryological parameters of infertility treatment using ART were analyzed. Key indicators of preimplantation embryo development (fertilization rate, blastocyst development, embryo cryopreservation) were higher in both experimental groups compared to control ones (standard semen processing). Embryos were obtained by fertilization with sperm which undergone microfluidic sorting. Embryo culture suggests a more physiological nature of gamete separation. During standard ejaculate processing centifugation is used to obtain a fraction of active sperm. However, it negatively affects the cytophysiology of male gametes and causes breaks in the DNA chain in the spermatozoid head.&#x0D; Conclusion. The authors observed the improvement in embryological parameters of in vitro embryos, which were obtained by means of microfluidic chip-based sperm selection. This method allows us to select cytophysiologically competent sperm and improve the embryo quality without increasing DNA fragmentation rate.

The effect of drugs used in the treatment of hemoblastosis on the morphofunctional state of spermatozoa

Introduction. The relevance of the study is due to the high frequency of complications after the use of drugs for treating malignant tumors, which is associated with the cytotoxic effect of chemotherapy drugs both on malignancy sites and on healthy tissues, including the cells of male gonads.Aim. To study the impact of drugs intended for the treatment of hemoblastoses on the total sperm count, their mobility, and the presence of pathological forms under experimental conditions.Materials and methods. A case-control study was conducted on 18 male rats aged 90 days. The control group consisted of male rats that did not receive hemoblastosis treatment drugs, and the second group was intraperitoneally injected with cyclophosphamide, hydroxydaunorubicin, vincristine, prednisolone (hereinafter referred to as CHOP). Mature spermatozoa were obtained from the dissected appendages of the testes, by opening them on a thermal stage. The contents of the rat's seminiferous tubules, in a volume of 0.02 ml, were diluted in 0.4 ml of 0.9% sodium chloride solution, preheated to 37°C. The total, absolute, and relative number of spermatozoa in a unit volume (0.4 ml) of epididymal suspension was counted, taking into account their mobility according to the generally accepted system. Actively mobile and weakly mobile were attributed to the fertile fraction, and "twitching" and immobile - to the infertile fraction of epididymal spermatozoa. Then, the fertility index was calculated, which represents the ratio of the number of fertile forms to infertile ones. To determine the viability of spermatozoa, their count with regard to mobility was conducted within the first hour every 15 minutes, and subsequently every 30 minutes until the complete cessation of all spermatozoa. To assess the pathological forms of spermatozoa, the absolute and percentage content of spermatozoa in a unit volume (0.4 ml) of epididymal suspension with defects in the head, neck, midpiece, and tail was counted under light microscopy.Results. The experimental exposure to CHOP group drugs had the following effects: a decrease in the total number of spermatozoa by 37% was observed in the male rats of the experimental group, accompanied by an increase in the number of their pathological forms by 26% compared to the control group.Conclusion. The increased risk of infertility, caused by the toxic effect of drugs intended for the treatment of hemoblastoses, is associated with a decrease in the total number of spermatozoa and an increase in pathological forms, leading to a reduction in the number of mobile cells.

Artificial insemination of all ejaculated sperm fractions accelerates embryo development and increases the uterine vascularity in the pig

The semen of boar is characterized by ejaculation in well-differentiated fractions with specific concentration, composition, and volume. The ‘sperm-rich fraction' (SRF), the most concentrated seminal fraction, is habitually collected in insemination centers to make artificial insemination (AI) doses. The absence of the other fractions in AI doses could alter the uterine reaction to AI and not trigger essential responses that could maximize fertility. Thus, there is an urge to ascertain the impact of different ejaculate fractions on the uterus after AI to optimize the semen doses. This work analyzed specific parameters related to fertility in pregnant artificially inseminated sows (n = 15) with ac-cumulative fractions of the semen of boars (n = 6): F1, composed of the sperm-rich fraction (SRF); F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF. Non-inseminated sows (n = 5) were included as control (C). The different types of seminal dose did not affect the number of ovulated follicles (CL; corpora lutea, p > 0.05) but did affect the embryo development (p < 0.05). The proportion of embryos in morula stages was significantly higher in AI-F1 sows (84.4%, p < 0.05). Morulas and blastocysts were balanced in AI-F2 or AI-F3 (p > 0.05). Independently of the type of seminal dose (F1, F2, or F3), we observed by immunohistochemistry that AI significantly increased uterine vascularization, although with some anatomical differences. The cranial region of the uterine horns was significantly more vascularized in AI-F1 or AI-F2 sows (26.7 ± 2.3 and 28.6 ± 2.0%, respectively), and AI-F3 showed significantly less vascularization at that point (17.8 ± 1.6%, p < 0.05). To summarize, the synergistic effect of all ejaculate fractions accelerates embryo development, at least during the preimplantation period, and increases the uterine reaction to AI in certain parts of the uterus.

Cryopreservation of equine spermatozoa reduces plasma membrane integrity and phospholipase C zeta 1 content as associated with oocyte activation.

Phospholipase C zeta (PLCZ1) is considered the major sperm-borne oocyte activation factor. Cryopreserved stallion spermatozoa are commonly used for intracytoplasmic sperm injection (ICSI). However, plasma membrane damage and protein modifications caused by cryopreservation could impair sperm structure and function, leading to a reduction of PLCZ1 and oocyte activation after ICSI. We compared membrane integrity and PLCZ1 abundance in populations for fresh, frozen, and refrozen stallion spermatozoa, either thawed and refrozen at room or low temperature; and examined the effect of relative PLCZ1 content on cleavage after ICSI. Western blotting, ELISA, and immunofluorescence were conducted in stallion spermatozoa, freezing extenders, and detergent-extracted sperm fractions to detect and quantify PLCZ1. Retrospectively, PLCZ1 content and cleavage rate were analyzed. Fresh, frozen, and refrozen at room and low temperatures spermatozoa were evaluated for acrosomal and plasma membrane integrity and PLCZ1 content using flow cytometry. Western blotting, ELISA, and immunofluorescence revealed significant reduction of PLCZ1 in spermatozoa after cryopreservation and confirmed PLCZ1 detection in extenders. After detergent extraction, a PLCZ1-nonextractable fraction remained in the postacrosomal region of spermatozoa. Plasma membrane integrity was significantly reduced after freezing. Acrosomal and plasma membrane integrity were similar between frozen and refrozen samples at low temperature, but both were significantly higher than samples refrozen at room temperature. Acrosomal and plasma membrane integrity significantly correlated to PLCZ1 content. Percentages of PLCZ1-labeled spermatozoa and PLCZ1 content were reduced after freezing but not after refreezing. Relative content and localization of PLCZ1 were associated with cleavage rates after ICSI. Sperm PLCZ1 content associates with cleavage rates after ICSI. Cryopreservation is detrimental to sperm plasma membrane integrity and PLCZ1 retention. However, refreezing did not result in additional PLCZ1 loss. Refreezing stallion spermatozoa at a low temperature resulted in better survival but did not improve PLCZ1 retention.

SUMO1 and Defective Spermatozoa Correlate with Endogenous Hydrogen Peroxide and Live Birth Outcome in Intrauterine Insemination Cycles for Unexplained Infertility.

This study aimed to investigate the correlation between hydrogen peroxide (H2O2), small ubiquitin-like modifier molecules (SUMO), and pregnancy outcomes in couples with unexplained infertility (UI) undergoing intrauterine insemination (IUI) treatment. We prospectively collected semen samples from 56 couples with UI and divided the spermatozoa into motile and immotile fractions by density gradient centrifugation (DSC). Immunofluorescence staining was used to examine the immunostaining and localization of nuclear pore complex (NPC), SUMO1, and SUMO2/3 in spermatozoa. We detected H2O2 levels by chemiluminescence methods. We found that H2O2 levels correlated with NPC (neck) (r = 0.400) and NPC (tail) (r = 0.473) in motile sperm fractions. In immotile fractions, H2O2 positively correlated with NPC (tail) (r = 0.431) and SUMO1 (neck) (r = 0.282). Furthermore, the positive NPC (tail) group had a significantly lower live birth rate than the negative NPC group (17.9% = 5/28 vs. 42.9% = 12/28). In conclusion, H2O2 positively correlated with SUMO1 (neck) and NPC (tail) in human spermatozoa. The DSC may partially eliminate defective spermatozoa (positive NPC staining); however, if defective spermatozoa remain in the motile fraction, this scenario is associated with a low live birth rate following IUI treatment.

Zinc protects against lead-induced testicular damage via modulation of steroidogenic and xanthine oxidase/uric acid/caspase 3-mediated apoptotic signaling in male Wistar rats

Aim This study evaluated the effect of lead, with or without zinc co-administration, on steroidogenic and xanthine oxidase (XO)/uric acid (UA)/caspase 3-mediated apoptotic signaling in the testis. Materials and methods Forty male Wistar rats were divided into four groups at random; vehicle-treated control, zinc-treated, lead-treated, and lead + zinc-treated groups. Results Lead exposure significantly lowered overall weight gain, testicular, epididymal, seminal vesicle, and prostate weights. Also, lead decreased sperm count, viability and motility but increased the fraction of sperm with aberrant morphology. In addition, lead caused a marked rise in the level of UA and XO activity but a decrease in nuclear factor erythroid 2-related factor 2 (Nrf2), reduced glutathione (GSH) as well as total antioxidant capacity (TAC) levels, and superoxide dismutase (SOD) and catalase activities. Furthermore, lead increased the testicular levels of nuclear factor kappa B (NFkB), interleukin-1beta (IL-1β), and tumour necrotic factor-alpha (TNF-α), which were associated with an increase in testicular caspase 3 activity and DNA fragmentation as well as a decline in circulating gonadotropin releasing hormone (GnRH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, and testicular 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD). These were associated with lead-induced degenerative changes in testicular tissues evidenced by shrunken seminiferous tubules, degeneration and sloughing of germ cells. Co-administration of zinc prevented lead-induced testicular injury by ameliorating oxidative stress, apoptosis, and inflammation through downregulation of XO/UA/caspase 3 pathway and upregulation of testicular 3β-HSD/17β-HSD. Conclusion This study demonstrated that zinc protected against lead-induced testicular toxicity via the downregulation of XO/UA/caspase 3 signaling.

O-233 Follicular fluid extracellular vesicles as a potential tool for human spermatozoa selection in vitro

Abstract Study question What characteristics and features of follicular fluid (FF) extracellular vesicles (EVs) of women of different ages can affect the functional characteristics of spermatozoa? Summary answer The size, lipid composition, and progesterone levels of the FF EVs change with a woman's age and affect the fertilizing ability of spermatozoa. What is known already Aging influences on molecular composition of the FF, reducing fertilization processes occurring in the fallopian tubes. FF is an important factor in activating spermatozoa during oocyte fertilization, enhancing the process of sperm hyperactivation and capacitation. FF progesterone is one of the main chemoattractants in the female genital tract, attracting sperm to the oocyte and starting the fertilization processes. Our previous studies have shown a significant effect of FF EVs of young women compared to advanced maternal age women (AMA) on the hyperactivation and motility of spermatozoa. The binding of FF EVs to the acrosoma of the sperm also was detected. Study design, size, duration FF EVs were obtained by sequential centrifugation at different rotational speeds. The sperm fraction was isolated, washed by differential centrifugation in a density gradient, suspended in the saline to a concentration of 10*6/ml and incubated with EVs (1:2) at 37 °C in CO2-incubator for 1 h. Next steps: immunofluorescent staining, test for acrosomal reaction of spermatozoa after incubation, NTA analysis, the lipid profile and progesterone measuring of FF EVs of women of different ages. Participants/materials, setting, methods All study participants signed a voluntary informed consent for the use of biological samples for research purposes. Sperm samples were isolated from seminal fluid (n = 21) aged 28-36. FF EVs were obtained from young (n = 14) aged 22-30 and AMA patients (n = 14) aged 42-47. The methods used in this work also include immunofluorescence microscopy, nanoparticle tracking analysis, flow cytometry, liquid chromatography–mass spectrometry, statistical data analysis. Lipids were extracted from EVs using a modified Folch extraction protocol. Main results and the role of chance Progesterone levels were significantly higher in FF EVs samples from young women compared to FF EVs samples from AMA women and controls (3951000 a.u., 598500 a.u., 6701 a.u.). This may explain the increase in sperm hyperactivation and motility after incubation with FF EVs. The difference in size (144.78 nm ± 9.92 VS 127.03 nm ± 17.17) and lipid composition of young women's FF EVs may explain the more significant functional and therapeutic effect on spermatozoa compared to AMA women FF EVs. A total of 389 vesicle lipids were identified. The level of cholesterol esters, cholesterol, lysophosphatidylcholines, phosphatidylcholines was 1,5 times higher in FF EVs of young women. The level of di- and triglycerides, ceramides, oxidized forms of phosphatidylcholines was higher in FF EVs of AMA women. The level of spontaneous acrosomal reaction was almost 3 times lower in sperm samples after incubation with FF EVs compared to the control (11,21% VS 32,88%). This supports the hypothesis that FF EVs may restrain premature acrosomal reaction in the fallopian tubes. Age-related changes are reflected in reproductive important biological processes, which leads to a change in the composition and functions of FF and affect the functional role of FF EVs. Limitations, reasons for caution Small amount of data. To be completely confident in the importance of the participation of FF EVs in the activation of spermatozoa, it is necessary to perform proteomic analyses of FF EVs to discuss the possible mechanisms underlying the effects of FF EVs of different age groups on sperm characteristics. Wider implications of the findings Further studies may help not only to complement the fundamental understanding of the processes of fertilization and the impact on spermatozoa morphofunctional characteristics, but also to develop a tool for improving ART outcomes in couples with male infertility. The solutions could be the use of FF EVs from young donors. Trial registration number not applicable

P-056 Oxidative stress and human semen quality: new results for diagnostic process of male infertility

Abstract Study question Is oxidative stress (OS) evaluated in human spermatozoa a predictive marker of better semen quality? Summary answer CellROX®Orange is a new fluorescent probe able to detect Reactive Oxygen Species (ROS) identifying a viable oxidized sperm fraction related to a better sperm performance. What is known already OS, defined as an unbalance between ROS production and antioxidant defences, is considered one of the causes of male infertility. OS evaluation in spermatozoa represents an important goal of research in this field because this parameter could be helpful to predict sperm fertilization ability to improve Assisted Reproductive Technology outcomes. By using different probes and methods for OS evaluation in spermatozoa or in semen, several studies were performed demonstrating a negative role of ROS on sperm functions. Such studies were not conclusive for the small number of included subjects, the high variability in the cohorts and the lack of validation. Study design, size, duration An observational study was conducted on 121 semen samples from patients undergoing routine semen analysis for couple infertility in the Andrology Laboratory of Careggi University Hospital of Florence from September 2021 to March 2022. Washed and Swim-up selected spermatozoa were incubated with two fluorescent probes, CellROX® Orange and Dihydroethidium (DHE), and revealed by flow cytometry. Participants/materials, setting, methods After routine semen analysis, the percentage of oxidized spermatozoa was evaluated with CellROX® Orange and DHE, and then correlated with standard semen parameters and sperm DNA fragmentation (sDF, evaluated by TUNEL/PI). Sperm kinematic parameters and hyperactivated motility were also assessed by C.A.S.A. system. Furthermore, CellROX® Orange positivity and phosphatidylserine membrane exposure (determined by Annexin V staining) or Caspase 3 and 7 activities (measured by FLICA™) were concomitantly evaluated. Main results and the role of chance We demonstrated that CellROX® Orange is able to detect hydrogen peroxide only in viable spermatozoa and the percentage of CellROX® Orange positive spermatozoa were positively associated with semen quality and negatively with sDF. To confirm these results, we performed the same experiments by using DHE, another probe which reveals hydrogen peroxide and superoxide anion in both viable and unviable cells. Similarly to CellROX® Orange, a positive correlation with semen quality and a negative one with sDF were found in viable spermatozoa. Conversely, in unviable spermatozoa, opposite associations were observed. These results indicate that oxidative status revealed by the two probes is related to a better sperm quality. To further investigate this possibility, we double labelled spermatozoa with CellROX® Orange and Annexin V (a marker of early signs of apoptosis) as well as with CellROX® Orange and FLICA™ (that detects caspase activity, a late sign of apoptosis). We found that CellROX® Orange mostly identifies spermatozoa without apoptotic features. Furthermore, we evaluated CellROX® Orange positivity in Swim-up selected spermatozoa, finding significantly higher levels in these samples respect to unselected samples, demonstrating once again that this probe identifies spermatozoa with better quality. Limitations, reasons for caution Our results indicate that within the viable oxidized spermatozoa there are cells with physiological and non-physiological intracellular ROS levels, however, they do not explain at what levels ROS cease to be functional and become deleterious for spermatozoa. Wider implications of the findings ROS evaluation in viable spermatozoa with the commercially available probes CellROX® Orange and DHE, allows the identification of the oxidized sperm fraction related to a sperm better performance. Therefore, the two probes may be useful to determine such fraction, likely improving the diagnostic process of male infertility. Trial registration number not applicable

Evaluation of a novel microfluidic chip-like device for purifying bovine frozen-thawed semen for in vitro fertilization

The aim of this study was to validate a novel sperm purification device, the VetCount™ Harvester, for use in bovine in vitro fertilization (IVF). The device's performance was compared to BoviPure™ gradient centrifugation, a commercially available and accepted routine technique. Semen quality parameters were assessed for frozen-thawed semen from six different bulls (n = 6) following sperm purification. For each bull two semen subsamples were purified utilizing BoviPure™ gradient centrifugation and the VetCount™ Harvester, including a third subsample as untreated control. Both treatments significantly increased the proportion of progressively motile sperm cells (84.4 ± 14.1% and 85.1 ± 7.8%, respectively) compared to the untreated semen (41.9 ± 18.8%). BoviPure™ gradient and VetCount™ Harvester selected predominantly viable acrosome intact (VAI) sperm cells with low membrane fluidity and low free intracellular calcium concentration [Ca2+]i (76.5 ± 4.4% and 78.6 ± 6.0%). Normalizing [Ca2+]i of VAI sperm cells (non-treated semen: [Ca2+]i = 1) VetCount™ Harvester purified spermatozoa (0.67 ± 0.10) showed significantly lower [Ca2+]i than BoviPure™ treated sperm (0.84 ± 0.14; P < 0.05). Subsequently, the fertilizing ability of the spermatozoa was evaluated performing a competitive fertilization assay. Sperm cells from both treatment groups were fluorescently labelled using different dyes and added in equal amounts to in vitro matured oocytes. After 18 h co-incubation, the origin of the fertilizing sperm cell was evaluated via fluorescence microscopy. In two bulls, VetCount™ Harvester selected sperm that fertilized significantly more oocytes then BoviPure™ treated sperm, in another bull it was the opposite. For three bulls no difference was observed.We conclude that the VetCount™ Harvester selects a high-quality, fertile sperm fraction from frozen-thawed bull semen. However, some considerations have to be kept in mind for the direct use of the isolated sperm fraction in IVF.

Seminal cadmium affects human sperm motility through stable binding to the cell membrane.

Environmental pollutants are claimed to be major factors involved in the progressive decline of the fertility rate worldwide. Exposure to the heavy metal Cadmium (Cd) has been associated with reproductive toxicity due to its ionic mimicry. However, the possible direct accumulation of Cd in human sperm cells has been poorly investigated. In this study, we aimed to clarify the possible direct effect of Cd exposure on sperm function through the analysis of its cell accumulation. Semen samples from 30 male subjects residing in high environmental impact areas and adhering to the "Exposoma e Plurifocalità nella Prevenzione Oncologica" campaign for testis cancer prevention were compared with semen samples from 15 males residing in low exposure areas. Semen levels and cell Cd content were quantified by inductively coupled plasma (ICP) spectroscopy. Cell Cd distribution was assessed by scanning electron microscopy coupled with energy dispersive spectroscopy (SEM-EDS). The impact of Cd on sperm function was evaluated by the in vitro exposure to the heavy metal, whilst possible scavenging approaches/agents were assessed. In addition to higher values of semen Cd, exposed subjects showed a reduction in total motile sperm fraction compared to not-exposed controls (59.6% ± 13.6% vs. 66.3% ± 7.3%, p = 0.037). Semen Cd levels were also significantly correlated with SEM-EDS signals of Cd detected on the head and neck of sperm (respectively p = 0.738, p < 0.001 and ρ = 0.465, p < 0.001). A total of 2h of in vitro exposure to 0.5μM Cd was associated with a significant reduction of sperm progressive motility. Scavenging approaches with either hypo-osmotic swelling or 10μM reduced glutathione were ineffective in blunting cell Cd and restoring motility. The reduction of exposure levels appears to be the main approach to reducing the reproductive issues associated with Cd.

Biochemical features of X or Y chromosome bearing spermatozoa for sperm sexing

This review presents information on the biochemical features of spermatozoa bearing an X or Y chromosome, enabling to obtain a sperm fraction with a pre-defined sex chromosome. Almost the only technology currently used for such separation (called sexing) is based on sperm fluorescence-activated cell sorting in regard to DNA content. Besides its applications, this technology made it possible to analyze the properties of isolated populations of spermatozoa bearing an X or Y chromosome. In recent years, a number of works have appeared proving the existence of differences between these populations at the transcriptome and proteome level. It is noteworthy that these differences are primarily related to energy metabolism and structural proteins of the flagellum. New methods of sperm enrichment with X or Y chromosome cells are based on the differences in motility between spermatozoa with different sex chromosomes. Sperm sexing is a widespread part of the protocol of artificial insemination of cows with cryopreserved semen, it allows to increase the proportion of offspring of the required sex. In addition, advances in the separation of X and Y spermatozoa may allow this approach to be applied in clinical practice to avoid sex-linked diseases.