Semen Liquefaction Research Articles

Synthesis and Optimization of Small Molecule Inhibitors of Prostate Specific Antigen.

Semen liquefaction is a postejaculation process that transforms semen from a gel-like (coagulated) form to a water-like consistency (liquefied). This process is primarily regulated by serine proteases from the prostate gland, most prominently, prostate-specific antigen (PSA; KLK3). Inhibiting PSA activity has the potential to impede liquefaction of human semen, presenting a promising target for nonhormonal contraception in the female reproductive tract. This study employed triazole B1 as a starting compound. Through systematic design, synthesis, and optimization, we identified compound 20 (CDD-3290) as a 216 nM inhibitor of PSA with better stability in media than triazole B1. Further, we also evaluated the selectivity profile of compound 20 (CDD-3290) by testing against closely related proteases and demonstrated excellent inhibition of PSA versus α-chymotrypsin and elastase and similar potency versus thrombin. Thus, compound 20 is an improved PSA inhibitor that can be tested for efficacy in vitro or in the female reproductive tract.

The effect of different concentrations of N-acetyl cysteine on liquefaction, quality and fertility of chilled semen in dromedary camel

The objectives of the current study were to evaluate the effects of N-acetyl cysteine (NAC) on semen liquefaction and sperm quality during short-term preservation in dromedary camel. In experiment 1, 9 ejaculates from 3 bulls were diluted in the base extender with different concentrations of NAC (0, 6, 12, and 24 mM). The viscosity reduction was investigated at 5, 15, 30, and 120 min and 24 h post-dilution. In the second experiment, 15 ejaculates from 5 dromedary bulls were diluted with an extender containing 0, 6, and 12 mM of NAC, and viability, motility, and kinematics, as well as functional and DNA integrity of spermatozoa, were evaluated at 2, 12, 24, and 48 h of chilled storage at 5 °C. Experiment 3 evaluated the effect of adding NAC in the semen extender by carrying out 60 inseminations with 26- and 48-hour chilled semen. The pregnancy rates were compared between NAC and the control extender. Adding NAC to the extender resulted in a significant drop in viscosity compared to the raw and extended samples at all evaluation time points with a dose-dependent effect (P 0.05). At the same time, concentrations greater than 6 mM lowered viability, motility, and functionality of sperm from 12 h post-dilution (P < 0.05). Chilled sperm in the EXT+NAC 6 mM showed a marked improvement in DNA integrity status from 2 h post dilution onwards, with better motility and functional integrity at 48 h compared to the control (P < 0.05). Pregnancy rates with 26 h of chilled semen were similar between NAC-treated sperm and the control (33.33% vs. 38.09%, respectively; P > 0.05), corroborating the safety of NAC regarding its effect on sperm fertilizing capacity. In conclusion, adding NAC to the semen extender at concentrations of 6 mM assists with the liquefaction of dromedary semen without compromising sperm quality during subsequent storage in chilled status.

Site-specific N-glycan changes during semen liquefaction

Normal liquefaction of semen is one of the key steps to ensure the smooth progress of fertilization, and glycosylation has been reported to be involved in the whole process of fertilization. Till now, it is still unclear whether and how glycosylation changes during the liquefaction process of semen. In this study, by performing a glycoproteomic analysis of human semen with the liquefaction process (liquefaction time of semen: 0 min vs 30 min) using our recently developed StrucGP software combined with the Tandem Mass Tags (TMT) based quantification, we identified 25 intact glycopeptides (IGPs) from 10 glycoproteins in semen that were significantly changed during liquefaction, including 23 up-regulated and two down-regulated. Among the 23 up-regulated glycopeptides, half were modified with sialylated glycans, suggesting that sialylated glycans may play a key role in the semen liquefaction process. The data provide an invaluable resource for further studies on the role of glycosylation during semen liquefaction.

Characterization of proteases in the seminal plasma and spermatozoa of llama

Camelids' semen has peculiar characteristics that differentiate it from other species, including the highly viscous aspect of seminal plasma that greatly difficult sperm manipulation and the development of techniques such as cryopreservation, artificial insemination, and/or in vitro fertilization. The presence of proteases in the seminal plasma is responsible for semen liquefaction, and sperm functionality to achieve fertilization. The enzymatic and molecular composition of the semen of llama remains unknown. Therefore, the goal of the study was to characterize the protease activity and composition of the seminal plasma and sperm of llama semen. The proteolytic activity was performed using gelatine zymography and the composition by mass-spectrometry.Metallo-proteases were the major source of gelatinolytic activity in seminal plasma, while serine-peptidases were the main enzymes of sperm cells. Matrix Metalloproteinase 2 (MMP2) was the most prominent metallo-protease of llama seminal plasma characterized under the exposure of different inhibitors (EDTA and benzamidine) and by a specific immunodetection. Moreover, the prostate and epididymis were identified as potential sites of its synthesis and secretion.Outstandingly, this metalloproteinase was undetectable in llama sperm. Regarding, the molecular composition of semen by mass-spectrometry, 4 metallo-, 9 serine-, 8 threonine-, and 1 aspartic-peptidases were identified alongside 15 regulators in the sperm cell; where 24 were directly or indirectly interacting. Whereas 6 metallo-, 12 serine-, 3 cysteine-, and 1 aspartic-peptidases were identified, besides 7 inhibitors and 5 regulators in llama seminal plasma where 30 of them were directly or indirectly interconnected. This is the first study describing a partial degradome of llama seminal plasma and spermatozoa suggesting significant differences especially the absence of MMP2 in spermatozoa in contrast to data observed in other species. The characterization of proteases in llama semen will provide a better understanding of the molecular mechanisms involved in the in vivo or in vitro fertilization process in this species.

COVID-19 and Semen Fluid Parameters, a Retrospective Study from Infertility Clinics.

The study of the effects of SARS-CoV-2 infection and/or vaccination on semen fluid analysis (SFA) parameters is still incomplete. The aim of this study is to assess the effect of COVID-19 infection and vaccination on sperm parameters for a sample of individuals visiting multi-infertility clinics in Jordan. SFA records were collected retrospectively between September and November 2021 and analyzed using Jamovi software (version 2.2.5 for Windows); p-values < 0.05 were considered statistically significant. Sperm concentration, progressive motility, normal morphology, and semen liquefaction time, volume, and viscosity were compared among two data categories. In the first category of data, SFA records from 354 participants were separated into four groups: only vaccinated, infected and vaccinated, neither infected nor vaccinated, and only infected. In the other category, SFA from 49 subjects before their infection and/or vaccination and after were classified into the same mentioned groups and analyzed. There were no statistically significant differences between the studied parameters in the SFA records in the first data category and the second. Nevertheless, the sperm concentration was higher among vaccinated subjects compared to unvaccinated ones (p = 0.04). It is concluded that SARS-CoV-2 infection and vaccines have no negative effects on SFA parameters.

Ejaculation: the Process and Characteristics From Start to Finish.

Semen analysis serves as the initial step in the evaluation of male infertility. However, given the difficulty in interpreting abnormal findings, physicians and patients often struggle with understanding the results. In this review, we aim to review the normal physiology of ejaculation and create an accessible resource for interpreting abnormal semen volume, viscosity, liquefaction, pH, appearance, and color. Emerging evidence has revealed that men with genitourinary tract infections have a greater number of seminal leukocytes, which may result in clumping of motile sperm and altered morphology. Hence, these patients may have abnormal sperm parameters secondary to their health status. Recent findings have further characterized the semen liquefaction process, suggesting that increased levels of semenogelin and decreased levels of proteases and plasminogen activators (e.g., urokinase and chymotrypsin) may be associated with the failure of semen to convert to a watery consistency. This article creates a resource which may be referenced when abnormalities in semen analysis are encountered. We offer a comprehensive overview of normal ejaculation physiology and abnormal variants in male ejaculate volume-including aspermia, anejaculation, retrograde ejaculation, and hypo- and hyperspermia-and their potential etiologies. Additionally, we discuss several processes (infection, inflammation, and dysfunction of male sex glands) which may affect semen viscosity, liquefaction, and pH. Finally, our discussion of the potential colors of male ejaculate is meant to reduce the anxiety of both patient and provider. Through a better understanding of the process and varying characteristics of ejaculation, physicians may adequately counsel their patients on abnormal findings and concerns regarding infertility.

Frequency of Abnormal Sperm Motility in Male Smokers with Primary Infertility

Objective: To assess the frequency of abnormal sperm motility and related factors in male smokers with primary infertility. Place and duration of the study: From 8th Feb 2021 to 8th Feb 2022 at Gynaecology Department of Bakhtawar Amin Trust Teaching Hospital Multan for 1 year Study design: A retrospective study Methodology: A total of 120 patients were included in the study among which 100 patients were smokers and 20 patients were non-smokers. For sample collection, patients were asked to masturbate and collect the first portion of ejaculate to ensure the maximum amount of sperm. Semen analysis was done on regular basis under a light microscope according to WHO guidelines. After the semen liquefaction, the 10μl of the sample was analyzed on a glass slide covered with a coverslip. For evaluating the motility, 200 spermatozoa were counted in 5 fields at 200x magnification. Results: Six non-smokers (30%) showed low motility. In comparison, 76 smokers (63.3%) showed low motility. Low motility was observed more in smokers as compared to smokers. The difference between motility in smokers and non-smokers was statistically significant (&lt;0.001) (Chi sq= 23.55). Cigarette smoking also affected the sperm count as 24 (20%) smokers had a low sperm count. Conclusion: Smoking affects vital sperm characteristics such as sperm count and motility and in turn, be a potential cause of subfertility or infertility. Keywords: Cigarette smoking, male fertility, sperm count, sperm motility.

The impact of bacterial prostatitis and IL-17 in male infertility

A case-control study aimed to assess the role of IL-17 serum level and bacterial prostatitis in development of male infertility. 120 patients from January to June 2021 included 60 prostatitis infertile patients, 30 prostatitis patients and 30 infertile patients as well as 30 healthy fertile male subjects. Blood and semen sample were collected from all subjects. 3 ml of venous blood was put in gel tube to separate serum estimate the IL-17 level by ELISA. A loop full of semen was taken to isolate bacterial causes and the remaining semen was analyzed to detect semen liquefaction, volume, appearance and basic sperm parameters (density, motility, viability and morphology). The results showed a significant increase (P&lt;0.05) in serum concentration of IL-17 in prostatitis infertile patients than prostatitis compared to infertile patients. According to a grade of the infertile male, the concentration of IL-17 increased with severity of disease and this is considered as a biomarker for progression. The results revealed that E.coli followed by S.aureus is the most common bacterial cause of prostatitis infection. The current study concluded that the infection with prostatitis disease affects the body's immune response represented by increased level of IL- 17 and correlated with infertility.

Human live spermatozoa morphology assessment using digital holographic microscopy

Digital holographic microscopy (DHM) was applied for the morphological assessment of live intact spermatozoa from fertile and infertile men directly after semen liquefaction. This method allowed us to study the sperm population directly from the sample droplet and not only from the focal plane of the microscope as in classical optical microscopy. The newly implemented 3-dimensional sperm morphological parameters (head height, acrosome/nucleus height, head/midpiece height) were included in morphological assessment of semen samples from fertile and infertile individuals. The values of the 3D parameters were less variable in fertile men than for infertile ones. DHM was also used to compare the morphological profiles of spermatozoa after applying the “swim-up” and gradient centrifugation techniques. During selection, the most statistically significant differences were observed after separation with a Percoll gradient of 90% and a 60-min “swim-up” procedure versus ‘native’ unfractionated samples. This shows that the developed methodology can be efficiently used for the selection of morphologically sound spermatozoa. The motility type for each spermatozoon was also assessed. The results indicate that the extension of the number of morphological parameters with new 3D parameters and the simultaneous assessment of sperm motility may be valuable addition to sperm examination.

Blocking serine protease activity prevents semenogelin degradation leading to hyperviscous semen in humans.

Prostate-specific antigen (PSA) is a prostate-specific serine protease enzyme that hydrolyzes gel-forming proteins (semenogelins) and changes the semen from gel-like to watery viscosity, a process called semen liquefaction. Highly viscous semen and abnormal liquefaction reduce sperm motility and contribute to infertility. Previously, we showed that nonspecific serine protease inhibitor (AEBSF) prevented proteolytic degradation of semenogelin in mice. However, it is unclear whether similar effect could be recapitulated in fresh human ejaculates. Therefore, in this study we evaluated the effect of AEBSF on the degradation of semenogelin (SEMG1) and its subsequent impact on semen liquefaction and sperm motility in fresh semen ejaculates collected from healthy men. We found that AEBSF showed a dual contraceptive action where it effectively 1) prevented degradation of SEMG1 resulting in viscous semen and 2) decreased sperm motility in human semen samples. However, the impact of AEBSF on sperm motility and viability could be due to its inhibitory activity toward other serine proteases or simply due to its toxicity. Therefore, to determine whether inhibition of PSA activity alone could disrupt SEMG1 degradation and contribute to hyperviscous semen, a neutralizing PSA antibody was used. We found that PSA antibody effectively prevented SEMG1 degradation with a subtle impact on sperm motility. These findings suggest that the target inhibition of PSA activity can prevent proteolytic degradation of SEMG1 and block liquefaction process, resulting in hyperviscous semen. As it is currently unknown if blocking semen liquefaction alone could prevent pregnancy, it needs further extensive studies before drawing any translational conclusions.

Morphological and ultrastructural changes in seminal coagulum of the squirrel monkey (Saimiri collinsi Osgood, 1916) before and after liquefaction

Studies with squirrel monkey semen are of special interest due to the large amount of coagulation that is a component of the semen, which is a problem that has to be overcome when the objective is harvesting of gametes. In the present study, there was characterization of the seminal coagulum of captive S. collinsi. Four samples of ejaculates were collected using electroejaculation procedures from four animals. The aim in conducting this study was to evaluate seminal coagulum of S. collinsi using histological and scanning electron microscopy (SEM) procedures before and after semen liquefaction in an ACP-118® extender. Seminal coagulum of S. collinsi was composed of a superficial plate (external), which coats the spongy seminal plasma matrix of S. collinsi. Additionally, there were sperm in the external and internal components of the coagulum with these gametes being isolated or grouped and with there being a heterogeneous distribution of gametes. The supplementation of semen with ACP-118® resulted in a partial dissolution of the seminal plate and spongy matrix portions of the seminal coagulum within the first hour of incubation.

Clinical Efficacy of Prodom-Assisted Urokinase in the Treatment of Male Infertility Caused by Impaired Semen Liquefaction.

Purpose To evaluate the clinical efficacy of prodom in the administration of urokinase in the vagina in couples with impaired semen liquefaction. Materials and Methods Overall, 261 patients with impaired semen liquefaction were randomly divided into prodom-assisted urokinase treatment (PAUT) group (n = 91), syringe-assisted urokinase treatment (SAUT) group (n = 86), and traditional treatment (TT) group (n = 84) in the first stage. If the first stage of treatment failed, other treatment methods were initiated instead and the patients were grouped according to the newer treatment method in the second stage. The pregnancy rate, time-to-conception, and treatment costs were evaluated in each group. Results In the first stage, the pregnancy rate in the PAUT, SAUT, and TT groups was 69.23%, 29.07%, and 22.62%, respectively; the time-to-conception was 2.66 ± 1.44, 3.69 ± 2.61, and 3.86 ± 3.00 months, respectively; the treatment costs were 658.18 ± 398.40, 666.67 ± 507.50, and 680.56 ± 480.94 $, respectively. The pregnancy rate and time-to-conception were different in the PAUT group compared with those in SAUT and TT groups (all P < 0.05). However, the difference in treatment costs was not significant (P = 0.717). In the second stage, 154 nonpregnant patients were divided into nine treatment groups, and the effects of changing TT to PAUT on the pregnancy rate, time-to-conception, and treatment costs were observed to be different from those of other treatments (all P < 0.05). Conclusion Prodom-assisted urokinase can effectively treat male infertility secondary to impaired semen liquefaction.

Purification of cryopreserved camel spermatozoa following protease-based semen liquefaction by lectin-functionalized DNA-defrag magnetic nanoparticles.

Although incorporating proteases into sperm medium is considered the most effective procedure to eliminate camel semen viscosity, it drastically affects viability, morpho-functional properties and, hence, fertilization potential of spermatozoa. The present work aimed at evaluating adequacy of employing magnetic nanoparticles-based sperm purification technique for eluting impaired and apoptotic camel spermatozoa from cryopreserved semen doses following protease-based semen liquefaction. Thirty cryopreserved semen doses (50 x 106 sperm/straw) representing the following liquefaction treatments: control (untreated), 0.1mg/ml papain or 5 U/ml bromelain were used (n=10 straws per treatment). Immediately after thawing (38°C for 40s), sperm concentration of each straw within treatment was readjusted to 15 x 106 sperm/mL by dilution in PBS (37°C). Sperm physical and cytological properties were then assessed (non-purified semen). Thereafter, each specimen was subjected to lectin-functionalized DNA-defrag magnetic nanoparticles sperm purification, and the same sperm traits were re-evaluated after undergoing purification (purified semen). Sperm DNA fragmentation level within each group, prior to and after magnetic nano-purification, was also determined by fluorescent imaging. The results showed a dramatic improvement (p<.05) in post-thaw motility (%), viability (%), normal sperm (%), intact acrosome (%) and HOST-reacted (%) spermatozoa in protease-liquefied semen following sperm magnetic nano-purification. Additionally, the highest (p<.05) DNA fragmentation level was recorded in all cryopreserved semen groups prior to purification, whereas the lowest (p<.05) was observed in the protease-treated specimens after magnetic nano-purification. These results indicate that protease-based semen liquefaction prior to cryopreservation in conjunction with magnetic nano-purification post-thawing holds potential for reducing the proportion of damaged and dead spermatozoa, hence improving camel sperm fertilization competence.

A NEW METHOD FOR ACCURATE PREDICTION OF SPERMATOGENESIS: FSH/T AND T IN SEMINAL PLASMA

A NEW METHOD FOR ACCURATE PREDICTION OF SPERMATOGENESIS: FSH/T AND T IN SEMINAL PLASMA

Mechanism of semen liquefaction and its potential for a novel non-hormonal contraception†.

Semen liquefaction is a proteolytic process where a gel-like ejaculated semen becomes watery due to the enzymatic activity of prostate-derived serine proteases in the female reproductive tract. The liquefaction process is crucial for the sperm to gain their motility and successful transport to the fertilization site in Fallopian tubes (or oviducts in animals). Hyperviscous semen or failure in liquefaction is one of the causes of male infertility. Therefore, the biochemical inhibition of serine proteases in the female reproductive tract after ejaculation is a prime target for novel contraceptive development. Herein, we will discuss protein components in the ejaculates responsible for semen liquefaction and any developments of contraceptive methods in the past that involve the liquefaction process.

Serine protease inhibitor disrupts sperm motility leading to reduced fertility in female mice†.

Inhibition of the sperm transport process in the female reproductive tract could lead to infertility. We previously showed that a pan-serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), blocked semen liquefaction in vivo and resulted in a drastic decrease in the number of sperm in the oviduct of female mice. In this study, we used a mouse model to test the efficacy of AEBSF as a reversible contraceptive, a sperm motility inhibitor, and a spermicide. Additionally, this study evaluated the toxicity of AEBSF on mouse vaginal tissues in vivo and human endocervical cells in vitro. We found that female mice treated with AEBSF had significantly less pups born per litter as well as fertilization rates in vivo compared to the vehicle control. We then showed that AEBSF reduced sperm motility and fertilization capability in vitro in a dose-dependent manner. Furthermore, AEBSF also exhibited spermicidal effects. Lastly, AEBSF treatment in female mice for 10 min or 3 consecutive days did not alter vaginal cell viability in vivo, similar to that of the vehicle and non-treated controls. However, AEBSF decreased cell viability of human ectocervical (ECT) cell line in vitro, suggesting that cells in the lower reproductive tract in mice and humans responded differently to AEBSF. In summary, our study showed that AEBSF can be used as a prototype compound for the further development of novel non-hormonal contraceptives for women by targeting sperm transport in the female reproductive tract.

Ramifications of protease-based liquefaction of camel semen on physical, kinematic and surface glyco-pattern of cryopreserved spermatozoa

The efficiency of incorporating different proteases in the diluent for reducing camel semen viscosity, and subsequent ramifications on morpho-functional and glycan surface properties of cryopreserved spermatozoa were investigated. Ejaculates (n = 48) were collected from three adult camels, Camelus dromedarius, during the breeding season (January - March). A portion of each raw ejaculate was evaluated for sperm physical and morphological traits, whereas the other portion was divided into three aliquots assigned for the following liquefaction treatments: control (untreated), 0.1 mg/mL papain or 5 U/mL bromelain. All samples were diluted with Tris-lactose diluent containing the anti-enzyme E-64 to neutralize both proteases before being processed for cryopreservation. Post-thaw physical and kinematic properties of spermatozoa were analyzed using a computer-assisted sperm analysis (CASA) system. The sperm surface glycocalyx pattern was evaluated with a panel of 14 fluorescent lectins. Although bromelain was more effective in elimination of semen viscosity, there was a negative correlation between bromelain supplementation and values for the variables: normal sperm, intact acrosome and intact sperm cell membrane. Bromelain supplementation, compared to papain-treated and control samples, was positively correlated with secondary sperm abnormalities, increased straight-line velocity (VSL, μm/s) and straightness (%) of spermatozoa. Results from the glycan analysis indicated that both proteases did not affect the N-linked glycan content of the entire sperm surface, whereas the treatment with proteases induced little change in N-acetylgalactosamine and fucose terminating glycans in the tail region of the sperm. Functional studies are needed to evaluate the sperm fertility rates of bromelain- and papain-treated semen for application in camel assisted reproductive technologies.

Semen levels of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) protein families members in men with high and low sperm DNA fragmentation

Matrix Metalloproteinases (MMPs) and their regulators – Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) – participate in extracellular matrix remodeling, fibrosis, and semen liquefaction, as well as to inflammatory activity. Seminal plasma has been shown to contain MMPs (MMP-2 and MMP-9) and TIMPs (TIMP-1 and TIMP-2). Also, a link between MMPs gene expression and excessive reactive oxygen species (ROS) has been established. In semen, ROS are associated with altered sperm function and increased DNA fragmentation. In this study, it is hypothesized that seminal MMPs and TIMPs levels are associated with sperm DNA fragmentation due to the fact that MMPs have been associated with semen quality. We also hypothesized that these proteins could predict DNA fragmentation status in sperm. Therefore, this study set out to verify if sperm DNA fragmentation levels relate to seminal levels of members of the MMP and TIMP protein families. The High sperm DNA fragmentation group presented lower seminal plasma levels of MMP-2, MMP-7, TIMP-1, TIMP-2 and TIMP-4 when compared to Low sperm DNA fragmentation group. Also, samples in the high sperm DNA fragmentation group presented higher acrosome integrity and lower mitochondrial activity levels when compared to low sperm DNA fragmentation samples. In the logistic regression analysis, MMP-2, MMP-7, and TIMP-4 classified samples as low and high sperm DNA fragmentation, with an overall model fit of 74.5%. Results from this study may demonstrate a specific inflammatory mechanism in samples with high sperm DNA fragmentation. This, in turn, can lead to the development of new studies regarding this mechanism and, in the future, create an opportunity to treat these patients for sperm DNA fragmentation by treating inflammatory seminal activity.

Study on Correlation between Serum Prostate Specific Antigen and Various Prostatic Pathology

Introduction: Prostatic enlargement that may due to any cause may give rise to bladder outlet obstruction. Prostatic specific antigen is the enzyme that is responsible for liquefaction of semen within a few minutes after it has clotted. Prostatic specific antigen is a widely used tumor marker for prostatic cancer. Prostatic specific antigen levels in the blood go up if the barrier between the lining epithelium and the blood stream is damaged. This study was done to determine the correlation between serum Prostatic specific antigen level and histological diagnosis in prostatic biopsy.Material and Methods: This is a one year prospective study carried out in the Department of Pathology, Nobel Medical College from August 16, 2016 to August 15 2017. A total of 175 cases were included in the study. Patient Prostatic specific antigen level were noted and biopsy specimen was collected after operation. Histopathological examination was done and correlation between HPE diagnosis and serum Prostatic specific antigen level was done.Results: Benign Prostatic Hyperplasia was the most common diagnosis that was encountered. Majority of the cases had a serum Prostatic specific antigen level less than 10 ng/ml. Serum Prostatic specific antigen level of more than 30 ng/ml was seen only in prostatic carcinoma.Conclusions: Serum Prostatic specific antigen is organ specific but not a disease for prostate. It can be used to monitor the carcinoma of the prostate rather than the diagnosis of the carcinoma.

Seminal GSTM3, CRISPLD2, and RARRES1 diagnose sperm functional alterations

Sperm functional alterations are considered the main cellular mechanisms of male infertility, observed in 25% of infertile men. We have previously shown, using a shotgun proteomics approach, that Glutathione S-transferase Mu3 (GSTM3), Cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) and Retinoic acid receptor responder protein 1 (RARRES1) seminal proteins are associated with sperm abnormalities. Current limitations in the quantitative aspect of a shotgun proteomics approach demand these results be validated in a larger cohort. In this study, thus, we sought to evaluate, using a confirmatory method, GSTM3, CRISPLD2 and RARRES1 levels in seminal plasma in association with sperm mitochondrial activity and DNA fragmentation. Prospective study. 197 normozoospermic men were recruited for this study. Semen was collected by masturbation following 2 to 5 days of ejaculatory abstinence. An aliquot was used for semen analysis, another for sperm functional evaluation, and the remaining semen volume was centrifuged for separation of seminal plasma. Sperm functional data were used to separate the experimental groups: High (control, n=27) and Low (study, n=27) sperm mitochondrial activity, and Low (control, n=29) and High (study, n=29) sperm DNA fragmentation. Seminal plasma of all samples was utilized to evaluate the levels of GSTM3, CRISPLD2 and RARRES1 by Western blotting. Data were normalized using total protein (Ponceau S staining) and multiplied by the ejaculate volume, to obtain the total protein levels in the ejaculate. Groups were compared using an unpaired Student’s t test (α=5%). GSTM3 was observed as two different bands (27 kDa, its expected molecular mass, and 38 kDa). Total levels of both bands were twice as high in samples with sperm mitochondrial alterations. CRISPLD2 was observed as three different bands (17, 28 and 38 kDa, expected molecular mass = 51 kDa), suggesting its digestion by seminal plasma proteases during semen liquefaction. Total seminal levels of the 17 and 28 kDa bands were decreased in samples with high sperm DNA fragmentation (1.6-fold and 2.6-fold decrease, respectively). Likewise, RARRES1 was identified as two bands (50 and 62 kDa, expected molecular mass = 33 kDa), suggesting post-translational modifications and/or dimer formation. The 50 kDa band was 1.9-fold decreased in the seminal plasma of men with sperm DNA fragmentation. Seminal plasma proteins directly reflect sperm functional alterations and, thus, male infertility. Seminal plasma proteins may be used as markers of functional alterations. In this study, we confirmed that this is the case for GSTM3 (sperm mitochondrial activity), and CRISPLD2 and RARRES1 (sperm DNA fragmentation).