Tratamiento antioxidante en hombres con infertilidad idiopática / Antioxidant treatment in men with idiopathic infertility

Objetivo. Los espermatozoides son vulnerables al estrés oxidativo, lo que ha llevado al uso de antioxidantes en hombres con infertilidad idiopática. Nuestro objetivo fue analizar los resultados del tratamiento antioxidante en hombres con infertilidad idiopática. Material y métodos. Estudio retrospectivo de 133 hombres con infertilidad idiopática que consultaron nuestro servicio entre 2010 y 2014. Evaluamos el número total de espermatozoides (NTE), formas progresivas (PR) y formas normales (FN) previo a tratamiento, tras 3 meses de tratamiento antioxidante y tras 3 meses más de tratamiento. Este consistió en ácido docosahexaenoico, coenzima Q10, zinc y selenio. Valoramos también la FSH e inhibinaB pretratamiento. Se utilizó el test de Wilcoxon para comparar los parámetros seminales antes y después del tratamiento y el coeficiente de correlación de Spearman para analizar la correlación entre variables pretratamiento y mejoría seminal. Resultados. La edad fue de 36,8±5,1 años y los valores de inhibinaB y FSH, de 128,5±66ng/l y 7±7U/l, respectivamente. Había 35 (26,3%) oligozoospérmicos, 99 (74,4%) astenospérmicos y 23 (17,2%) teratospérmicos. Encontramos mejoría significativa en el NTE, las PR y las FN tras 3 meses de antioxidantes respecto a los valores pretratamiento. Valores pretratamiento más bajos de NTE, PR y FN se correlacionaron con una mayor mejoría en el NTE (rs: −0,31; p=0,004), PR (rs: −0,27; p=0,002) y FN (rs: −0,48; p<0,001), respectivamente. No hubo correlación entre los valores hormonales y los resultados del tratamiento. Conclusiones. Los hombres con infertilidad idiopática pueden beneficiarse de 3 meses de tratamiento antioxidante. Aquellos con mayores alteraciones en el seminograma podrían obtener un mayor beneficio del tratamiento (AU)

Objective. Spermatozoa are susceptible to oxidative stress, which has lead to the use of antioxidants in men with idiopathic infertility. Our objective was to analyze results of antioxidant treatment in men with idiopathic infertility. Material and methods. Retrospective study of those 133 consecutive men with idiopathic infertility who attended our department between 2010-2014. We collected data about total sperm number (TSN), progressive forms (PF) and normal forms (NF) previous to treatment, after 3 months of antioxidant treatment and after 3 more months of treatment. We also assessed FSH and inhibinB levels before treatment. Antioxidant treatment was based on docosahexaenoic acid, coenzyme Q10, zinc and selenium. Wilcoxon test for paired samples was performed to compare semen parameters before and after treatment and Spearman's rank correlation coefficient to analyze any correlation between pretreatment variables and seminal improvement. Results. Mean age was 36.8±5.1 years. InhibinB and FSH values were 128.5±66ng/l and 7±7U/l, respectively. There were 35 (26.3%) oligozoospermic, 99 (74.4%) asthenospermic and 23 (17.2%) teratospermic patients. We found significant improvement in TSN, PF and NF after 3 months compared to before treatment. Lower pretreatment levels of TSN, PF percentage and NF percentage significantly correlated with a greater improvement of TSN (rs: −0.31; p=0.004), PF percentage (rs: −0.27; p=0.002) and NF percentage (rs: −0.48; p<0.001), respectively. No correlation was found between pretreatment hormonal values and results of treatment. Conclusions. Men with idiopathic infertility can benefit from 3 months of antioxidants. Those with greater deficiencies in sperm analysis could benefit the most from treatment (AU)

Human sperm tail proteome suggests new endogenous metabolic pathways.

Proteomic studies are contributing greatly to our understanding of the sperm cell, and more detailed descriptions are expected to clarify additional cellular and molecular sperm attributes. The aim of this study was to characterize the subcellular proteome of the human sperm tail and, hopefully, identify less concentrated proteins (not found in whole cell proteome studies). Specifically, we were interested in characterizing the sperm metabolic proteome and gaining new insights into the sperm metabolism issue. Sperm were isolated from normozoospermic semen samples and depleted of any contaminating leukocytes. Tail fractions were obtained by means of sonication followed by sucrose-gradient ultracentrifugation, and their purity was confirmed via various techniques. Liquid chromatography and tandem mass spectrometry of isolated sperm tail peptides resulted in the identification of 1049 proteins, more than half of which had not been previously described in human sperm. The categorization of proteins according to their function revealed two main groups: proteins related to metabolism and energy production (26%), and proteins related to sperm tail structure and motility (11%). Interestingly, a great proportion of the metabolic proteome (24%) comprised enzymes involved in lipid metabolism, including enzymes for mitochondrial beta-oxidation. Unexpectedly, we also identified various peroxisomal proteins, some of which are known to be involved in the oxidation of very long chain fatty acids. Analysis of our data using Reactome suggests that both mitochondrial and peroxisomal pathways might indeed be active in sperm, and that the use of fatty acids as fuel might be more preponderant than previously thought. In addition, incubation of sperm with the fatty acid oxidation inhibitor etomoxir resulted in a significant decrease in sperm motility. Contradicting a common concept in the literature, we suggest that the male gamete might have the capacity to obtain energy from endogenous pools, and thus to adapt to putative exogenous fluctuations.

Efecto de la administración de un complejo de antioxidantes en pacientes afectos de astenoteratozoospermia idiopática / Effect of the administration of an antioxidant complex in patients with idiopathic asthenoteratozoospermia

Introducción: El factor masculino se observa en casi el 50% de las parejas que acuden a consulta por dificultades reproductivas, y puede ser consecuente a una disminución en el número de espermatozoides (oligozoospermia) o a una diminución en su calidad por defectos en su movilidad (astenozoospermia) o en su morfología (teratozoospermia). Existen diversos estudios que relacionan estos defectos de calidad espermática con alteraciones en la fragmentación del ADN espermático y plantean el posible efecto beneficioso de tratamientos antioxidantes, si bien existe una gran disparidad en los resultados obtenidos. Objetivos: A fin de contribuir a esclarecer el posible efecto beneficioso de la administración empírica de antioxidantes, en el presente trabajo abordamos el estudio del efecto de la administración de un complejo antioxidante (Androferti® Laboratorios Q Pharma), en pacientes afectos de astenoteratozoospermia idiopática. Pacientes y métodos: Para ello, se incluyeron un total de 69 varones a los que se administró el tratamiento antioxidante, a dosis de 1,5 g/día, durante tres meses. Concluido el tratamiento, se detectó una mejoría significativa en la movilidad (la movilidad global a+b pasó de 22,04 a 28,95%; p = 0,001) y en la morfología (las formas normales pasaron de 9,86 a 14,78%; p < 0,001). Discusión: Numerosas publicaciones refieren efectos beneficiosos, mediante la administración de sustancias antioxiadantes, sobre la fragmentación de ADN espermático, lo que se podría traducir en una mejoría de la calidad seminal y embrionaria, en caso de gestación. Nuestro estudio se halla en la misma línea, mostrando una mejoría en la mayoría de los parámetros seminales como consecuencia de la administración de L-carnitina asociada a otras sustancias de acción antioxidante, tratamiento que parece ser especialmente eficaz en pacientes más jóvenes. Conclusiones: Nuestros resultados confirman el efecto beneficioso de la administración de un complejo de antioxidantes en pacientes afectos de astenoteratozoospermia idiopática mejorando la calidad seminal y, en consecuencia, favoreciendo la deseada gestación (AU)

Introduction: The male factor is seen in almost 50% of the couples attending a reproductive clinic, and may be a result of a decrease in sperm count (oligozoospermia) or in sperm quality due to mobility defects (asthenozoospermia) or in its morphology (teratozoospermia). There are several studies that related the these sperm quality defects with the presence of sperm DNA fragmentation and suggest the possible beneficial effect of antioxidant treatments, although there is wide disparity in the results obtained. Objectives: We have studied the effect of the administration of an antioxidant complex (Androferti® Laboratory Q Pharma) in patients with idiopathic asthenoteratozoospermia in order to contribute to the clarification of the possible beneficial effect of the empirical administration of antioxidants, in the present work. Patients and methods: A total of 69 males in whom the antioxidant treatment was administered at a day dose of 1.5 g for 3 months were included. After completing the treatment, significant improvement was detected in sperm mobility (global mobility a+b improved from 22.04% to 28.95%; p = 0.001) and sperm morphology (the normal forms increased from 9.86 % to 14.78%, p <0.001). Discussion: Numerous publications have reported the beneficial effects of administering antioxidant substances on sperm DNA fragmentation. These effects could be translated into an improvement in semen and embryo quality, in case of pregnancy. Our study is in line with these finding, showing an improvement in most of the seminal parameters resulting from the administration of L-carnitine in association with other antioxidants, a treatment that appears to be particularly effective in younger patients. Conclusions: Our results confirm the beneficial effect of the administration of an antioxidant complex in patients with idiopathic asthenoteratozoospermia, this improving their sperm quality, consequently favoring the desired pregnancy (AU)

Proteomic characterization of the human sperm nucleus.

Generating a catalogue of sperm nuclear proteins is an important first step towards the clarification of the function of the paternal chromatin transmitted to the oocyte upon fertilization. With this goal, sperm nuclei were obtained through CTAB treatment and isolated to over 99.9% purity without any tail fragments, acrosome or mitochondria as assessed by optical microscopy and transmission electron microscopy. The nuclear proteins were extracted and separated in 2-D and 1-D gels and the 2-D spots and 1-D bands were excised and analysed to identify the proteins through LC-MS/MS. With this approach, 403 different proteins have been identified from the isolated sperm nuclei. The most abundant family of proteins identified are the histones, for which several novel members had not been reported previously as present in the spermatogenic cell line or in the human mature spermatozoa. More than half (52.6%) of the proteins had not been detected in the previous human whole sperm cell proteome reports. Of relevance, several chromatin-related proteins, such as zinc fingers and transcription factors, so far not known to be associated with the sperm chromatin, have also been detected. This provides additional information about the nuclear proteins that are potentially relevant for epigenetic marking, proper fertilization and embryo development.

Protamine 2 precursors (Pre-P2), protamine 1 to protamine 2 ratio (P1/P2), and assisted reproduction outcome.

OBJECTIVE: To determine whether the presence of protamine 2 precursors (pre-P2/P2 ratio) and the protamine 1 to protamine 2 ratio (P1/P2) are related to the assisted reproduction outcome. DESIGN: Prospective study. SETTING: Assisted Reproduction Unit and University laboratory. PATIENT(S): One hundred two infertile patients undergoing treatment at the Assisted Reproduction Unit of the Hospital Clinic of Barcelona. INTERVENTION(S): Intracytoplasmic sperm injection (ICSI) and/or IVF treatment of the infertile patients, sperm protamine analysis through electrophoresis and densitometry, and pre-P2 analysis through Western blot. MAIN OUTCOME MEASURE(S): The presence of protamine 2 precursors (pre-P2/P2 ratio), sperm P1/P2 ratio, fertilization rates by IVF and/or ICSI, and pregnancy outcome. RESULT(S): Pre-P2/P2 and P1/P2 ratios are positively associated with the pregnancy rate. In addition, the P1/P2 ratio is positively associated with the proportion of embryos obtained by IVF, but not by ICSI. The pre-P2/P2 ratio was not related to the fertilization rate. CONCLUSION(S): Decreased pre-P2/P2 and P1/P2 ratios are related to a poor pregnancy outcome, but not with the proportion of embryos obtained after ISCI.

Atypical XX male with the SRY gene located at the long arm of chromosome 1 and a 1qter microdeletion.

Male individuals with a 46,XX karyotype have been designated as XX males. In 80% of the cases, the presence of Yp sequences, including the male sex-determining gene, SRY, has been demonstrated by molecular and/or fluorescence in situ hybridization (FISH) analyses. In most cases, Yp sequences are located on the short arm of the X chromosome, resulting from unequal recombination between Yp and Xp during paternal meiosis. Much less frequent in XX males is the localization of the SRY gene to an autosome. Here we report on the genetic investigation of an atypical XX male in which the SRY gene was located at the end of the long arm of chromosome 1. The patient, with a normal male phenotype, was referred for azoospermia. Conventional cytogenetic analysis showed a 46,XX karyotype. Molecular-cytogenetics (FISH) and molecular (PCR and MLPA) studies identified not only Yp-specific sequences located on the distal long arm of chromosome 1 but also the deletion of the subtelomeric 1qter region. A specific phenotype has been reported for a deletion of the 1qter region associated with mental retardation. The molecular investigation of the 1qter region showed that in our patient the microdeletion is more telomeric than in patients reported with mental retardation. To our knowledge, this is the first report of a XX male with the Yp region transferred to the terminal long arm of chromosome 1. This is also the first microdeletion of the subtelomeric 1qter region not associated with mental retardation.

A common protamine 1 promoter polymorphism (-190 C->A) correlates with abnormal sperm morphology and increased protamine P1/P2 ratio in infertile patients.

It is known that targeting the protamine 1 gene in mice leads to infertility, abnormal chromatin packaging, and abnormal sperm morphology. Because many infertile patients also have an abnormal sperm morphology and chromatin packaging, the human protamine 1 gene (PRM1) is an important candidate to screen for potential mutations. In this work, we have screened the PRM1 gene in search of potential mutations and determined the sperm morphology and the ratio between protamine 1 and protamine 2 (P1/P2 ratio). Direct sequencing of the PRM1 promoter led to the identification of a common single-nucleotide polymorphism (SNP; -190 C-->A). The -190 AA genotype was detected at a higher frequency (13.8%) in patients with markedly altered sperm morphology (A change was also consistently higher (.331) in infertile patients with a markedly altered morphology compared with population controls (.178; P < .01). Additionally, we have determined that the P1/P2 ratio is significantly increased in patients with the PRM1 -190 AA genotype compared with patients with the CA or CC genotypes (P = .006, Mann-Whitney). These findings indicate that the common PRM1 -190 C-->A polymorphism identified is associated with abnormal sperm head morphology and abnormal P1/P2 ratio in infertile patients.

Identification of proteomic differences in asthenozoospermic sperm samples.

BACKGROUND: Asthenozoospermia is one of the most common findings present in infertile males, but its aetiology remains unknown in most cases. Present proteomic tools now offer the opportunity to identify proteins which are differentially expressed in asthenozoospermic semen samples and potentially involved in infertility. METHODS: We compared the expression of 101 sperm protein spots in 20 asthenozoospermic samples to that of 10 semen donor controls using two-dimensional proteomic analysis. RESULTS: Seventeen protein spots have been identified at different amounts in the asthenozoospermic samples compared with controls. These are cytoskeletal actin-B, annexin-A5, cytochrome C oxidase-6B, histone H2A, prolactin-inducible protein and precursor, calcium binding protein-S100A9 (2 spots), clusterin precursor, dihydrolipoamide dehydrogenase precursor, fumarate hydratase precursor, heat shock protein-HSPA2, inositol-1 monophosphatase, 3-mercapto-pyruvate sulfurtransferase/dienoyl-CoA isomerase precursor, proteasome subunit-PSMB3, semenogelin 1 precursor and testis expressed sequence 12. The detected amount of these proteins enabled the grouping of asthenozoospermic sperm samples in an unsupervised clustering analysis. CONCLUSIONS: We have identified several proteins present at different amount in asthenozoospermic sperm samples. These proteins could be candidates towards the development of diagnostic markers, and open up the opportunity to gain further insight into the pathogenic mechanisms involved in asthenozoospermia.

Marked correlations in protein expression identified by proteomic analysis of human spermatozoa.

The present work was started to explore whether a correlation could be detected among proteomic expression, protamine content and DNA integrity in human sperm cells. Towards this goal, we extracted the proteins present in the sperm cells from 47 sperm samples from infertile patients and from ten semen donors, analysed each sample by 2-D gel electrophoresis, and quantified the expression of 101 spots identified by MALDI-TOF analysis. Additionally, the protamine content and DNA integrity were also determined. Several interesting proteins such as transcription factors, prohibitin, heat shock and proteasome proteins have been identified. We have found that the expression of an important number of proteins (58 different 2-D spots) is correlated in independent sperm samples at high statistical significance (p<0.001 and r>0.5). Additionally, eight proteins have also been found to correlate with DNA integrity and seven with protamine content (p<0.05). To our knowledge, this is the first report describing the correlation between proteomics, DNA integrity and protamine content. It also sheds new light into the fundamental aspects of the human sperm and points to new potential proteins involved in male infertility.

Human sperm DNA fragmentation: correlation of TUNEL results as assessed by flow cytometry and optical microscopy.

An association between DNA fragmentation in sperm determined by the terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay and the incidence of reproductive failure has been reported, either using flow cytometry or optical microscopy. However, the results obtained using each of these two approaches are different. Since there is a relative lack of studies standardizing these two approaches, the direct comparison of the results described in the different articles is difficult at present. To allow the comparison of the TUNEL results obtained using flow cytometry and optical microscopy, we applied these two approaches in a total of 66 human sperm samples. A positive correlation is detected in the TUNEL results as measured by flow cytometry and optical microscopy (Spearman; r = 0.720, P < 0.001). The percentage of TUNEL-positive spermatozoa assessed by flow cytometry is 2.6 times higher than that detected in optical microscopy (39.7% +/- 23.1% versus 15.3% +/- 10.3%). Although there is a good correlation of the TUNEL results obtained by flow cytometry and optical microscopy, the percentages obtained with either technique are different. Therefore, the TUNEL results described in the present work should be valuable to compare the results described in many independent articles, using either optical microscopy or flow cytometry.

Proteomics of human spermatozoa, protamine content and assisted reproduction outcome.

It is well known that alterations in the expression of the major sperm nuclear proteins (protamines) are related to infertility in man. In addition, other minor proteins extracted from human spermatozoa are being analysed by 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and identified by MALDI-TOF MS analysis. The function of the identified proteins turns out to be energy production, transcription, protein synthesis, transport, folding and turnover, cell cycle, apoptosis and oxidative stress, signal transduction, cytoskeleton, flagella and cell movement, cell recognition, metabolism and unknown function. Many of the proteins identified using MALDI-TOF had not been yet been described as being expressed in human spermatozoa. Substantial differences have been detected in the levels of some of the newly identified human sperm proteins in the different groups of infertile patients. Present research efforts are targeting the potential correlations among changes in the proteomic composition, protamine content, DNA integrity and assisted reproduction outcome.

Proteomic identification of human sperm proteins.

Conventional 1-DE has in the past provided a wealth of information concerning the major sperm proteins. However, so far there are relatively few reports exploiting the potential of the present proteomic tools to identify and to study additional yet-unidentified important proteins present in human spermatozoa. In the present work, 2-DE of proteins extracted from human normozoospermic spermatozoa led to the resolution of over 1000 spots. Subsequent excision from the gels of 145 spots and MALDI-TOF MS analysis allowed the identification of 98 different proteins. The function of these proteins turned out to be energy production (23%), transcription, protein synthesis, transport, folding and turnover (23%), cell cycle, apoptosis and oxidative stress (10%), signal transduction (8%), cytoskeleton, flagella and cell movement (10%), cell recognition (7%), metabolism (6%) and unknown function (11%). As many as 23% of the proteins identified have not been previously described as being expressed in human spermatozoa. The present data provide an important clue towards determining the function of these proteins and opens up the possibility to perform additional experiments.

Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients.

BACKGROUND: The protamine 1-to-protamine 2 ratio (P1/P2) is altered in the sperm cells of some infertile patients. Also, evidence for increased protamine 2 precursors (pre-P2) in a few patients has been reported. But so far, there have been no studies measuring simultaneously these two variables in a large number of patients. METHODS: We measured the P1/P2 ratio and the presence of pre-P2 using, for the first time, an antibody specific to the precursor pre-P2, together with other sperm parameters in 224 infertile patients. Additionally, the DNA integrity was assessed by terminal transferase dUTP nick-end labelling (TUNEL) in a subset of the samples. RESULTS: Pre-P2 levels show a significant positive correlation with the P1/P2 ratio, with the presence of other proteins and, at low pre-P2 levels, with TUNEL-positive sperm. An inverse correlation with sperm count, normal morphology and motility was detected. CONCLUSIONS: The levels of pre-P2 may provide clues into the pathogenic mechanisms of infertility. The increased proportion of pre-P2 in some patients with increased P1/P2 ratio suggests an involvement of pre-P2 processing. The positive correlation between TUNEL-positive sperm and pre-P2 at low pre-P2/P2 ratios also suggests a link between deficient protamine processing and decreased DNA integrity.

High frequency of gr/gr chromosome Y deletions in consecutive oligospermic ICSI candidates.

BACKGROUND: The Y chromosome gr/gr microdeletion eliminates two copies of the DAZ gene and several additional transcriptional units and has been associated as a risk factor for infertility. Our objective was to study the presence of the gr/gr deletion in ICSI candidates in our population and to determine whether the laboratory, clinical and ICSI outcome were different in the gr/gr deleted patients. METHODS: Two hundred and eighty-three ICSI candidates were studied. Semen analysis, serum FSH, LH, testosterone, inhibin B, karyotype and detection of sequence tagged sites in the Y chromosome were performed. RESULTS: gr/gr deletions were detected in 11 (5.07%) of 217 oligospermic and in one (1.52%) of 66 azoospermic consecutive ICSI candidates, but in none of 232 controls (P=0.002). The fertility rate was not different in the four patients of the gr/gr deleted group treated by ICSI (64.38%; 47/73) as compared to average results at our center (65.49%; 2393/3654). CONCLUSIONS: gr/gr deletions are a risk factor for spermatogenic failure at our population, but the prognosis of the four patients of the gr/gr deleted group treated by ICSI is not different from that of other ICSI patients.