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Sperm capacitation is a critical process for male fertility. It involves a series of biochemical and physiological changes that occur in the female reproductive tract, rendering the sperm competent for successful fertilization. The precise mechanisms and, specifically, the role of mitochondria, in sperm capacitation remain incompletely understood. Previously, we revealed that in mouse sperm mitochondrial activity (e.g., oxygen consumption, membrane potential, ATP/ADP exchange, and mitochondrial Ca2+ ) increases during capacitation. Herein, we studied mitochondrial function by high-resolution respirometry (HRR) and reactive oxygen species production in capacitated (CAP) and non-capacitated (NC) human spermatozoa. We found that in capacitated sperm from normozoospermic donors, the respiratory control ratio increased by 36%, accompanied by a double oxygen consumption rate (OCR) in the presence of antimycin A. Extracellular hydrogen peroxide (H2 O2 ) detection was three times higher in CAP than in NC sperm cells. To confirm that H2 O2 production depends on mitochondrial superoxide ( O 2 · - $$ {\mathrm{O}}_2^{\cdotp -} $$ ) formation, we evaluated mitochondrial aconitase (ACO2) amount, activity, and role in the metabolic flux from the sperm tricarboxylic acid cycle. We estimated that CAP cells produce, on average by individual, (59 ± 22)% more O 2 · - $$ {\mathrm{O}}_2^{\cdotp -} $$ in the steady-state compared to NC cells. Finally, we analyzed two targets of oxidative stress: lipid peroxidation by western blot against 4-hydroxynonenal and succinate dehydrogenase (SDH) activity by HRR. We did not observe modifications in lipoperoxidation nor the activity of SDH, suggesting that during capacitation, the increase in mitochondrial H2 O2 production does not damage sperm and it is necessary for the normal CAP process.

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Introduction: Advances in cancer treatments have determined an increase in survival rates. However, these lifesaving therapies may have a negative impact on reproductive health. To diminish the infertility risk; different fertility preservation strategies have been designed. Sperm freezing is the gold standard fertility preservation method in the case of post-pubertal men. The main objective of this study is to evaluate the fertility status of Uruguayan male cancer survivors who have gone through sperm freezing, as well as to assess oncofertility counseling received by these patients. Methods: This is a descriptive, cross-sectional, observational, and transversal study. A survey was conducted on male cancer survivors who cryopreserved sperm between 1985 and 2021 in "Reprovita Lab and Biobank" which is the only sperm bank in this country. Results: One hundred thirty-five participants answered the survey. At the time of diagnosis, the mean age of patients was 28.8 ± 6.4 years old. Testicular was the most frequent type of cancer (64%). Only, 12% (n = 15) already had children at the time of diagnosis. Among the interviewed survivors, 50% (n = 62) attempted to conceive after cancer treatment, and 68% (n = 42) achieved natural pregnancy. Patients who did not achieve spontaneous conception (n = 11), used their cryopreserved samples, and 45.4% achieved pregnancy. About 86% (n = 107) of survivors believed that the timing of oncofertility referrals was appropriate and 97% considered that having the possibility of protecting their fertility was very important. Eighty percent (n = 101), were advised by their attending physicians, 14% (n = 18) sought advice from family members or friends, and 4% (n = 5) from oncofertility specialists. Discussion: To our knowledge, this is the first study evaluating the reproductive outcomes of male cancer survivors in our country and the region. Most of the interviewed survivors considered fertility preservation as a positive initiative, independent of their reproductive outcomes, reflecting the importance of fertility preservation counseling as one of the most important aspects for futurequality of life of young cancer patients.

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BACKGROUND: With regard to the origin of its population and microevolutionary processes, Uruguay exhibits distinctive features that distinguish it from other countries in Latin America, while at the same time sharing several similarities. In this article, we will focus on the variability of paternal genetic lineages in two geographical regions with different histories that can be considered as examples of distinct populations for the continent. In general terms, the genetic diversity is a result of different demographic processes related to the American conquest and colonisation. These resulted in distinct ancestral components which vary geographical and depend on the distribution by sex within these components. In Uruguay, native maternal haplogroups are significantly more frequent in the North. Although there are several studies about the geneticvariability of Uruguay, little is known about male genetic lineages. AIMS: The aim of this work is to present an updated study of the male genetic variability of the Uruguayan population. METHODS: We analyzed 13 biallelic markers and 27 STRs located in the male-specific region of the Y chromosome for 157 males: 98 from the capital, Montevideo, and 59 from Tacuarembó. RESULTS: Almost all haplogroups found in both locations are European (99% and 93.2% respectively). One Sub-Saharan African haplogroup was found in Montevideo (1%) and 2 in Tacuarembó (3%), while Native haplogroups were found only in Tacuarembó, evidencing a strong sex-biased admixture. By crossing genetic and genealogical information we could relate European haplogroups with different waves and times of migrations. DISCUSSION: Network analysis indicated a very diverse male population, suggesting that European migrants came from heterogeneous geographic locations and in different waves. Tacuarembó has closer population affinities with Iberian populations while Montevideo is more diverse. Male population expansion expansion, can be explained by the large number of migrants that arrived during the XIX century and the first half of the XX century. CONCLUSIONS: The Uruguayan male gene pool is the result of several migration waves with diverse origins, with strong sex-biased admixture that can be explained by the European migration, the violence against the indigenous males, and the segregation of the Africansadmixture that can be explained due to European migration, violence against Natives, and segregation against African males.admixture that can be explained due to European migration, violence against Natives, and segregation against African males.admixture that can be explained due to European migration, violence against Natives, and segregation against African males.admixture that can be explained due to European migration, violence against Natives, and segregation of hte Africans.

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The diagnosis of male infertility is based essentially on the patient's medical history and a standard semen analysis. However, the latter rarely provides information on the causes of a possible infertility, emphasizing the need to extend the analysis of the sperm function. Mitochondrial function has been associated with sperm function and dysfunction, the latter primarily through the production of excessive amounts of reactive oxygen species (ROS). We hypothesized that analysis of sperm mitochondrial metabolism together with sperm ROS production could be an additional tool to improve routine semen analysis, after appropriate validations. To test our hypothesis, we performed several experiments using a non-routine method (high-resolution respirometry, HRR) to access mitochondrial function. First, we investigated whether mitochondrial function is related to human sperm motility and morphology. When mitochondrial metabolism was challenged, sperm motility decreased significantly. Additionally, morphological abnormalities in the sperm mid-piece and mitochondria were associated with global sperm defects evaluated by routine methods. Subsequently, sperm mitochondrial function was assessed by HRR. Respiratory control ratio (RCR) was determined and evaluated in the context of classical sperm analysis. In parallel, sperm hydrogen peroxide (H2O2) production and seminal plasma (SP) antioxidant capacity were measured. The percentage of sperm with progressive motility correlated positively with RCR, SP antioxidant capacity, and negatively with the concentration of extracellular H2O2 production ([H2O2]). The percentage of normal sperm morphology correlated positively with RCR and negatively with [H2O2]. Sperm morphology did not correlate with seminal plasma antioxidant capacity. Furthermore, Receiver Operating Characteristic curves were used for the first time to test the diagnostic ability of RCR, [H2O2], and SP antioxidant capacity as binary classifiers. An RCR cut off value of 3.2 was established with a sensitivity of 73% and a specificity of 61%, using reference values considered normal or abnormal in routine semen analysis. The cut off value for [H2O2] was 0.2 µM/106 sperm (sensitivity = 65%, specificity = 60%). There were no reference values for SP antioxidant capacity that distinguished between abnormal and normal sperm samples. We conclude that sperm mitochondrial function indices in combination with [H2O2] may be useful tools to complement the routine semen analysis.

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BACKGROUND: Over the last years, there has been an increasing concern about a global decline in men's fertility. Specifically, some evidence indicates that sperm quality has decreased over the last years. However, reports showing the changes in sperm quality with time are inconsistent. Part of the contradictions between studies is attributed to geographical differences. Surprisingly, few studies include data from South American countries, creating a bias in the conclusions. This study aims to determine how sperm quality has evolved over the past 30 years in Uruguay. For this purpose, 317 medical records from allegedly healthy sperm donor candidates, aged between 18 and 36 years old, who voluntarily requested to be considered as sperm donors between 1988 and 2019, were analyzed. The studied variables were the following sperm parameters: semen volume, sperm cell concentration, total sperm number, progressive motility, vitality, and sperm morphology. A correlative statistical analysis was performed between seminal parameter values and the year data were collected. RESULTS: We found a statistically significant decrease in sperm concentration and normal sperm morphology during the studied period. There was no decrease in vitality, seminal volume, and total progressive motility. Semen parameters were not associated with tobacco, drugs, or alcohol consumption. CONCLUSIONS: We conclude that the sperm quality of donor candidates in Uruguay decreased during this period. Further studies should be carried out to verify the occurrence of this phenomenon in the general population and find its possible causes.

RéSUMé: CONTEXTE: Au cours des dernières années, on s'est de plus en plus inquiété d'une baisse de la fertilité masculine. Plus précisément, certaines données indiquent que la qualité du sperme a diminué au cours des dernières années. Cependant, les rapports montrant les changements dans la qualité du sperme avec le temps sont incohérents. Une partie des contradictions entre les études est attribuée à des différences géographiques. Étonnamment, peu d'études ont inclue des données provenant de pays d'Amérique du Sud, ce qui crée un biais dans les conclusions. Cette étude vise à déterminer comment la qualité du sperme a évolué au cours des 30 dernières années en Uruguay. À cette fin, 317 dossiers médicaux de candidats donneurs de sperme prétendument en bonne santé, âgés de 18 à 36 ans, qui ont volontairement demandé à être considérés comme donneurs de sperme entre 1988 et 2019, ont été analysés. Les variables étudiées étaient les paramètres suivants du sperme: volume de sperme, concentration de cellules spermatiques, nombre total de spermatozoïdes, motilité progressive, vitalité, et morphologie des spermatozoïdes. Une analyse statistique corrélative a été effectuée entre les valeurs des paramètres séminaux et l'année de recueil des données. RéSULTATS: Nous avons trouvé une diminution statistiquement significative de la concentration de spermatozoïdes et de la morphologie normale des spermatozoïdes pendant la période étudiée. Il n'y a eu aucune diminution de la vitalité, du volume de sperme, et de la motilité progressive totale. Les paramètres du sperme n'étaient pas associés à la consommation de tabac, de drogues ou d'alcool. CONCLUSIONS: Nous concluons que la qualité du sperme des candidats donneurs en Uruguay a diminué au cours de cette période. D'autres études devraient être menées pour vérifier l'apparition de ce phénomène dans la population générale et en trouver les causes possibles.

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BACKGROUND: Preterm birth (PTB), defined as infant delivery before 37 weeks of completed gestation, results from the interaction of both genetic and environmental components and constitutes a complex multifactorial syndrome. Transcriptome analysis of PTB has proven challenging because of the multiple causes of PTB and the numerous maternal and fetal gestational tissues that must interact to facilitate parturition. The transcriptome of the chorioamnion membranes at the site of rupture in PTB and term fetuses may reflect the molecular pathways of preterm labor. METHODS: In this work, chorioamnion membranes from severe preterm and term fetuses were analyzed using RNA sequencing. Functional annotations and pathway analysis of differentially expressed genes were performed with the GAGE and GOSeq packages. A subset of differentially expressed genes in PTB was validated in a larger cohort using qRT-PCR and by comparing our results with genes and pathways previously reported in the literature. RESULTS: A total of 270 genes were differentially expressed (DE): 252 were upregulated and 18 were down-regulated in severe preterm births relative to term births. Inflammatory and immunological pathways were upregulated in PTB. Both types of pathways were previously suggested to lead to PTB. Pathways that were not previously reported in PTB, such as the hemopoietic pathway, appeared upregulated in preterm membranes. A group of 18 downregulated genes discriminated between term and severe preterm cases. These genes potentially characterize a severe preterm transcriptome pattern and therefore are candidate genes for understanding the syndrome. Some of the downregulated genes are involved in the nervous system, morphogenesis (WNT1, DLX5, PAPPA2) and ion channel complexes (KCNJ16, KCNB1), making them good candidates as biomarkers of PTB. CONCLUSIONS: The identification of this DE gene pattern will help with the development of a multi-gene disease classifier. These markers were generated in an admixed South American population in which PTB has a high incidence. Since the genetic background may differentially impact different populations, it is necessary to include populations such as those from South America and Africa, which are usually excluded from high-throughput approaches. These classifiers should be compared to those in other populations to obtain a global landscape of PTB.

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The cilia and flagella of eukaryotic cells serve many functions, exhibiting remarkable conservation of both structure and molecular composition in widely divergent eukaryotic organisms. SPAG6 and SPAG16 are the homologous in the mice to Chlamydomonas reinhardtii PF16 and PF20. Both proteins are associated with the axonemal central apparatus and are essential for ciliary and flagellar motility in mammals. Recent data derived from high-throughput studies revealed expression of these genes in tissues that do not contain motile cilia. However, the distribution of SPAG6 and SPAG16 in ciliated and non-ciliated tissues is not completely understood. In this work, we performed a quantitative analysis of the expression of Spag6 and Spag16 genes in parallel with the immune-localization of the proteins in several tissues of adult mice. Expression of mRNA was higher in the testis and tissues bearing motile cilia than in the other analyzed tissues. Both proteins were present in ciliated and non-ciliated tissues. In the testis, SPAG6 was detected in spermatogonia, spermatocytes, and in the sperm flagella whereas SPAG16 was found in spermatocytes and in the sperm flagella. In addition, both proteins were detected in the cytoplasm of cells from the brain, spinal cord, and ovary. A small isoform of SPAG16 was localized in the nucleus of germ cells and some neurons. In a parallel set of experiments, we overexpressed EGFP-SPAG6 in cultured cells and observed that the protein co-localized with a subset of acetylated cytoplasmic microtubules. A role of these proteins stabilizing the cytoplasmic microtubules of eukaryotic cells is discussed.

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BACKGROUND: Infertility affects 15% of human couples, with men being responsible in approximately 50% of cases. Moreover, the aetiology of male factor infertility is poorly understood. The majority of male factor infertility remains idiopathic and potentially genetic in origin. The association of the Y chromosome and mitochondrial haplogroups with male infertility has been previously reported. This association differs between studied populations and their geographical distributions. These effects have been only rarely analysed in mixed populations, such as South Americans. METHODS: In this study, we analysed the contributions of the Y chromosome and mitochondrial haplogroups to male infertility in a mixed population. A case control study was conducted. Regular PCR and high-resolutionmelting- real-time PCR were performed to type haplogroups from fertile and infertile men. The sperm parameters from infertile men were compared in each haplogroup by logistic regression analysis and ANOVA. RESULTS: The genotyping confirmed the known admixture characteristic of the Uruguayan population. The European paternal contribution was higher than the maternal contribution in both fertile and infertile men. Neither maternal nor paternal ancestry presented differences between the cases and controls. Men belonging to the Y chromosome haplogroup F(xK) more frequently presented with an abnormal sperm morphology than men from other haplogroups. The sperm parameters were not associated with the mitochondrial haplogroups. CONCLUSIONS: The data presented in this study showed an association between male infertility and ancestry in the Uruguayan population. Specifically, abnormal sperm morphology was associated with the Y chromosome haplogroup F(xK). Since the Y chromosome lacks recombination, these data suggest that some genes that determine sperm morphology might be inherited in blocks with the region that determines specific haplogroups. However, the possible association between the Y chromosome haplogroup F(xK) and sperm morphology requires further confirmatory testing. Data linking infertility with ancestry are needed to establish the possible causes of infertility and define male populations susceptible to infertility. Whether the admixed characteristics of the Uruguayan population exert any pressure on male fertility potential must be further investigated.

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Infertility affects about 15% of couples of reproductive age. The male factor is involved in nearly 50% of infertility cases. Defective human sperm function has been associated with evidence of high levels of reactive oxygen species (ROS) and a resultant loss of fertilizing potential in vivo and in vitro. Analogous to what has been observed in somatic cells, mitochondria are likely the major sources of ROS in sperm cells. In this study, we analyzed mitochondrial function using high-resolution respirometry, ROS production, and footprints of oxidative and nitrative stress processes in intact human sperm cells. We showed that mitochondrial dysfunction (measured through the respiratory control ratio) was correlated with a decrease in human sperm motility. The samples analyzed presented nitro-oxidative modifications of proteins, such as protein 3-nitrotyrosine, that were observed mainly in the mid-piece (where mitochondria are localized) and in the sperm head. Semen samples presenting lower percentage of motile sperm showed higher amounts of nitro-oxidative protein modifications than those with larger quantities of motile sperm. When spermatozoa were exposed to inhibitors of the respiratory mitochondrial function, in the presence of a nitric oxide flux, sperm produced potent nitro-oxidative species (i.e., peroxynitrite). This effect was observed in more than 90% of intact living sperm cells and in sperm mitochondrial fractions. These data suggest that dysfunctional mitochondria in sperm cells produce oxidants that may contribute to male infertility. These data provide the rationale for testing the potential of compounds that improve sperm mitochondrial function to treat male infertility.

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BACKGROUND: Complex traits like cancer, diabetes, obesity or schizophrenia arise from an intricate interaction between genetic and environmental factors. Complex disorders often cluster in families without a clear-cut pattern of inheritance. Genomic wide association studies focus on the detection of tens or hundreds individual markers contributing to complex diseases. In order to test if a subset of single nucleotide polymorphisms (SNPs) from candidate genes are associated to a condition of interest in a particular individual or group of people, new techniques are needed. High-resolution melting (HRM) analysis is a new method in which polymerase chain reaction (PCR) and mutations scanning are carried out simultaneously in a closed tube, making the procedure fast, inexpensive and easy. Preterm birth (PTB) is considered a complex disease, where genetic and environmental factors interact to carry out the delivery of a newborn before 37 weeks of gestation. It is accepted that inflammation plays an important role in pregnancy and PTB. METHODS: Here, we used real time-PCR followed by HRM analysis to simultaneously identify several gene variations involved in inflammatory pathways on preterm labor. SNPs from TLR4, IL6, IL1 beta and IL12RB genes were analyzed in a case-control study. The results were confirmed either by sequencing or by PCR followed by restriction fragment length polymorphism. RESULTS: We were able to simultaneously recognize the variations of four genes with similar accuracy than other methods. In order to obtain non-overlapping melting temperatures, the key step in this strategy was primer design. Genotypic frequencies found for each SNP are in concordance with those previously described in similar populations. None of the studied SNPs were associated with PTB. CONCLUSIONS: Several gene variations related to the same inflammatory pathway were screened through a new flexible, fast and non expensive method with the purpose of analyzing their association to PTB. It can easily be used for simultaneously analyze any set of SNPs, either as the first choice for new association studies or as a complement to large-scale genotyping analysis. Given that inflammatory pathway is in the base of several diseases, it is potentially useful to analyze a broad range of disorders.

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A specific cause of infertility cannot be identified in at least 25% of men referred to a specialized clinic. Diagnosis of infertile men is based mainly on standard semen analysis and the observation of sperm under light microscope. The aim of our study was to find the subcellular sperm characteristics that could explain infertility in a group of teratozoospermic infertile men. Morphological characteristics of sperm from non-teratozoospermic (control donors) and teratozoospermic infertile men were analyzed by transmission electron microscopy (TEM) and quantified. Our analysis showed that sperm cells from control donors presented a higher number of normal heads and tails than infertile men. Regarding subcellular characteristics of nucleus and tails, only the percentage of vacuolated nucleus, the absence of at least one pair of microtubules of the axoneme and the total distortion of the tail were statistically higher in infertile men than in control donors. There were no differences in the number of normal acrosomes between the groups. Although the ultrastructural sperm defects overlapped between control donors and infertile men, TEM permits the identification and differentiation of a larger amount of defects than light microscopy. Vacuolated nucleus and gross alterations of the tail are the major sperm defects that seem to have prognostic value in teratozoospermic men.

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