N = 598 healthy participants, the different variables were tested for associations by means of linear regression models and group comparisons . Confirming previous findings from larger cohorts with shallow phenotyping, we found that birth factors do not explain a substantial amount of variance in functional asymmetries. Likewise, functional asymmetries did not seem to have comprehensive predictive power concerning clinical outcomes in our healthy participants. Future studies may further investigate postulated relations in healthy and clinical samples while acknowledging deep phenotyping of laterality.The development of handedness and other form of functional asymmetries is not yet understood in its critical determinants. Early life factors  have been discussed to contribute to individual manifestations of functional asymmetries. However, large-scale data such as the UK Biobank suggest that the variance in handedness that is explained by early life factors is minimal. Additionally, atypical handedness has been linked to clinical outcomes such as neurodevelopmental and psychiatric disorders. Against the background of this triad, the current study investigated associations between different forms of functional asymmetries and (a) early life factors as well as (b) clinical outcomes. Functional asymmetries were determined by means of a deep phenotyping approach which notably extends previous work. In our final sample of  For humans, the most obvious form of lateralization is handedness. Importantly, human handedness is not only asymmetric on an individual level but also on a population level. More precisely, a recent meta-analysis estimated that 10.6% of the population is left-handed7. Despite such clear evidence on the phenotypic level, it is still largely unknown in how far genetic and environmental factors contribute to the development of individual handedness and other forms of functional asymmetries8. Similarly, it is an open question how far the ontogenesis of a lateralized brain may overlap with developmental pathways of psychopathology. In this regard, several neurodevelopmental and psychiatric disorders have been associated with atypical lateralization9. Unraveling causal mechanisms in the development of structural and functional asymmetries may hence have clinical relevance.Functional asymmetries are widespread across species11 have been refuted as being too simplistic12. Likewise, candidate genes initially found to show associations with handedness could often not be replicated13. Along these lines, twin studies confirm that genetic factors explain about a quarter of the variance in human handedness15. Twin studies estimate the additive genetic heritability of a trait by comparing phenotypic concordance between monozygotic and dizygotic twins. Genome-wide association studies (GWAS) take a molecular approach in that millions of single nucleotide polymorphisms (SNPs) are tested for an association with the phenotype of interest. The largest GWAS on handedness so far  suggested common SNPs account for only 3.45–5.9% of the variance16. Interestingly, significant loci were located in genes playing a role in microtubule formation and regulation. Microtubules are protein complexes that form the cellular cytoskeleton and support neurogenesis, neuronal migration and axonal transport17. Noteworthy, mutations of microtubule-related genetic variants have also been linked to neurodevelopmental19 and neurodegenerative disorders19. Handedness as considered on a population level probably disposes on a strong genetic basis which is clearly evident in the overwhelming bias towards right-handedness. That is, right-handedness likely reflects a common manifestation of brain asymmetries that already develop in the majority of fetuses21. Still, in summary, existing literature points towards a small role of genetic factors in the development of individual handedness deviating from this population-level bias .Early (mono-)genetic theories on the development of handedness22. Since Medland et al.14 found no evidence for shared environmental variance to play a role in handedness development, it has often been concluded that non-shared environmental variance must account for the remaining proportion of unexplained variance in handedness23. For example, de Kovel et al.24 investigated large-scale data from the UK Biobank for an association between early life factors  and adult left-handedness. Indeed, adult left-handedness was shown to correlate with birth year and birth location—an effect that de Kovel et al.24 attributed to cultural artifacts. This is in line with studies indicating handedness to reflect interactions between genetic factors and cultural influences in terms of parenting, teaching, and implicit model learning26. Moreover, in the study by de Kovel et al.24, birthweight, multiple status, season of birth, maternal breastfeeding, and the participant’s sex were associated with left-handedness. However, even the combined predictive power of these factors was only marginal. McManus23 hence put forward that virtually none of the 75% of variance remaining unexplained from above-mentioned twin studies can be explained by environmental factors. Therefore, it has been argued that unexplained variance in handedness or other forms of functional asymmetries may not be environmental in the stricter sense but includes developmental noise, randomness23 and measurement error27. Graham22 continues that some portion of randomness may not be solved by applying stochastic rules to the behavior of involved biological agents but that other laws of dynamical systems such as deterministic chaos may play a role. Similarly, de Kovel et al.24 suggested that probabilistic randomness in terms of a ‘random model of early embryonic development’ might also contribute to the ontogenesis of individual left-handedness. That is, gene expression might underlie some sort of gradient which may be lateralized across embryonic brains on average29, but also includes symmetry or a reversal of asymmetry on the individual level.For a long time, phenotypic variation has been assumed to arise from genetic or non-genetic sources while the latter component was defined as being environmental30 reviewed the evidence for environmental factors previously associated with handedness  and their potential to induce epigenetic modifications. Similar to GWAS, epigenome-wide association studies (EWAS) aggregate epigenetic markers across the whole epigenome. A large EWAS that tested numerous cytosine-phosphate-guanine nucleotide base pairings (CpGs) for an association with left-handedness was recently published17. Meta-analysis of 3914 whole-blood samples from adult subjects showed that left-handedness was associated with CpGs located nearby SNPs that are known to explain phenotypic variance in handedness. However, overall, very little variance was explained by DNA methylation. Given that the authors also reported temporal instability of associations across different types of tissue, it was concluded that brain tissue rather than peripheral one may be better suited for future approaches.Epigenetic regulation has been suggested as a mechanism linking environmental factors and phenotypic outcomes in that environmental factors can modulate gene expression without modifying the actual nucleotide sequence. With respect to handedness, Schmitz et al.32, dyslexia34, and autism spectrum disorder (ASD)35. Recent meta-analyses further confirmed this pattern for post-traumatic stress disorder (PTSD)36, but not for depression37. Still, for depression38 as well as for schizophrenia40, and dyslexia42, studies have shown a higher frequency of atypical language lateralization. Since stress is considered a crucial factor in the ontogenesis and progression of such disorders, it has been argued that disturbed asymmetries may mediate the development of psychopathological outcomes in diathesis-stress models43. However, it remains debated how far specific alterations in lateralization represent a distinct diagnostic feature of certain mental disorders and how atypical lateralization does relate to observed symptoms. In this context, Mundorf et al.9 discuss three different kinds of associations that may characterize the relation between atypical asymmetries and psychopathological outcomes: (a) There are factors that simultaneously contribute to diffuse atypical lateralization on a whole-brain level and to a generic risk for psychopathology in a transdiagnostic way (non-specific association). (b) There are factors that contribute to function-specific atypical lateralization and to a risk for a specific diagnosis (diagnosis-specific association). (c) There are factors that contribute to a specific symptomatology in a transdiagnostic way on the level of lateralization as well as on the level of psychopathology (symptom-specific association). Importantly, these three feasible associations pose different predictions for atypical lateralization patterns as observed across different mental disorders that should be tested by means of empirical research9.As already introduced above, unraveling factors that play a role in the ontogenesis of asymmetries may also have clinical relevance. Non-right-handedness  as well as atypical lateralization of other forms of functional asymmetries has been extensively studied in relation to neurodevelopmental, psychiatric, and mental disorders. Amongst others, a heightened prevalence of non-right-handedness was found in schizophrenia3, shallow phenotyping can be understood as a waiver to conceptualize  and to measure (dimension of measurement) a phenotype with sufficient complexity. Regarding the dimension of conceptualization in the case of functional asymmetries, it becomes apparent that most studies only assess handedness but largely neglect other forms of functional lateralization. However, other kinds of functional asymmetries may be better suited than handedness for some research interests. For instance, we endorsed the integration of social laterality phenotypes such as hugging since these allow to capture different evolutionary pressures and may be better suited for research across species3. From a dimension of measurement, it may be claimed that handedness, the most common proxy for hemispheric asymmetries, is commonly not assessed accurately. In contrast, handedness is often only deduced from a unidimensional measure. In its most extreme, this rationale can be found in the assessment of handedness in terms of only one item that typically asks for writing hand. Thereby handedness is treated as a binary concept and only refers to one manual task . Of note, many studies in the field of laterality have recognized this issue and satisfy an assessment of handedness using several items. For instance, many researchers use the Edinburgh Handedness Inventory (EHI44), a questionnaire that queries hand preference for various manual tasks . Such an approach ultimately allows the calculation of a laterality quotient (LQ) which satisfies handedness as a continuous variable. Still, for the EHI as well as for the simple assessment of writing hand, research broadly relies on self-reported preference measures. Only a few studies further integrate performance measures that assess hand skill. A promising example of such a performance measure is the Pegboard task45, which requires participants to place several pegs initially stuck in a straight row of holes on a board in a second parallel row of other holes as quickly as possible. As this is done with both hands consecutively while reaction time is taken, it is possible to compare performance of the left and the right hand. Using the Pegboard task rather than a shallow handedness phenotype has resulted in the identification of the first genetic variants associated with handedness in GWAS46. Ideally, to come to a preferably differentiated picture of an individual’s lateralization, studies may combine different measures of self-reported hand preference and measures of hand skill as well as measures of other forms of functional asymmetries . This notion may be especially relevant considering that different performance measures of handedness (the Pegboard task amongst them) have been revealed to show only small correlations among each other and may reflect distinct dimensions of asymmetries47. Undoubtedly, a comprehensive assessment of functional asymmetries—which we refer to as deep phenotyping—may not be applicable for larger-scale studies. However, even though the rigid focus on handedness as a sole proxy for functional asymmetries alongside its unidimensional measurement may have become some kind of common minimal standard, a deeper phenotyping of functional lateralization may be indicated to achieve further progress in laterality research3.One severe limitation that does apply to the majority of cited studies is the issue of shallow phenotyping. As we outlined in more depth in a recent opinion paper24. Second, we aimed at understanding associations between handedness and other forms of functional lateralization with subclinical tendencies of several mental disorders in a healthy sample.Capturing functional lateralization phenotypes by means of deep phenotyping, this study aims to further accumulate knowledge on how environmental factors  play a role in the ontogenesis of handedness and other forms of functional lateralization. Therefore, the first part of the study may be considered a replication approach of the findings by de Kovel et al.N = 631 participants.We recruited healthy participants between 18 and 35 years with German language skills sufficient for understanding questionnaires and instructions given in our study. Moreover, all participants were of Central European ancestry. Ancestry was assessed by means of self-report inquiring the country of descent of participants as well as all parents and grandparents. Individuals reporting Central European ancestry for all three generations were eligible to participate. In our definition, Central European ancestry covered all Northern, Western and Southern Europe, including Spain, while Portuguese descent was excluded. We included Polish and Russian ancestry, but excluded individuals of Southeast European descent . With respect to handedness, we had no specific inclusion and exclusion criteria but we aimed for a balanced ratio of all handedness categories so that we specifically enrolled left- and mixed-handed individuals. Thus, we over-selected participants with atypical handedness in order to improve statistical power and approach variance homogeneity in statistical analyses. Study advertisement only indicated that the study investigated handedness and did not reference handedness and mental health. Since we also excluded participants reporting psychopathological conditions, we do not believe the results of the current study to be biased by participant recruitment and advertising. In total, we tested This study was approved by the local ethics committee of the Faculty of Psychology at Ruhr University Bochum, Bochum, Germany. All participants gave written informed consent and were treated in accordance with the declaration of Helsinki.44) and the Waterloo Footedness Questionnaire (WFQ48) as self-report asymmetry measures of handedness and footedness, respectively. Moreover, participants completed validated German versions of the following clinical questionnaires: the Beck’s Depression Inventory , the Adult ADHD Self-Report Scale Symptom Checklist , the State-Trait Anxiety Inventory—Trait , the Childhood Trauma Questionnaire  and the Schizotypal Personality Questionnaire . Finally, participants performed various hand skill tasks including the Pegboard task45, the Alphabet test59, and the Tapley–Bryden test60.Data collection took place between 11/04/2018 and 14/10/2022. Having given informed consent, participants completed an online survey asking for above-mentioned inclusion and exclusion criteria as well as for several factors surrounding their birth . Eligible participants were then invited for testing at Ruhr University Bochum. Testing sessions started with a second online survey including the Edinburgh Handedness Questionnaire (EHI61) and lateralization for visual attention/visuo-spatial perception was assessed using a Line bisection task62. At the end of the testing session, participants were compensated with 20 euros or course credit.Moreover, language lateralization was assessed using a Dichotic listening task  were calculated by means of the following formula: LQ = [(right − left)/(right + left)] × 100. Using the EHI and the WFQ to create categories of left-, mixed-, and right-handedness/-footedness, we defined scores of < = − 60 as left-handed/left-footed and scores of > = + 60 as right-handed/right-footed. Participants scoring between these cutoffs were classified as mixed-handed/mixed-footed. Behavioral asymmetry tasks  as well as clinical questionnaires were analyzed according to corresponding manuals.63. R scripts used for analysis can be retrieved from the Open Science Framework (https://osf.io/nkem6/).Statistical analysis was conducted in R version 4.1.2 (2021-11-01) and RStudio. The manuscript was prepared using the papaja package64, which tests different normalizing procedures and applies the one with the best outcome. For details, see 65 for each set of variables. Table We grouped measured variables in three conceptual categories: 1) birth factors, (2) asymmetry measures, and (3) clinical questionnaires. Quantitative asymmetry measures, birth factors, and clinical questionnaire scores were transformed to normality using the bestNormalize function birth fa24. We did so by modeling writing hand measured by the first EHI item  as a function of significant predictor variables in the study by de Kovel et al.24, specifically birth weight, birth size, breastfeeding, twin status, and the presence of any birth complication.We applied different statistical models to analyze hypothesized relations between these variables. After descriptive statistics (Part 1), we modeled asymmetry measures as a function of birth factors Parts 2–5). Subsequently, we modeled clinical questionnaires as a function of asymmetry measures (Parts 6 and 7). Therefore, different variables  served either as predictor or outcome variables (Table –5. Subsep < 0.05) linear regression models (Parts 2 and 6) by means of visual inspection of the following residual plots: correct specification of the model , normality of residuals , homoscedasticity of residuals  and existence of outliers or influential data points .We checked statistical assumptions of nominally significant (p < 0.05) ANOVAs , we checked required assumptions by means of visual inspection of the following residual plots: correct specification of the model  and normality of residuals . Moreover, we performed Levene’s tests in order to test for homogeneity of variance.For nominally significant  born between 1984 and 2003. Mean age was 23.61 years . On average, participants had 13.51 years of education .The final sample comprised Table When applying a L–M–R handedness categorization based on the EHI, 101 participants (17%) were left-handed (EHI LQ < − 60), 82 participants 14%) were mixed-handed (− 60 < EHI LQ < 60), and 415 participants (69%) were right-handed (EHI LQ > 60) , 309 (52%), and 229 (38%) participants were left- (WFQ LQ < − 60), mixed- (− 60 < WFQ LQ < 60), and right-footed (WFQ LQ > 60), respectively Table . In the t-tests for quantitative birth factors and clinical questionnaires between left- (EHI LQ < = 0) and right-handers (EHI LQ > 0). Table Table p > 0.05).We ran linear regression models for seven outcomes (quantitative asymmetry measures) and five predictors (quantitative birth factors), applying FDR correction for 20 independent tests (Table p < 0.05). Pegboard LQ showed weak associations with the variables “any birth complications”, We ran ANOVAs for seven outcomes (quantitative asymmetry measures) and six predictors (binary birth factors), applying FDR correction for 25 independent tests (Table p = 0.017) and right-handed participants (p = 0.049). Right- and mixed-handed participants did not differ from each other regarding maternal age at birth (Table p = 0.056).We ran ANOVAs for two outcomes  and seven predictors (quantitative birth factors), applying FDR correction for 4 independent tests Table . Figure th Table , model 5N = 598) = 6.21, p = 0.045, V = 1.76, but did not survive FDR correction (p = 0.224).For Part 5a, we ran Chi square tests for two outcomes  and six predictors (binary birth factors), applying FDR correction for 5 independent tests Table . Full re24 by running a logistic regression analysis on a binary handedness measure . This analysis included N = 150 left-handers and N = 435 right-handers . Birth weight, birth size, breastfeeding, twinning, and the presence of any birth complications were included as predictors. None of the predictors reached significance .We ran linear regression models for five outcomes  and seven predictors (quantitative asymmetry measures), applying FDR correction for 15 independent tests Table . Figure p = 0.129).Moreover, there were weak nominally significant associations between BDI score and Alphabet LQ, p = 0.015) indicated violation of the assumption of variance homogeneity (Table p = 0.003).Finally, we ran ANOVAs for five outcomes  and two predictors , applying FDR correction for 3 independent tests Table . Figure p = 0.011). The WFQ categories further showed associations with the STAI-T score, p = 0.013, 0.024, and 0.028, respectively).Moreover, the WFQ categories were associated with the SPQ score, p = 0.021) (Table p = 0.022).The EHI categories showed an association with the SPQ score, To sum up, effects for ANOVAs on clinical questionnaires as a function of categorial asymmetry measures seem to be driven by the middle (mixed-handed/mixed-footed) category mainly.43.In this study, we considered associations between functional hemispheric asymmetries and early life factors as well as subclinical symptoms of psychopathology in healthy participants. This triad is interesting from a conceptual point of view: it relates to the idea that the ontogenesis of functional hemispheric asymmetries may be disturbed by certain birth factors resulting in atypical lateralization that sets a greater vulnerability or risk for psychopathological outcomes, or actually mediates their development24 relied on handedness as a single, categorical index of functional asymmetries which was inquired with only one item asking for hand preference. In this regard, the current study is unique since different forms of functional hemispheric asymmetries  were approached by means of deep phenotyping with different measures . Birth factors and clinical questionnaires were assessed by means of self-report. Thereby, our approach allowed for more nuanced statistical tests and also acknowledged the fact that different laterality phenotypes may differ in their strength of association with our predictors  as well as in their sensitivity for showing these associations in our statistical analyses.Previous studies such as de Kovel et al.Few associations reached statistical significance and most did not survive correction for multiple testing. In Parts 2 to 5, we modelled birth factors as a function of functional asymmetries but did not find any significant effects in Part 2. For Part 3, we found the occurrence of birth complications and higher birth order position to model the LQ as calculated for the Pegboard task in that birth complications and higher birth order position were associated with lower Pegboard LQs . For Part 4, handedness categories (left-handed/mixed-handed/right-handed) were identified as a function of maternal age at birth in that left-handed participants reported higher maternal age at birth as the other two handedness categories (which did not differ amongst each other). For Part 5a, prevalence of handedness categories (left-handed/mixed-handed/right-handed) significantly differed between participants of different birth order positions. In Parts 6 and 7, we modelled functional asymmetries as a function of clinical questionnaires. For Part 6, scores on clinical questionnaires were identified as a function of LQs as calculated for different tasks. The EHI LQ as well as the Alphabet LQ were shown to model the SPQ score in that higher SPQ scores were associated with lower LQs in these tasks . Similarly, higher BDI scores predicted lower Alphabet LQs while higher CTQ scores predicted lower DLT LQs, and higher ASRS scores predicted lower WFQ LQs. Therefore, in Part 6, higher scores on clinical questionnaires were uniformly predicted by more leftward lateralization. For Part 7, we found few significant differences for categorical asymmetry measures with respect to clinical questionnaires, which remained significant after correction for multiple testing. In detail, mixed-footed (WFQ) as well as mixed-handed (EHI) participants showed higher SPQ scores than right-footed (WFQ) and right-handed (EHI) participants. Hence, these effects seemed to be driven by the middle (mixed-footed/mixed-handed) category in large parts. The WFQ was further associated with the STAI-T and the BDI scores but post-hoc tests did not reach significance.Noteworthy, most effects only reached statistical significance when not controlling for multiple comparisons. We consider it important to highlight the small effect sizes of basically all observed effects. That is, eta squared was 24 also report only few statistically significant associations with negligible effect sizes between adult left-handedness and a plentitude of birth factors. Amongst the few factors that turned out to be significant in the publication by de Kovel et al.24, we also assessed birth weight, birth size, breastfeeding, twin status, and birth complications. Therefore, in Part 5b we attempted to directly replicate the findings of de Kovel et al.24 in modelling handedness as classified by the writing hand item of the EHI as a function of these predictors. Noteworthy, none of the predictors reached significance. Failure to replicate the results by de Kovel et al.24 may be attributed to the fact that our sample size was substantially smaller. Importantly, de Kovel et al.24 used large-scale data from the UK Biobank covering ~ 500,000 participants. However, having said that most of the associations tested in our data did not reach statistical significance nor convincing effect sizes, one may conclude that they lack decisive importance at the population level. However, in the larger study by de Kovel et al.24 effects reached statistical significance and probably did not so by chance. It is rather conceivable that effects are significant and thus important for the single individual. For instance, being part of a multiple birth may be of great importance in triggering the development of atypical brain asymmetry in some individuals, but not in others.The prevailing pattern of a large number of non-effects alongside small effect sizes of nominally significant results in the current study is in accordance with existing literature. Most prominently, de Kovel et al.24 conclude that the current literature does not support the notion that specific environmental variables (in their as well as in our study) may fill the gap between variance explained by genetic factors and so-far unexplained variance in handedness . This is the case for many clinical phenotypes in the epidemiological literature, for which twin studies show a substantial amount of variance explained by non-shared environmental factors. However, the role of non-shared environmental factors is likely to be heavily overestimated (and overinterpreted), as it is based on simple subtraction; it equals the variance not explained by additive genetic and shared environmental factors. Therefore, what is typically called non-shared environmental variance not only includes measurement error and gene-environment interaction, but also chance or random events27. De Kovel et al.24 also accounted for this perspective in discussing their findings in the context of randomness in fetal brain development as already elucidated in the introduction. Notably, for the birth factors it has been proposed that their effect is mediated via epigenetic mechanisms. However, a large EWAS found only little handedness variance to be captured by epigenetic modifications of DNA17, casting doubt not only on strong associations between birth factors and functional asymmetries, but also on strong associations between epigenetic factors and functional asymmetries.However, de Kovel et al.24 only tested for associations with self-reported writing hand, we included diverse functional laterality phenotypes and assessed them by means of deep phenotyping. Since we replicated the gross pattern of non-effects found by de Kovel et al.24 for self-reported hand preference, deep phenotyping of multiple asymmetry measures does not seem to enhance the power of unraveling relations for the research question at hand. However, it should be noted that birth factors as well as clinical questionnaires in the current study were also assessed by means of self-report. Self-reports are typically prone to certain biases as well as reporting errors. Especially for the birth factors collected as self-report , we had to exclude several data points based on plausibility .32, ASD35, PTSD36). Therefore, one might conclude there is some sort of rubicon covering noticeable qualitative differences between healthy and clinical samples regarding asymmetry measures. Regarding the different kinds of associations that may possibly link atypical functional lateralization and psychopathological outcomes9, non-occurrence of effects in the context of our healthy sample may rather point towards a broader, generic and transdiagnostic effect of atypical lateralization on psychopathology, if any.Similarly, one might question whether the clinical questionnaires used in the current study were actually suitable in our healthy sample. Since participants were only included reporting no mental, psychiatric, or neurological disorder, at best we might have covered preclinical manifestations of the psychopathological constructs. As a result, the variance in our clinical questionnaires might not have been sufficient enough to unravel putatively existing effects. This assumption gains further plausibility considering the fact that clinical samples frequently produce large effects with respect to laterality measures. For instance, as already mentioned in the introduction, meta-analyses univocally confirm a certain relation between atypical handedness or other forms of functional lateralization and diverse clinical diagnoses  rather than a reversal (such as left-handedness) is of relevance for clinical outcomes36.Still, for the results that reached statistical significance for a categorical operationalization of asymmetry measures in the current study, it is striking that they often concerned the “middle” category. Indeed, effects often seemed to be driven by mixed-handed/mixed-footed participants while more extreme forms of lateralization towards the left or the right side of the continuum did not seem to be influential. Hence, one might speculate that it is not left- but mixed-handedness/-footedness that shows the closest association with clinical constructs. Indeed, this is in line with several meta-analyses suggesting that disorders such as PTSDIn conclusion, the current study further confirms previous findings of mostly negligible associations between birth factors and functional asymmetry measures in healthy individuals. Deep phenotyping did not lead to any substantial changes in this overall results pattern, confirming the robustness of previous findings using shallower phenotyping. Likewise, effects between functional lateralization and diverse psychopathological outcomes did not achieve noticeable predictive power in our sample of healthy individuals. Further research might identify qualitative differences between healthy and clinical samples as studying the latter typically renders strong effects for lateralization indices. Future studies might also benefit from the inclusion of social laterality phenotypes and biological markers as well as from the application of longitudinal approaches.Supplementary Information.


Background: Infertility remains a global health problem with male-factor infertility accounting for around 50% of cases. Understanding the molecular markers for the male contribution of live birth success has been limited. Here, we evaluated the expression levels of seminal plasma extracellular vesicle (spEV) non-coding RNAs (ncRNAs) in men of couples in relation with those with and without a successful live birth after infertility treatment.Method: Sperm-free spEV small RNA profiles were generated from 91 semen samples collected from male participants of couples undergoing assisted reproductive technology (ART) treatment. Couples were classified into two groups based on successful live birth  and . Mapping of reads to human transcriptomes followed the order: miRNA > tRNA > piRNA > rRNA> “other” RNA > circRNA > lncRNA. Differential expression analysis of biotype-specific normalized read counts between groups were assessed using EdgeR (FDR<0.05).Result: We found a total of 12 differentially expressed spEV ncRNAs which included 10 circRNAs and two piRNAs between the live birth groups. Most (n = 8) of the identified circRNAs were downregulated in the no live birth group and targeted genes related to ontology terms such as negative reproductive system and head development, tissue morphogenesis, embryo development ending in birth or egg hatching, and vesicle-mediated transport. The differentially upregulated piRNAs overlapped with genomic regions including coding PID1 genes previously known to play a role in mitochondrion morphogenesis, signal transduction and cellular proliferation.Conclusion: This study identified novel ncRNAs profiles of spEVs differentiating men of couples with and without live birth and emphasizes the role of the male partner for ART success. Infertility is the inability to conceive within 1 year of regular unprotected sexual intercourse. It remains a global reproductive problem affecting 15% of heterosexual couples and nearly 190 million individuals with the desire to conceive . The diain vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI)  . Howevern (ICSI) . This man (ICSI) . Thus, uAs such, profiling seminal plasma to identify novel markers of reproductive success has been proposed . SeminalRecent findings from our group  complemeStudy population. The study population includes men (n = 91) of couples receiving fertility treatment at Baystate Medical Center located in Springfield, Massachusetts who agreed to participate in the Sperm Environmental Epigenetics and Developments Study (SEEDS). SEEDS is prospective observational cohort study designed to investigate the associations of male preconception endocrine disrupting chemical exposure with sperm epigenetics and subsequent early-life development. The inclusion criteria were male partners of at least 18 years old without vasectomy and fresh ejaculate sperm used for ART treatment while females were included if 40 years or younger. Relevant demographics , lifestyle factors , and medical history (diagnoses of infertility) data were collected by clinic personnel during the ART cycle for both partners. Male fertility diagnoses were based on semen quality parameters according to World Health Organization (WHO) reference values  gradient fractionation method  according to manufacturer’s instructions. All steps including polyadenylated RNA library preparation, reverse transcription cDNA synthesis, and 6 cycles of PCR amplification followed modified SMARTer smRNA-Seq protocol (Clontech). SPRI beads were used for library cleanup, and size selection and quality assessment were performed using AMPure X beads and fragment analyzer, respectively. Multiplexed samples were loaded onto the flow cell using Illumina barcodes (1—24) and sequencing of 50-bp single-end reads was performed on an Illumina HiSeq 4000. Libraries construction and sequencing were conducted at the UMass Medical Genomics Core Facility.small RNA sequencing analysis. The quality of the raw reads was assessed using FastQC (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE203494. Details of the count output were reported in our previous publication . All analyses were performed in R (v4.1.0) software.lues TMM  and speclues TMM  and werelues TMM . ncRNA sGene target determination and functional annotation. BEDTools (https://www.gencodegenes.org/human/release_38lift37.html). Based on evidence suggesting shared gene-regulating mechanisms between piRNA and miRNA (https://metascape.org/gp/index.html#/main/step1)  .n = 91) are presented in 2) of 36.2 ± 6.0 and 29.9 ± 5.5, respectively. n = 28; no live birth, n = 63). There was no statistical difference (p > 0.05) in the couple demographics, infertility diagnoses and ART outcomes such as the number of oocytes retrieved and fertilized eggs between the two groups. As expected, the total counts of day 3 high quality embryos were significantly higher (p < 0.05) in the group with livebirth relative to the group without successful livebirth. While similar observation was also observed in day 5 high quality embryos, it did not achieve statistical significance (p > 0.05).The overall demographic and clinical characteristics of the participants , of which the majority (80%) had lower expression in spEVs among men of couples without successful live birth compared to those with live birth. The remaining two differentially expressed spEV ncRNAs were annotated as piRNAs (piR-28478 and piR-1077), in which both were over-expressed in men without live birth compared to men of couples with a successful live birth.First, we sought to identify spEV derived differentially expressed ncRNAs in men without successful live births compared to men with live births. We found a total of 12 ncRNAs were differentially expressed in men without live birth . The mosPID1) and processed pseudogene (RP11-1315F8), respectively. The piRNAs were also predicted to target 470 unique genes  that were differentially expressed (q < 0.05) between live birth groups, in which predominantly (66.7%) were downregulated among men of couples without successful live birth. Finally, gene targets of the identified circRNAs and piRNAs were enriched in functional terms related to cellular communications, signaling, organ morphogenesis, and early embryonic development and birth.Infertility treatment for couples has become prevalent especially in high income countries. However, the success of a live birth from one ART cycle has not improved over decades and results in multiple attempts that are accompanied by significant financial and emotional costs . In atteOther epigenetic mechanisms, such as EV-derived ncRNAs, are implicated in remodeling sperm RNAs during epididymal maturation and ejaculation  and havecircRNAs are single-stranded RNAs produced from alternative backsplicing events of corresponding messenger RNA (mRNA) resulting in a covalently closed-loop structures without 5′caps and 3′poly tails . DespitePID1 and the pseudogene, RP11-1315F8, respectively. PID1, also known as NYGGF4, encodes for a protein that contains a phosphotyrosine-interacting domain containing 1 and plays a role in diverse physiological processes including mitochondrion morphogenesis, signal transduction and glucose uptake (PID1 has been suggested to differ based on cellular context, and its silencing in cell lines resulted in reduced proliferation rate (The biological role of EV piRNAs in human body fluids in disease outcomes has been well documented . Howevere uptake . The funion rate . While tThis study has notable strengths. We were the first to utilize high throughput small RNA sequencing to identify spEV ncRNAs that distinguished couples undergoing ART treatment that resulted with and without live birth. Additionally, the seminal plasma utilized in our study was collected from the same semen sample used for IVF treatment; thus, our prospective study design allowed us to connect spEV RNA cargo obtained during fertility treatment with subsequent live birth outcomes. We also acknowledge that this study has several limitations. First, spEV ncRNAs were compared between men of couples seeking infertility treatment; thus selection bias and the generalizability of our findings should be noted. Also, given there were no differences in couple demographic and male infertility status between the two live birth groups, we did not include these covariates in our statistical models. However, we cannot rule out the potential for other unmeasured confounders such as differences in nutrition, environmental exposure patterns or underlying health conditions. While the adaptor-ligation free protocol used for generating our small RNA libraries exhibits less sensitivity to modifications to RNA nucleotides resulting in more diverse libraries , distingIn conclusion, through the use of small RNA-sequencing, our study is the first, to our knowledge, to identify spEV ncRNA cargo that distinguished men of couples with and without successful live birth during infertility treatment. These findings highlight that spEVs, derived from several accessory sex glands, contain important molecular cargo that may impact the trajectory of early-life development and subsequent reproductive success, and moreover, the importance of the male partner for reproductive success.


In these settings, perinatal distress, i.e., high symptoms of anxiety, depression, and/or stress during pregnancy, may be linked with adverse birth outcomes. However, few prospective studies have investigated the impact of perinatal distress on adverse birth outcomes such as preterm birth , low birth weight (<2.5 kg), and small for gestational age birth  and 33.9 (SD = 1.1) weeks of gestation. Distress was measured using the Edinburgh Postnatal Depression Scale, the anxiety subscale of the Hospital Anxiety and Depression Scale, and the Perceived Stress Scale. To determine birth outcomes, gestational age was estimated from the last menstrual period, fundal palpation, and/or ultrasound, while birth weight was obtained from delivery records and measured within three days after birth for those delivered at home. Logistic regression and mediation analysis were employed to evaluate the impact of perinatal distress on adverse birth outcomes.Perinatal anxiety , depression , stress , and total distress  were all associated with low birth weight, and small for gestational age birth but none did with preterm birth. Mediation analysis demonstrated that perinatal distress was a mediator in the pathway between socioeconomic adversity and adverse birth outcomes.Our study revealed that perinatal distress was linked with adverse birth outcomes and acted as a mediator between socioeconomic adversity and these outcomes. Our findings highlight the importance of screening women for distress and providing appropriate interventions, focusing on women experiencing socioeconomic adversity. Integrating mental health services into primary maternal care in low-income countries could be an effective approach to achieve this. For womDistress was assessed at inclusion, i.e., at or before 20 weeks of gestation and at 32 to 36 weeks of gestation. As the “perinatal period” often refers to the period from 22 weeks of gestation up to the end of the first week post-partum, we distinguish perinatal distress, referring to the measure of distress at 32 to 36 weeks of gestation, from early antenatal distress measured at inclusion. Anxiety was measured using the seven-item anxiety subscale of the Hospital Anxiety and Depression Scale (HADS-A), each item rated from 0–3. The HADS-A score was dichotomized at ≥8, a cut-off associated with clinically significant anxiety symptoms . DepressOverall distress was defined as high symptoms in at least one of the three domains of distress, i.e., anxiety, depression, or stress. In addition, presence of high symptoms in one, two, or three domains were considered to indicate the level of distress. Likewise, overall distress over time and level of distress over time were generated to show the change during pregnancy from inclusion to 32 to 36 weeks of gestation. As a continuous outcome, a total distress score was obtained by summing the standardized anxiety, depression, and stress scores. Finally, a change in the scores of each measure of distress over the course of pregnancy was also computed by subtracting the scores at inclusion from the corresponding values at 32 to 36 weeks of gestation.The internal consistency of the scales was assessed using Cronbach’s alpha, which yielded 0.87 for the anxiety scale, 0.71 for perceived stress, and 0.78 for depression at inclusion. At 32 to 36 weeks of gestation, the internal consistency remained almost same for the anxiety and depression scales while it increased to 0.77 for perceived stress.th percentile for gestational age at birth, and sex was classified as small for gestational age (SGA) according to international standards proposed by the INTERGROWTH-21st project  was approved by the Institutional Research Review Board of College of Health Science, Aksum University, Ethiopia. Verbal consent was obtained from each participant prior to data collection.Inclusivity in global research. Additional information regarding the ethical, cultural, and scientific considerations specific to inclusivity in global research is included in the In total, 934 of the 991 included women were followed until delivery and had completed measures of distress with at least one known birth outcome . The chaAs seen in All continuous measures of perinatal distress—anxiety, depression, stress, and total distress—were associated with low birth weight and small for gestational age, while none of them did with preterm birth. Of the change in measures of distress over time, none of the changes in measures of distress over time was associated with the adverse birth outcomes . AdditioIn the logistic regression models, perinatal anxiety , stress , depression , and total distress  were significantly associated with higher odds of low birth weight, and small for gestational age. None of the measures of perinatal distress was associated with preterm birth. Also, level of perinatal distress was not associated with any of the adverse birth outcomes. Moreover, overall distress and level of distress over time during the course of pregnancy were not associated with any adverse outcome. From the socioeconomic adversities, only intimate partner violence was associated with small for gestational age .In the present study, we have investigated the influence of different measures of perinatal distress on adverse birth outcomes. We determined that perinatal distress was associated with adverse birth outcomes in this cohort of primarily rural Ethiopian women. Indeed, perinatal distress was a mediator in the pathway between socioeconomic adversity and adverse birth outcomes. Given the negative health effects of the adverse birth outcomes in early childhood, in later life, and future generations , 25, ourAll continuous measures of perinatal distress were associated with low birth weight and/or small for gestational age, consistent with several studies –62. The Furthermore, perinatal distress was found to be a mediator in the pathway between socioeconomic adversity and low birth weight and/or small for gestational age. This finding implies that socioeconomically disadvantaged women will likely experience distress, leading to adverse birth outcomes. Therefore, our path analysis findings may highlight the importance of targeted screening and management of distress, focusing on women experiencing socioeconomic adversity. In low-income countries like Ethiopia, the screening and management of distress can be facilitated by integrating mental health better within primary maternal health care services.Unlike birth size, we did not detect an influence by any measure of distress on preterm birth, which was consistent with birth-cohort studies in several low income countries, including the subgroup analyses of a recent meta-analysis , 37, 64.Interestingly, our data also showed that none of the changes in distress measures over time were associated with adverse birth outcomes. The difference in each measure of distress over time during pregnancy, however, was small. Thus, the insignificant change in scores might indicate persistent high distress and suggest the need for maternal mental health interventions starting from early pregnancy.One of the major strengths of our study is that our data considered a broad range of distress measurements in both early and late pregnancy and their influence on adverse birth outcomes. However, our study is limited in that bio-specimens were not collected. Hence, we could not measure biomarkers such as cortisol or norepinephrine, validated markers of distress and could be seen as surrogate markers of possible changes in oxygen and nutrient supply to the fetus, linked to adverse birth outcomes. Also, the gestational age estimated by ultrasound was not available for most women, so it is possible that our small for gestational age data is not sufficiently accurate. Additionally, our study design was not suitable to rule out the probable reverse causality between distress and adverse birth outcomes due to the two-way conversation between the fetus and the mother. Finally, distress was assessed via an interviewer-administered questionnaire, and our data may be subjected to misclassification bias.Our study revealed that perinatal distress was linked with adverse birth outcomes and acted as a mediator between socioeconomic adversity and these outcomes. Our findings highlight the importance of screening women for distress and providing appropriate interventions, focusing on women experiencing socioeconomic adversity. Integrating mental health services into primary maternal care in low-income countries could be an effective approach to achieve this.S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Dataset(DTA)Click here for additional data file.S1 Checklist(DOCX)Click here for additional data file.S2 Checklist(DOCX)Click here for additional data file.


Primary cardiac hydatid cyst is a rare and fatal pathology, especially wheninvolving the left ventricular free wall. A 44-year-old male was diagnosed withlarge intramural left ventricular hydatid cyst with wall thickness of 6 mm atthe thinnest point. Cyst was accessed through pleuropericardial approach  which resulted in easy entry intothe cyst, mitigating the risk of mechanical injury. This case report highlightsthat with detailed evaluation, cardiac hydatidosis can be addressed withoff-pump technique, reducing the anaphylaxis risks and cardiopulmonarybypass-related effects. Clinicalmanifestation of echinococcosis depends on the involved organ along with number andsize of the cysts.Hydatid cyst disease or .Cardiac component as a part of multivisceral involvement is observed in < 2% ofcases, with primary infection of the heart being an exceedingly rare condition (<0.2%). Isolated primary cardiac hydatidosis being rare can be mistaken forventricular aneurysm, atrial myxoma, or simple epicardial cyst. Cardiac hydatidcysts can be pericardial, endocardial, or, very rarely, intramural. Left sided cystsusually tend to grow subepicardially whereas right sided cysts have tendency to growsubendocardially and intracavitarily,4. Release of cystcontents can induce a life-threatening allergic reaction, which might also beencountered during surgical excision of hydatid cysts.The symptoms range from being asymptomatic to having a life-threatening course. As inour case, cardiac hydatid disease presented with chest pain and shortness of breath.Additionally, palpitations and recurrent syncope may also occur and are related tounderlying cardiac arrhythmias or mechanical effect. Intracardiac rupture of cystcan result in pulmonary embolism or stroke,7.Surgical excision, usually with cardiopulmonary bypass (CPB) support, remains themainstay of treatment even in asymptomatic patients due to risk of rupture, butexcision needs to be therapeutically supplemented by anthelminthic medications inthe preoperative and postoperative periods to prevent recurrencesThe aim of our case report is to present a successful resection of giant leftventricular intramural hydatid cyst on beating heart and to outline importantconsiderations during off-pump surgical excision.A 44-year-old non-diabetic, normotensive male presented with history of left sidedchest pain and cough with progressive dyspnea for the previous two months. Thepatient was examined and initially sent for high-resolution computed tomography(HRCT) scan and echocardiography. HRCT revealed large well encapsulatedmultiloculated mass measuring around 13.9 × 10 × 12.9 cm in sizewithin the myocardium of the left ventricular free wall along with mildcalcification of medial wall. HRCT also showed the mechanical effect causingreduction in left ventricular intraluminal volume and diaphragmatic depression.Cardiac magnetic resonance imaging (MRI) was done in order to delineate the extentand tissue penetration. MRI showed “beak sign” on the medial side of cyst indicatingits myocardial origin and attenuated left ventricular wall thickness of 6-7 mm atthe thinnest portion and 11-12 mm at the thickest portion. Reduction in leftventricular volumes was also noted .CoronarAfter two weeks of prior albendazole therapy, the patient was prepped for surgery.Sternotomy was done, and, as anticipated, the pericardium was found adherent towhole epicardial surface  and 2B. After resection, echocardiography showed moderate mitral regurgitation possibly dueto decompression of the ventricular cavity, but the patient remained hemodynamicallystable . PostopeThe heart is a rare but potentially fatal site for hydatid cyst, especially in theleft ventricular free wall. A commonly involved site is the left ventricle, followedby right ventricle, interventricular septum, atria, and pulmonary artery. Inadvanced cases, rupture is a lethal complication of cardiac hydatid cyst, especiallyif not treated timely following the initial diagnosis or if the patient is presentedbelatedly.We report our experience with a giant intramural left ventricular hydatid cyst whichwas successfully resected on beating heart. Cardiac intramural hydatid cyst is arare entity, and its management should be based on multidisciplinary approachinvolving collaboration between surgeons, radiologists, and infectious diseasespecialists. To our knowledge, this is the largest and first intramural ventricularhydatid cyst removal done without CPB. Cardiac hydatid cyst resection is usuallyperformed under CPB support, but detailed preoperative evaluation and carefulintraoperative assessment can result in extirpation without CPB support. This canaid in avoiding the CPB-related effects and possible risk of anaphylactic shock hadthere been aspiration of fluid contents into the circulation.Certain factors seemed pivotal for off-pump cyst resection with successfuloutcome:Left ventricular wall thickness ≥ 6 mm adjacent to cyst with noapparent breach in myocardial wall on preoperative diagnostic modalities andintraoperative transesophageal echocardiography assessment.Clear fluid on aspiration along with partial decompression of the cyst priorto incision helps in avoiding any inadmissible complication or spillage.Once the cyst has been cleared off its contents, use of protoscolicidal agentfollowed by excision of germinative layer and resection of free cystwall.Steady progression of the pathology and inflammation is the most likely reason foradhesions between cyst and pericardium. Pleuropericardial approach (entering intothe cyst across the pericardium) mitigates the risk of mechanical injury and suddenrupture in an attempt to clear adhesions from pericardium. Covering the pleural andpericardial surface with betadine-soaked sponges before entering the cyst cavityhelps in avoiding contamination.In conclusion, primary hydatid cyst of the heart, specifically the intramural type,is rare. Detailed and meticulous assessment should be considered in largeventricular hydatid cyst in order to delineate the extent and to choose the surgicalstrategy with lesser risks and invasiveness. We are reporting this case to underlinethat cardiac hydatidosis can be addressed with off-pump technique approach ascompared to empty beating on-pump technique and thereby reducing the risk ofanaphylaxis and CPB-related effects.