Paula Toni Del Giudice

Effect of varicocele on sperm function and semen oxidative stress

Study Type – Aetiology (case control)

Adolescent varicocele: improved sperm function after varicocelectomy

OBJECTIVE To assess the effect of varicocelectomy on sperm function (DNA integrity and mitochondrial activity) and levels of lipid peroxidation in seminal plasma of adolescents. DESIGN Prospective study. SETTING Patients recruited from a local public school. PATIENT(S) Adolescents (14-19 years old), Tanner stages IV or V with varicocele grades II or III, attending a local public school. INTERVENTION(S) Two semen collections with a one week interval between collections before bilateral varicocele repair using subinguinal microsurgical varicocelectomy, and two semen collections with a one week interval between collections three months after the surgery. MAIN OUTCOME MEASURE(S) Rate of sperm DNA fragmentation as assessed by the Comet assay and categorized as classes I (no DNA fragmentation) to IV (high DNA fragmentation). Rate of mitochondrial activity as assessed by the diaminobenzidine assay and categorized as grades I (all mitochondria active) to IV (all mitochondria inactive). Levels of lipid peroxidation in seminal plasma by a colorimetric method that quantifies a lipid peroxidation subproduct (malondialdehyde). RESULT(S) Concerning DNA integrity, the samples after varicocelectomy showed more spermatozoa with intact nuclear DNA (grade I) and less spermatozoa with Comet grades II, III, and IV. Regarding mitochondrial activity, the samples after varicocelectomy showed less cells with inactive mitochondria (class III). No differences were observed in classes I, II, and IV. Concerning lipid peroxidation, no significant differences were observed. CONCLUSION(S) This study was able to demonstrate that varicocelectomy in adolescents is associated with increased sperm DNA integrity and mitochondrial activity. However, levels of seminal products of lipid degradation (malondialdehyde) are not different.

Effect of leukocytospermia and processing by discontinuous density gradient on sperm nuclear DNA fragmentation and mitochondrial activity

PurposeTo assess the effect of leukocytospermia and semen processing on sperm DNA and mitochondria.MethodsTwenty-two patients with and 41 without leukocytospermia were included. Sperm DNA fragmentation was assessed by the Comet assay, and mitochondrial activity by a colorimetric method for active mitochondria. Semen was processed using Percoll, and motility, DNA fragmentation, and mitochondrial activity were analyzed pre- and post-processing.ResultsNo differences were observed in age, abstinence, volume, sperm morphology, progressive motility, concentration, and vitality (p > 0.10). Variables were grouped according to time (pre- vs post-processing) and group (leukocytospermia vs non-leukocytospermia) because no interactions could be observed. Leukocytospermia was associated to increased DNA fragmentation, while semen processing led to a decrease in DNA fragmentation and to increased mitochondrial activity.ConclusionWhile semen processing selects sperm with higher rates of DNA integrity independent of the presence or absence of leukocytes in semen, samples without leukocytospermia present more sperm without DNA fragmentation. Semen processing also selects sperm with higher mitochondrial activity.

Proteomic analysis of seminal plasma in adolescents with and without varicocele

OBJECTIVE To compare proteomic profiles of seminal plasma from adolescents with varicocele and changes in semen quality with the plasma from adolescents with varicocele without seminal changes and from adolescents without varicocele. DESIGN Observational study. SETTING Patients in an academic research environment. PATIENT(S) Adolescents without varicocele (control group), adolescents with varicocele and normal semen quality (VNS group), adolescents with varicocele and abnormal semen quality (VAS group). INTERVENTION(S) Two semen collections at 1-week interval. Protein separation by two-dimensional protein electrophoresis, analysis by gel densitometry, and identification by mass spectrometry. MAIN OUTCOME MEASURE(S) Overexpressed proteins in each group, observed by increased densitometric signal in gels, and exclusively identified proteins in each group. RESULT(S) No differences were observed among the three groups regarding clinical parameters. In semen analysis, the VAS group presented lower sperm concentration, motility, and morphology compared with the VNS and control groups. Forty-seven protein spots of interest were submitted to mass spectrometry identification. Apoptosis regulation proteins were overexpressed in the VAS group, whereas spermatogenesis proteins were overexpressed in the VNS group. Controls presented proteins related to homeostasis. CONCLUSION(S) Changes in the proteomic profile of adolescents with varicocele and normal semen parameters (VNS group) indicate that normal semen analysis may not reflect alterations in proteins in seminal plasma. Implementation of proteomics will help characterize proteins identified in seminal plasma and will facilitate detection of new proteins associated with spermatogenesis and sperm function.

Proteomic analysis of follicular fluid from women with and without endometriosis: new therapeutic targets and biomarkers.

Endometriosis is a gynecological disease that affects women of reproductive age. The protein profiles of women with endometriosis who were able or unable to achieve pregnancy and women without endometriosis who did achieve pregnancy were compared in this study. The follicular fluid was collected from 21 patients undergoing in vitro‐fertilization treatment, according to the following groups: nine women in the control group (Group C), four women with endometriosis who achieved pregnancy (Group E.P), and eight women with endometriosis who did not achieve pregnancy (Group E.NP). Follicular fluid proteins were separated using 2D‐electrophoresis,and their spots were compared, excised, and submitted to LC–ESI‐MS/MS for proteins identification. The analysis showed 29 differentially expressed spots among the groups, and from these, 21 proteins were identified. Analysis showed some functional enrichment in the E.P group, including response to oxidative stress and apoptosis, while the E.NP group showed functions related to response to reactive oxygen species and positive regulation of apoptosis. These data suggest that endometriosis leads to differential protein expression in the follicular fluid, which can influences the outcome of pregnancy. These proteins may be potential targets for better diagnostics and new therapeutic intervention in affected women, as well as assisting in comprehending the physiopathologic mechanisms underlying endometriosis. Mol. Reprod. Dev. 80: 441–450, 2013.

Unraveling the sperm proteome and post-genomic pathways associated with sperm nuclear DNA fragmentation

PurposeSperm DNA fragmentation has been suggested as a marker for infertility diagnosis and prognosis. Hence, understanding its impact on male physiology and post-genomic pathways would be clinically important. We performed the proteomics and functional enrichment analyses of viable spermatozoa from ejaculates with low and high sperm DNA fragmentation to identify protein expression and pathways altered in association with sperm DNA fragmentation.MethodsSperm DNA fragmentation using the Comet assay and the Komet 6.0.1 software was assessed in raw samples from 89 subjects from a human reproduction service. The Low and High sperm DNA fragmentation groups were formed according to the Olive Tail Moment variable. Spermatozoa proteins from these groups were pooled and analyzed by a shotgun proteomic approach (2D nanoUPLC-ESI-MSE). Differentially expressed proteins were used for a functional enrichment study.ResultsTwo hundred and fifty-seven proteins were identified or quantified in sperm from the Low and High sperm DNA fragmentation groups. Of these, seventy-one proteins were exclusively or overexpressed in the Low group, whereas twenty-three proteins were exclusively or overexpressed in the High group. One hundred and sixty-three proteins were conserved between these groups. We also functionally related the differentially expressed proteins in viable spermatozoa from the groups. Processes such as triacylglycerol metabolism, energy production, protein folding, response to unfolded proteins, and cellular detoxification were found to be altered in these cells.ConclusionsSperm DNA fragmentation is associated with differential protein expression in viable spermatozoa. These proteins may potentially be used as biomarkers for sperm DNA integrity.

DIFFERENTIAL SEMINAL PLASMA PROTEOME ACCORDING TO SEMEN RETRIEVAL IN MEN WITH SPINAL CORD INJURY

OBJECTIVE To evaluate protein expression profile and to quantify proteins present in seminal plasma from men with spinal cord injury (SCI) and healthy men without SCI. DESIGN Experimental study. SETTING University hospital. PATIENT(S) Twelve SCI patients divided into two groups, six who underwent electroejaculation (EEJ) and six who underwent penile vibratory stimulation (PVS); and ten control subjects presenting normal sperm motility and concentration. INTERVENTION(S) EEJ and PVS. MAIN OUTCOME MEASURE(S) The seminal plasma protein profile was analyzed by two proteomic strategies: data-independent label-free quantitative proteomics (MS(E)) and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). RESULT(S) A total of 638 different proteins were identified by MS(E) and 18 by 2D SDS-PAGE followed by tandem mass spectrometry. Interactome analysis showed key reproductive biologic processes-insemination, sperm and oocyte fusion, and acrosome reaction-related to all groups, as were triglyceride stimuli. Processes related to actin and muscle function and to iron oxidation, transportation, and homeostasis were found only in the EEJ and PVS groups; response to hydrogen peroxide and increased immune response was found only in the PVS group. CONCLUSION(S) This study was able to demonstrate differential protein expression among control, PVS, and EEJ groups; SCI is responsible for alterations in seminal plasma protein profile leading to a deviation from homeostasis; proteins reported in both PVS and EEJ groups correlate with the pathophysiology of SCI-related infertility.

Changes in the seminal plasma proteome of adolescents before and after varicocelectomy

OBJECTIVE To compare seminal plasma protein profiles before and after varicocele correction to assess if surgical intervention alters the protein profile. DESIGN Prospective study. SETTING Academic research environment. PATIENT(S) Nineteen adolescent boys with varicocele grades II or III. INTERVENTION(S) Two semen samples were collected before bilateral subinguinal microsurgical varicocelectomy, and two semen samples were collected 3 months after surgery. Seminal plasma protein profiles were determined with the use of two-dimensional gel electrophoresis. Proteins were separated in 18-cm 3-10 pH strips and 10%-17.5% gradient gels. Gels were stained, scanned, and compared with the use of Imagemaster 2D platinum 7.0. Spots of interest were removed from gels, and protein digestion was performed with the use of trypsin. Digests were identified with the use of electrospray ionization-quadrupole/time-of-flight tandem mass spectrometry (ESI-QTOF MS/MS), and spectra were analyzed with the use of the Mascot software. MAIN OUTCOME MEASURE(S) Proteins uniquely or overexpressed in each period (before or after varicocelectomy). RESULT(S) Nineteen spots were differentially expressed between pre- and postsurgery samples. Identified proteins were albumin, proteasome subunit alpha type 6, alpha-1-antitrypsin, fibronectin, CD177, prostatic acid phosphatase, specific prostatic antigen, alpha-2-antiplasmin, vitamin D-binding protein, gastricsin, clusterin, semenogelin-1, semenogelin-2, superoxide dismutase, protein-glutamine gamma glutamyltransferase-4, and prolactin-inducing protein. CONCLUSION(S) Varicocelectomy is associated with changes in the seminal plasma protein profile. Understanding specific pathways leading to male infertility may further assist physicians in demonstrating deviation from homeostasis in male infertility. In addition, it may be possible to observe if surgical intervention does indeed revert altered pathways toward a homeostatic state.

Sperm nuclear DNA fragmentation rate is associated with differential protein expression and enriched functions in human seminal plasma.

To analyse the proteomic profile of seminal plasma with the aim of identifying the proteins and post‐genomic pathways associated with sperm DNA fragmentation.