R.P. Bertolla

Single embryo and oocyte lipid fingerprinting by mass spectrometry

Methods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid (represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species.

Clinical outcome of intracytoplasmic sperm injection in infertile men with treated and untreated clinical varicocele.

PURPOSE We evaluated the impact of varicocelectomy on intracytoplasmic sperm injection outcomes in infertile men with clinical varicocele. MATERIALS AND METHODS We studied 242 infertile men with a history of clinical varicocele who underwent intracytoplasmic sperm injection. Of the men 80 underwent prior subinguinal microsurgical varicocelectomy (treated group 1) and 162 had any grade of clinical varicocele (untreated group 2) at sperm injection. We compared semen analysis results before and after varicocelectomy, and the sperm injection procedure outcomes. Mean time from surgery to sperm injection was 6.2 months. Logistic regression was done to verify whether varicocelectomy influenced the odds of clinical pregnancy, live birth and miscarriage. RESULTS We noted an improved total number of motile sperm (6.7 × 10(6) vs 15.4 × 10(6), p <0.01) and a decreased sperm defect score (2.2 vs 1.9, p = 0.01) after vs before varicocele repair. The clinical pregnancy (60.0% vs 45.0%, p = 0.04) and live birth (46.2% vs 31.4%, p = 0.03) rates after the sperm injection procedure were higher in the treated than in the untreated group. The chance of achieving clinical pregnancy (OR 1.82; 95% CI 1.06-3.15) and live birth (OR 1.87, 95% CI 1.08-3.25) by the sperm injection procedure were significantly increased while the chance of miscarriage was decreased (OR 0.433, 95% CI 0.22-0.84) after varicocele was treated. CONCLUSIONS Results suggest that varicocelectomy improves clinical pregnancy and live birth rates by intracytoplasmic sperm injection in infertile couples in which the male partner has clinical varicocele. The chance of miscarriage may be decreased if varicocele is treated before assisted reproduction.

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.

Does varicocele grade determine extent of alteration to spermatogenesis in adolescents

OBJECTIVE To determine whether grade of varicocele determines extent of alterations to semen quality in adolescents. DESIGN Prospective study. SETTING Patients recruited from a local public school. PATIENT(S) Adolescents (14 to 18 y of age) attending a local public school. INTERVENTION(S) Scrotal palpation in a temperature-controlled room, testicular volume assessment with a Prader orchidometer, and semen analysis according to World Health Organization guidelines, with morphology by Krugers strict criteria. MAIN OUTCOME MEASURE(S) Presence, and grade, or absence of varicocele; testicular volume (assessed with a Prader orchidometer); semen analysis results; and prevalence of testicular asymmetry. RESULT(S) Among the adolescents, 27.8% (95% confidence interval [CI]: 23.2, 32.4) presented varicocele grades II and III, and 7.8% (95% CI: 5.0, 10.6) presented with a grade III varicocele. There was a high prevalence of testicular asymmetry in adolescents with left grade II (41.7%) and III varicocele (51.9%), whereas adolescents without varicocele showed very low testicular asymmetry (11.0%). Testicular asymetry was significantly less prevalent in adolescents without varicocele. Sperm progressive motility and concentration were lower in the two varicocele groups but were not different according to grade. However, the total number of progressively motile sperm in the ejaculate was lower in the varicocele grade II and III groups, and patients with varicocele grade III presented lower values than those with grade II. CONCLUSION(S) Grades II and III varicocele cause a decrease in testicular volume and in semen quality that is independent of grade, but when assessing the total number of progressively motile sperm in the ejaculate, grade III varicoceles place these adolescents very close to the World Health Organization cutoff rate, and thus, current guidelines for treating the adolescent varicocele may need to be revised.

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.

Quality and functional aspects of sperm retrieved through assisted ejaculation in men with spinal cord injury

OBJECTIVE To assess semen quality, sperm DNA fragmentation, and mitochondrial activity in fertile men as well as in men with spinal cord injury who were collecting semen through different methods. DESIGN Prospective controlled study. SETTING Academic research environment. PATIENT(S) Men with spinal cord injury who achieved ejaculation through electroejaculation (n = 12) and penile vibratory stimulation (n = 10); 30 fertile control men without spinal cord injury. INTERVENTION(S) Electroejaculation or penile vibratory stimulation, semen analysis according to World Health Organization guidelines, morphology by Krugers strict criteria. MAIN OUTCOME MEASURE(S) Semen was analyzed according to World Health Organization guidelines; morphology was analyzed according to Krugers strict criteria. Sperm DNA fragmentation, as assessed by the TUNEL technique, was classified as percentage positive. Mitochondrial activity was assessed by incorporation of diaminobenzidine by mitochondria. Cells were classified as I (all active) to IV (all inactive). RESULT(S) The control group presented a statistically significantly higher percentage of sperm with active mitochondria and a statistically significantly lower percentage of sperm with inactive mitochondria. Although sperm DNA fragmentation was not significantly different when considering collection method (electroejaculation: 30; 8.4; penile vibratory stimulation: 31.2; 8), both groups presented statistically significantly higher DNA fragmentation than did controls (11.8; 4.5). A strong inverse correlation was observed between sperm DNA fragmentation (assessed by in situ DNA nick end labeling) and mitochondrial activity in the case of electroejaculation (r = -0.714), but not in the case of penile vibratory stimulation (r = 0.060). CONCLUSION(S) Spinal cord injury led to a decrease in sperm mitochondrial activity and an increase in sperm DNA fragmentation, and the latter is a sign of testicular alterations. Studies should focus on improving the testicular environment in these men.

Differences in the seminal plasma proteome are associated with oxidative stress levels in men with normal semen parameters

OBJECTIVE To study the seminal plasma proteome in association with semen lipid peroxidation levels in men with normal semen parameters. DESIGN Cross-sectional study. SETTING University andrology and research laboratories. PATIENT(S) A total of 156 normozoospermic men. INTERVENTION(S) Seminal lipid peroxidation levels were assessed in individual samples through thiobarbituric acid reactive substances quantification. Subsequently, lipid peroxidation data were used to divide the samples into the experimental groups: low lipid peroxidation levels (control group, bottom 15%, n = 23) and high lipid peroxidation levels (study group, top 15%, n = 23). Seminal plasma proteins from these groups were pooled (four pools per group, with biological variation between the pools) and used for a shotgun proteomic analysis using a liquid chromatography-tandem mass spectrometry approach. Quantitative data were used for univariate (unpaired Students t test) and multivariate (partial least-squares discriminant analysis, logistic regression, and discriminant analyses) statistical analyses. Significant proteins were also used for functional enrichment analysis. MAIN OUTCOME MEASURE(S) Seminal plasma protein profile and postgenomic pathways of seminal plasma are associated with seminal lipid peroxidation levels. RESULT(S) In total, 629 proteins were quantified in seminal plasma. Of these, 23 proteins were absent or underexpressed and 71 were exclusive or overexpressed in the study group. The main enriched functions in association with seminal lipid peroxidation were unsaturated fatty acids biosynthesis, oxidants and antioxidants activity, cellular response to heat stress, and immune response. Moreover, we suggested mucin-5B as a potential biomarker of semen oxidative stress. CONCLUSION(S) The seminal plasma proteome does reflect semen lipid peroxidation status and, thus, oxidative stress.

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.