BACKGROUND: DNA methylation plays a critical role in the dynamics of gene expression regulation and the development of various disorders. Whole-genome bisulfite sequencing can provide single-base resolution of CpG methylation levels and is the “gold standard” for DNA methylation quantification, but its high cost limits its widespread application. In contrast, targeted sequencing provides an optimal, cost-effective solution when focusing on specific candidate regions while providing sufficient sequencing depth. Here, we present a targeted bisulfite sequencing approach in which nanopore sequencing is used to study the methylation status of regions of interest. METHODS: We applied this workflow to study the promoters of candidate genes associated with severe preterm delivery in a Latin American population. We amplified fragments greater than 1 kilobase in length from 12 genes via long PCR. Each sample was barcoded and pooled for sequencing in MinION flow cells. RESULTS: This approach achieves high sequencing depths, ensuring robust DNA methylation (DNAm) estimates. We detected significant hypomethylation of MIR155HG and hypermethylation of the ANKRD24 gene promoter in severe preterm birth samples, which is concordant with previously reported gene expression changes. CONCLUSIONS: This approach represents a scalable and cost-effective method suitable for targeted promoter methylation profiling across several samples. Our study provides a proof-of-concept for larger studies, demonstrating the broad applicability and scalability of our assay to any locus of interest. These features render the method especially apt for clinical diagnostics and precision medicine.

IntroductionThe male factor is responsible for 50% of infertility cases. Numerous studies have explored the relationship between human sperm morphology assessed via optical and electron microscopy and reproductive outcomes. In the sperm midpiece, mitochondria are arranged in a helical shape, forming a compact sheath. Disruptions in this precise mitochondrial structure, size, or organization may contribute to infertility. However, despite established links between abnormal sperm morphology and pathology, mitochondrial abnormalities in sperm remain relatively understudied.MethodsIn this study, we employed computational image analysis and fluorescence labelling to quantitatively assess morphometric changes in the sperm midpiece and correlate these findings with mitochondrial ultrastructure in fertile and infertile men.ResultsOur results revealed a significant increase in midpiece area, width, and roundness in sperm from men with teratozoospermia. These findings were further validated by electron microscopy. The ultrastructural morphometric analysis demonstrated disassembled, enlarged, and irregularly shaped mitochondria in sperm from infertile men. Additionally, we applied ultrastructural morphometric analyses to apoptotic sperm samples, observing similar qualitative and quantitative mitochondrial alterations, particularly in those from infertile individuals.DiscussionTraditional sperm morphology assessments are inherently subjective, but this limitation can be addressed through quantitative morphometric analysis. Enhancing the objectivity and precision of such evaluations is essential for elucidating the biological mechanisms of male infertility and optimizing assisted reproductive technologies. In our study, spermatozoa with poor morphology (<4%) and proximal flagellar abnormalities displayed significantly shorter and wider midpieces. Ultrastructural analysis further revealed that mitochondria in sperm from infertile men were significantly larger and more irregular in shape compared to those from fertile men. These findings indicate an association between altered midpiece morphometry, mitochondrial ultrastructure, and male infertility. The integration of computational tools for automated detection and quantification of these morphological changes offers a promising avenue to improve diagnostic accuracy and deepen our understanding of male reproductive disorders.

In mammalian sperm, mitochondria are very densely packed and form a helical sheath in the midpiece of the flagellum. Mitochondria from somatic cells can rapidly change shape to adapt to environmental conditions. During capacitation, mammalian spermatozoa undergo morphological and physiological changes to acquire fertilization ability, evidenced by changes in sperm motility patterns (hyperactivation) and the ability to perform the acrosome reaction. Whether there are changes in sperm mitochondrial morphology during capacitation is unknown. This work aimed to quantify morphometric changes in the sperm midpiece during capacitation. Using mitochondrial fluorescent probes and a combination of freely available software, we quantified the dimensions and fluorescence intensity of the midpiece. After capacitation, the area occupied by the mitochondria decreased due to a reduction in the width but not the length of the midpiece. The decrease in the area of the midpiece occurred in spermatozoa that underwent the acrosome reaction, suggesting a reorganization of the mitochondria during capacitation. Ultrastructural analysis supported these results. The application of image processing to fluorescence microscopy images may help to identify morphological changes during capacitation.

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 (H2O2) detection was three times higher in CAP than in NC sperm cells. To confirm that H2O2 production depends on mitochondrial superoxide (O2 center dot-$$ {\mathrm{O}}_2<^>{\cdotp -} $$) 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 O2 center dot-$$ {\mathrm{O}}_2<^>{\cdotp -} $$ 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 H2O2 production does not damage sperm and it is necessary for the normal CAP process. During human sperm capacitation, mitochondrial function increases, accompanied by a three times higher extracellular hydrogen peroxide production. Hyperactivation and acrosome reaction occur, without the presence of oxidative stress damage in the spermatozoa. The source of part of the measured hydrogen peroxide is sperm mitochondrial radical superoxide. The data indicates that an increase in mitochondrial function plays a role in generating this radical species, with this mechanism contributing to the modulation of sperm capacitation.image

DNA methylation plays a critical role in the dynamics of gene expression regulation and the development of various disorders. Whole-genome bisulfite sequencing can provide single base resolution of CpG methylation levels and is the “gold standard” for DNA methylation quantification, but it also has a high cost. In contrast, targeted sequencing is optimal when focusing on specific candidate regions, while providing sufficient sequencing depth. Here, we present a targeted bisulfite sequencing approach to study the methylation status of regions of interest. We amplify selected regions from bisulfite-treated DNA and sequence them using Nanopore sequencing. In this work, we applied this workflow to candidate gene promoters for severe premature labor in a Latin American population.We successfully amplified fragments over 1 Kb in length using long PCR conditions for 12 genes that were barcoded per sample and pooled to be sequenced on MinION flow cells. This approach achieved high sequencing depths, ensuring reliable DNAm estimation. We found significant hypomethylation of the MIR155HG gene promoter in severe preterm birth samples, which is concordant with reported gene expression changes.We demonstrate that combining bisulfite DNA treatment with pooled long-read sequencing is a cost- and time-effective method to evaluate DNAm in several targeted regions and several samples in parallel. This study provides proof-of-concept for larger studies, demonstrating the applicability and high scalability of our assay to any locus of interest. Our experience suggests that this approach can be easily transferred to other diagnostic questions.### Competing Interest StatementThe authors have declared no competing interest.

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.

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm mitochondrial activity, so the measurement of sperm mitochondrial function is an indicator of sperm quality. High-resolution respirometry is a method of measuring the oxygen consumption of cells or tissues in a closed-chamber system. This technique can be implemented to measure respiration in human sperm and provides information about the quality and integrity of the sperm mitochondria. High-resolution respirometry allows the cells to move freely, which is an a priori advantage in the case of sperm. This technique can be applied with intact or permeabilized spermatozoa and allows for the study of intact sperm mitochondrial function and the activity of individual respiratory chain complexes. The high-resolution oxygraph instrument uses sensors to measure the oxygen concentration coupled with sensitive software to calculate the oxygen consumption. The data are used to calculate respiratory indices based on the oxygen consumption ratios. Consequently, the indices are the proportions of two oxygen consumption rates and are internally normalized to the cell number or protein mass. The respiratory indices are an indicator of sperm mitochondrial function and dysfunction.

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.

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.

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.

In this work, we analyze the performance of several tracking methods in the scenarios of low temporal sampling acquisition setup in fluorescence microscopy. Machine Learning methods were applied to classify and analyze the extracted trajectories of sperm samples and their motion parameters. The results were compared with the most widely used sperm motility classification methods. Analyzed image sequences include real sequences acquired by confocal fluorescence microscopy and synthetic sequences generated by in-house software. The complete framework runs as a standalone application and can be used with minimal training by users with no programming skills.

To fertilize an oocyte, sperm must undergo several biochemical and functional changes known as capacitation. A key event in capacitation is calcium influx through the cation channel of sperm (CatSper). However, the molecular mechanisms of capacitation downstream of this calcium influx are not completely understood. Capacitation is also associated with an increase in mitochondrial oxygen consumption, and several lines of evidence indicate that regulated calcium entry into mitochondria increases the efficiency of oxidative respiration. Thus, we hypothesized that calcium influx through CatSper during capacitation increases mitochondrial calcium concentration and mitochondrial efficiency and thereby contributes to sperm hyperactivation and fertilization capacity. To test this hypothesis, we used high-resolution respirometry to measure mouse sperm mitochondrial activity. We also measured mitochondrial membrane potential, ATP/ADP exchange during capacitation, and mitochondrial calcium concentration in sperm from wild-type and CatSper knockout mice. We show that the increase in mitochondrial activity in capacitated wild-type sperm parallels the increase in mitochondrial calcium concentration. This effect is blunted in sperm from CatSper knockout mice. Importantly, these mechanisms are needed for optimal hyperactivation and fertilization in wild-type mice, as confirmed by using mitochondrial inhibitors. Thus, we describe a novel mechanism of sperm capacitation. This work contributes to our understanding of the role of mitochondria in sperm physiology and opens the possibility of new molecular targets for fertility treatments and male contraception.

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.

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.

Table S1. Primers used for quantitative PCR analysis. Sequences of the primers used for qRT-PCR. (XLSX 4 kb)

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 remainsidiopathic and potentially genetic in origin. The association of the Y chromosome and mitochondrial haplogroupswith male infertility has been previously reported. This association differs between studied populations and theirgeographical distributions. These effects have been only rarely analysed in mixed populations, such as SouthAmericans.METHODS:In this study, we analysed the contributions of the Y chromosome and mitochondrial haplogroups tomale 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 Europeanpaternal 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 chromosomehaplogroup 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 theUruguayan 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.

Context: Preterm birth (PTB) is a complex disease in which medical, social, cultural, and hereditary factors contribute to the pathogenesis of this adverse event. Interactions between genes and environmental factors may complicate our understanding of the relative influence of both effects on PTB. To overcome this, we combined data obtained from a cohort of newborns and their mothers with, multiplex analysis of inflammatory-related genes and several environmental risk factors of PTB to describe the environmental-genetic influence on PTB.Objective: The study aimed to investigate the association between maternal and fetal genetic variations in genes related to the inflammation pathway with PTB and to assess the interaction between environmental factors with these variations.Study design: We conducted a case-control study at the Pereira Rossell Hospital Center, Montevideo, Uruguay. The study included 143 mother-offspring dyads who delivered at preterm (gestational age < 37 weeks) and 108 mother-offspring dyads who delivered at term. We used real-time PCR followed by a high-resolution melting analysis to simultaneously identify gene variations involved in inflammatory pathways in the context of environmental variables. The genes analyzed were: Toll-like receptor 4 (TLR4), Interleukin 6 (IL6), Interleukin 1 beta (IL1B) and Interleukin 12 receptor beta (IL12RB).Results: We detected a significant interaction between IL1B rs16944 polymorphism in maternal samples and IL6 rs1800795 polymorphism in newborns, emphasizing the role of the interaction of maternal and fetal genomes in PTB. In addition, smoke exposure and premature rupture of membranes (PROM) were significantly different between the premature group and controls. IL1B and IL6 polymorphisms in mothers were significantly associated with PTB when controlling for smoke exposure. TLR4 polymorphism and PROM were significantly associated with PTB when controlling for PROM, but only in the case of severe PTB.Conclusions: Interactions between maternal and fetal genomes may influence the timing of birth. By incorporating environmental data, we revealed genetic associations with PTB, a finding not found when we analyzed genetic data alone. Our results stress the importance of studying the effect of genotype interactions between mothers and children in the context of environmental factors because they substantially contribute to phenotype variability. (C) 2016 Elsevier Ireland Ltd. All rights reserved.