Optimizing Biocompatibility and Gene Delivery with DMAEA and DMAEAm: A Niacin-Derived Copolymer Approach.

Gene therapy is pivotal in nanomedicine, offering a versatile approach to disease treatment. This study aims to achieve an optimal balance between biocompatibility and efficacy, which is a common challenge in the field. A copolymer library is synthesized, incorporating niacin-derived monomers 2-acrylamidoethyl nicotinate (AAEN) or 2-(acryloyloxy)ethyl nicotinate (AEN) with N,N-(dimethylamino)ethyl acrylamide (DMAEAm) or hydrolysis-labile N,N-(dimethylamino)ethyl acrylate (DMAEA). Evaluation of the polymers' cytotoxicity profiles reveals that an increase in AAEN or DMAEA molar ratios correlates with improved biocompatibility. Remarkably, an increase in AAEN in both DMAEA and DMAEAm copolymers demonstrated enhanced transfection efficiencies of plasmid DNA in HEK293T cells. Additionally, the top-performing polymers demonstrate promising gene expression in challenging-to-transfect cells (THP-1 and Jurkat cells) and show no significant effect on modulating immune response induction in ex vivo treated murine monocytes. Overall, the best performing candidates exhibit an optimal balance between biocompatibility and efficacy, showcasing potential advancements in gene therapy.

Improving Gene Delivery: Synergy between Alkyl Chain Length and Lipoic Acid for PDMAEMA Hydrophobic Copolymers.

In the field of gene delivery, hydrophobic cationic copolymers hold great promise. They exhibit improved performance by effectively protecting genetic material from serum interactions while facilitating interactions with cellular membranes. However, managing cytotoxicity remains a significant challenge, prompting an investigation into suitable hydrophobic components. A particularly encouraging approach involves integrating nutrient components, like lipoic acid, which is known for its antioxidant properties and diverse cellular benefits such as cellular metabolism and growth. In this study, a copolymer library comprising 2-(dimethylamino)ethyl methacrylate (DMAEMA) and lipoic acid methacrylate (LAMA), combined with either n-butyl methacrylate (nBMA), ethyl methacrylate (EMA), or methyl methacrylate (MMA), is synthesized. This enables to probe the impact of lipoic acid incorporation while simultaneously exploring the influence of pendant acyclic alkyl chain length. The inclusion of lipoic acid results in a notable boost in transfection efficiency  while maintaining low cytotoxicity. Interestingly, higher levels of transfection efficiency are achieved in the presence of nBMA, EMA, or MMA. However, a positive correlation between pendant acyclic alkyl chain length and cytotoxicity is observed. Consequently, P(DMAEMA-co-LAMA-co-MMA), emerges as a promising candidate. This is attributed to the optimal combination of low cytotoxic MMA and transfection-boosting LAMA, highlighting the crucial synergy between LAMA and MMA.

Tailoring Gene Transfer Efficacy through the Arrangement of Cationic and Anionic Blocks in Triblock Copolymer Micelles.

The arrangement of charged segments in triblock copolymer micelles affects the gene delivery potential of polymeric micelles and can increase the level of gene expression when an anionic segment is incorporated in the outer shell. Triblock copolymers were synthesized by RAFT polymerzation with narrow molar mass distributions and assembled into micelles with a hydrophobic core from poly(n-butyl acrylate). The ionic shell contained either (i) an anionic segment followed by a cationic segment (HAC micelles) or (ii) a cationic block followed by an anionic block (HCA micelles). The pH-responsive anionic block contained 2-carboxyethyl acrylamide (CEAm), while the cationic block comprised 3-guanidinopropyl acrylamide (GPAm). Increasing the molar content of CEAm in HAC and HCA micelles from 6 to 13 mol % improved cytocompatibility and the endosomal escape property, while the HCA micelle with the highest mol % of anionic charges in the outer shell exhibited the highest gene expression. It became evident that improved membrane interaction of the best performing HCA micelle contributed to achieving high gene expression.

Optimization of Mixed Micelles Based on Oppositely Charged Block Copolymers by Machine Learning for Application in Gene Delivery.

The COVID-19 mRNA vaccines represent a milestone in developing non-viral gene carriers, and their success highlights the crucial need for continued research in this field to address further challenges. Polymer-based delivery systems are particularly promising due to their versatile chemical structure and convenient adaptability, but struggle with the toxicity-efficiency dilemma. Introducing anionic, hydrophilic, or "stealth" functionalities represents a promising approach to overcome this dilemma in gene delivery. Here, two sets of diblock terpolymers are created comprising hydrophobic poly(n-butyl acrylate) (PnBA), a copolymer segment made of hydrophilic 4-acryloylmorpholine (NAM), and either the cationic 3-guanidinopropyl acrylamide (GPAm) or the 2-carboxyethyl acrylamide (CEAm), which is negatively charged at neutral conditions. These oppositely charged sets of diblocks are co-assembled in different ratios to form mixed micelles. Since this experimental design enables countless mixing possibilities, a machine learning approach is applied to identify an optimal GPAm/CEAm ratio for achieving high transfection efficiency and cell viability with little resource expenses. After two runs, an optimal ratio to overcome the toxicity-efficiency dilemma is identified. The results highlight the remarkable potential of integrating machine learning into polymer chemistry to effectively tackle the enormous number of conceivable combinations for identifying novel and powerful gene transporters.

Stimuli-Responsive Non-viral Nanoparticles for Gene Delivery.

Considering nucleic acids as the language of life and the genome as the instruction manual of cells, their targeted modulation promises great opportunities in treating and healing diseases. In addition to viral gene transfer, the overwhelming power of non-viral mRNA-based vaccines is driving the development of novel gene transporters. Thereby, various nucleic acids such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA, gRNA, or ASOs) need to be delivered, requiring a transporter due to their high molar mass and negative charge in contrast to classical agents. This chapter presents the specific biological hurdles for using nucleic acids and shows how new materials can overcome these.

Application of specific ELISAs for BMP15 and GDF9 to cumulus cell extracts from infertile women.

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific paracrine factors which regulate ovarian cumulus cell (CC) functions. This study aimed to investigate if BMP15 and GDF9 bound to CCs can be characterized, quantified, and show an association with IVF outcomes in infertile women. BMP15 and GDF9 ELISAs were validated and applied to discarded CC extracts. Pooled CCs from individual patients were collected from 120 (cohort 1; BMP15 only) and 81 infertility patients (cohort 2; BMP15 and GDF9) undergoing superovulation. BMP15 and GDF9 levels expressed per CC DNA were correlated with maternal age, clinical and embryology data. Total BMP15 and GDF9 were highly correlated with each other (r = 0.9, p < 0.001). The GDF9:BMP15 ratio was unrelated to oocyte number or age. BMP15/CC DNA and GDF9/CC DNA were unaffected by the type of superovulation and were not related to oocyte/embryo outcomes.

Genomic signatures and correlates of widespread population declines in salmon.

Global losses of biodiversity are occurring at an unprecedented rate, but causes are often unidentified. Genomic data provide an opportunity to isolate drivers of change and even predict future vulnerabilities. Atlantic salmon (Salmo salar) populations have declined range-wide, but factors responsible are poorly understood. Here, we reconstruct changes in effective population size (Ne) in recent decades for 172 range-wide populations using a linkage-based method. Across the North Atlantic, Ne has significantly declined in >60% of populations and declines are consistently temperature-associated. We identify significant polygenic associations with decline, involving genomic regions related to metabolic, developmental, and physiological processes. These regions exhibit changes in presumably adaptive diversity in declining populations consistent with contemporary shifts in body size and phenology. Genomic signatures of widespread population decline and associated risk scores allow direct and potentially predictive links between population fitness and genotype, highlighting the power of genomic resources to assess population vulnerability.

Exploiting whole genome sequence data to fine map and characterize candidate genes within a quantitative trait loci region affecting androstenone on porcine chromosome 5.

Male piglets are routinely castrated to eliminate boar taint. However, this treatment is undesirable, and alternative approaches, including genetic strategies to reduce boar taint, are demanded. Androstenone is one of the causative agents of boar taint, and a QTL region affecting this pheromone has previously been reported on SSC5: 22.6-24.8 Mb in Duroc. The QTL region is one of the few reported for androstenone that does not simultaneously affect levels of other sex steroids. The main objective of this study was to fine map this QTL. Whole genome sequence data from 23 Norwegian Duroc boars were analyzed to detect new polymorphisms within the QTL region. A subset of 161 SNPs was genotyped in 834 Duroc sires and analyzed for association with androstenone in adipose tissue and testosterone, estrone sulphate and 17ß-estradiol in blood plasma. Our results revealed 100 SNPs significantly associated with androstenone levels in fat (P < 0.001) with 94 of the SNPs being in strong linkage disequilibrium in the region 23.03-24.27 Mb. This haplotype block contains at least four positional candidate genes (HSD17B6, SDR9C7, RDH16 and STAT6) involved in androstenone biosynthesis. No significant associations were found between any of the SNPs and levels of testosterone and estrogens, confirming previous findings. The amount of phenotypic variance explained by single SNPs within the haplotype block was as high as 5.4%. As the SNPs in this region significantly affect levels of androstenone without affecting levels of other sex steroids, they are especially interesting as genetic markers for selection against boar taint.

Endoscopy-assisted subfascial anterior transposition of the ulnar nerve for the treatment of cubital tunnel syndrome.

BACKGROUND: Many techniques have been described to relieve the compression and reduce subluxation of the ulnar nerve following surgery. The subfascial anterior transposition of the ulnar nerve (SfATUN) is one described technique, but involves a long scar, risk of injury to the medial antebrachial cutaneous nerve, and possible nerve ischemia from anterior transposition. We assessed a more refined approach of endoscopy-assisted SfATUN for the treatment of cubital tunnel syndrome. METHODS: A consecutive case series of 21 patients (15 males and 6 females) with evidence of nerve subluxation after ulnar nerve decompression were operated using an endoscopy-assisted SfATUN. Each patient was assessed with pre- and postoperative nerve conduction studies, McGowan grading, and recovery of grip strength. RESULTS: The average age of patients was 54 years (range 23-74 years), and they were followed up for a mean of 9 months (range 3-22 months). Preoperative McGowan grades were eight grade II and 13 grade III. Eighteen of the 21 patients showed improvement, including improvement by two McGowen grades in 8 patients and improvement by one grade in 10 patients. Three grade III patients did not show improvement in grading after surgery. A proportion of 90% of patients showed significant improvements in motor nerve conduction velocity of the ulnar nerve across the elbow (p < 0.001), and all showed some improvement in grip strength (p < 0.001). One patient underwent redo neurolysis. CONCLUSION: A combination of endoscopy-assisted SfATUN allows for decompression transposition and reduced strain on the ulnar nerve through a small scar. This is now our standard approach for cubital tunnel syndrome and the "unstable" nerve.

Identification of quantitative genetic components of fitness variation in farmed, hybrid and native salmon in the wild.

Feral animals represent an important problem in many ecosystems due to interbreeding with wild conspecifics. Hybrid offspring from wild and domestic parents are often less adapted to local environment and ultimately, can reduce the fitness of the native population. This problem is an important concern in Norway, where each year, hundreds of thousands of farm Atlantic salmon escape from fish farms. Feral fish outnumber wild populations, leading to a possible loss of local adaptive genetic variation and erosion of genetic structure in wild populations. Studying the genetic factors underlying relative performance between wild and domesticated conspecific can help to better understand how domestication modifies the genetic background of populations, and how it may alter their ability to adapt to the natural environment. Here, based upon a large-scale release of wild, farm and wild x farm salmon crosses into a natural river system, a genome-wide quantitative trait locus (QTL) scan was performed on the offspring of 50 full-sib families, for traits related to fitness (length, weight, condition factor and survival). Six QTLs were detected as significant contributors to the phenotypic variation of the first three traits, explaining collectively between 9.8 and 14.8% of the phenotypic variation. The seventh QTL had a significant contribution to the variation in survival, and is regarded as a key factor to understand the fitness variability observed among salmon in the river. Interestingly, strong allelic correlation within one of the QTL regions in farmed salmon might reflect a recent selective sweep due to artificial selection.

Mapping and validation of a major QTL affecting resistance to pancreas disease (salmonid alphavirus) in Atlantic salmon (Salmo salar).

Pancreas disease (PD), caused by a salmonid alphavirus (SAV), has a large negative economic and animal welfare impact on Atlantic salmon aquaculture. Evidence for genetic variation in host resistance to this disease has been reported, suggesting that selective breeding may potentially form an important component of disease control. The aim of this study was to explore the genetic architecture of resistance to PD, using survival data collected from two unrelated populations of Atlantic salmon; one challenged with SAV as fry in freshwater (POP 1) and one challenged with SAV as post-smolts in sea water (POP 2). Analyses of the binary survival data revealed a moderate-to-high heritability for host resistance to PD in both populations (fry POP 1 h(2)~0.5; post-smolt POP 2 h(2)~0.4). Subsets of both populations were genotyped for single nucleotide polymorphism markers, and six putative resistance quantitative trait loci (QTL) were identified. One of these QTL was mapped to the same location on chromosome 3 in both populations, reaching chromosome-wide significance in both the sire- and dam-based analyses in POP 1, and genome-wide significance in a combined analysis in POP 2. This independently verified QTL explains a significant proportion of host genetic variation in resistance to PD in both populations, suggesting a common underlying mechanism for genetic resistance across lifecycle stages. Markers associated with this QTL are being incorporated into selective breeding programs to improve PD resistance.

The development and characterization of a 57K single nucleotide polymorphism array for rainbow trout.

In this study, we describe the development and characterization of the first high-density single nucleotide polymorphism (SNP) genotyping array for rainbow trout. The SNP array is publically available from a commercial vendor (Affymetrix). The SNP genotyping quality was high, and validation rate was close to 90%. This is comparable to other farm animals and is much higher than previous smaller scale SNP validation studies in rainbow trout. High quality and integrity of the genotypes are evident from sample reproducibility and from nearly 100% agreement in genotyping results from other methods. The array is very useful for rainbow trout aquaculture populations with more than 40 900 polymorphic markers per population. For wild populations that were confounded by a smaller sample size, the number of polymorphic markers was between 10 577 and 24 330. Comparison between genotypes from individual populations suggests good potential for identifying candidate markers for populations' traceability. Linkage analysis and mapping of the SNPs to the reference genome assembly provide strong evidence for a wide distribution throughout the genome with good representation in all 29 chromosomes. A total of 68% of the genome scaffolds and contigs were anchored through linkage analysis using the SNP array genotypes, including ~20% of the genome assembly that has not been previously anchored to chromosomes.

Genetic differentiation of brackish water populations of cod Gadus morhua in the southern Baltic, inferred from genotyping using SNP-arrays.

The Baltic is a semi-enclosed sea characterised by decreasing salinity in the eastern and northern direction with only the deeper parts of the southern Baltic suitable as spawning grounds for marine species like cod. Baltic cod exhibits various adaptations to brackish water conditions, yet the inflow of salty North Sea water near the bottom remains an influence on the spawning success of the Baltic cod. The eastern Baltic population has been very weakly studied in comparison with the western population. The aim of this study is to demonstrate for the first time genetic differentiation by the use of a large number of SNPs between eastern and western Baltic populations existing in differentiated salinity conditions. Two cod samples were collected from the Bay of Gdansk, Poland and one from the Kiel Bight, Germany. Samples were genotyped using a cod derived SNP-array (Illumina) with 10 913 SNPs. A selection of diagnostic SNPs was performed. A set of 7944 validated SNPs were analysed to assess the differentiation of three samples of cod. Results indicated a clear distinctness of the Kiel Bight from the populations of the eastern Baltic. FST comparison between both eastern samples was non-significant. Clustering analysis, principal coordinates analysis and assignment test clearly indicated that the eastern samples should be considered as one subpopulation, well differentiated from the western subpopulation. With the SNP approach, no differentiation between groups containing 'healthy' and 'non-healthy' cod individuals was observed.

Genetic differentiation of southeast Baltic populations of sea trout inferred from single nucleotide polymorphisms.

Sea trout (Salmo trutta m. trutta) is a migratory form of brown trout common in the Baltic Sea. Nine populations from the southeast Baltic (Poland; Lithuania; Denmark, Bornholm; Estonia and Russia) were genotyped using iPLEX Gold technology (Sequenom) with 62 informative SNPs. A diagnostic panel of 23 SNPs was applied to estimate genetic differentiation and assess the population structure of Baltic sea trout. The highest level of pairwise FST differences was observed between the Russian (East Gulf of Finland) and Polish (Baltic main basin) populations. The lowest differences were between the two Polish and the Polish and Lithuanian populations. A genetic similarity was noted between the Estonian Riguldi River and Danish Bornholm populations, and this finding was supported by a Bayesian and factorial correspondence analysis. Diversity within populations was highest for populations from Estonia and lowest for the Lithuanian population. Genetic structure analysis indicated that individuals from the nine populations were clustered into four groups.

The characterization of low pathogenic avian influenza viruses isolated from wild birds in northern Vietnam from 2006 to 2009.

Due to concerns that wild birds could possibly spread H5N1 viruses, surveillance was conducted to monitor the types of avian influenza viruses circulating among the wild birds migrating to or inhabiting in northern Vietnam from 2006 to 2009. An H5N2 virus isolated from a Eurasian woodcock had a close phylogenetic relationship to H5 viruses recently isolated in South Korea and Japan, suggesting that H5N2 has been shared between Vietnam, South Korea, and Japan. An H9N2 virus isolated from a Chinese Hwamei was closely related to two H9N2 viruses that were isolated from humans in Hong Kong in 2009, suggesting that an H9N2 strain relevant to the human isolates had been transmitted to and maintained among the wild bird population in Vietnam and South China. The results support the idea that wild bird species play a significant role in the spread and maintenance of avian influenza and that this also occurs in Vietnam.

Self-perceived health and return to work following work-related hand injury.

BACKGROUND: Return to work is an important outcome following traumatic work-related hand injuries. It is unclear how psychosocial factors affect the time to return to work following traumatic work-related hand injury. AIMS: To investigate the relationships between hand injury severity, self-perceived health, demographics and time off work (TOW) following traumatic work-related hand injuries and the influence of psychosocial factors on the readiness of return to work. METHODS: Data from 120 traumatic work-related hand injured patients were gathered. The Modified Hand Injury Severity Score (MHISS) and Short Form Health Survey (SF-36) were used to assess the severity of hand trauma and self-perceived health, respectively. The relationships between MHISS, SF-36, demographics and TOW were analysed by multiple regression analysis. RESULTS: Mean duration of TOW was 127 days for patients with a mild MHISS, 108 days for a moderate score, 160 days for a severe score and 236 days for those with a major score. A positive correlation between MHISS and duration of TOW was identified. Self-perceived physical functioning was found to have a negative correlation with TOW, whereas self-perceived mental health was positively correlated with TOW. CONCLUSIONS: This study highlights the importance of self-perceived health in considering return to work following traumatic work-related hand injury.

Molecular epidemiology of avian influenza viruses circulating among healthy poultry flocks in farms in northern Vietnam.

Repeated epizootics of highly pathogenic avian influenza (HPAI) virus subtype H5N1 were reported from 2003 to 2005 among poultry in Vietnam. More than 200 million birds were killed to control the spread of the disease. Human cases of H5N1 infection have been sporadically reported in an area where repeated H5N1 outbreaks among birds had occurred. Subtype H5N1 strains are established as endemic among poultry in Vietnam, however, insights into how avian influenza viruses including the H5N1 subtype are maintained in endemic areas is not clear. In order to determine the prevalence of different avian influenza viruses (AIVs), including H5N1 circulating among poultry in northern Vietnam, surveillance was conducted during the years 2006-2009. A subtype H5N1 strain was isolated from an apparently healthy duck reared on a farm in northern Vietnam in 2008 and was identified as an HPAI. Although only one H5N1 virus was isolated, it supports the view that healthy domestic ducks play a pivotal role in maintaining and transmitting H5N1 viruses which cause disease outbreaks in northern Vietnam. In addition, a total of 26 AIVs with low pathogenicity were isolated from poultry and phylogenetic analysis of all the eight gene segments revealed their diverse genetical backgrounds, implying that reassortments have occurred frequently among strains in northern Vietnam. It is, therefore, important to monitor the prevalence of influenza viruses among healthy poultry between epidemics in an area where AIVs are endemic.

Quantitative trait loci for clinical mastitis on chromosomes 2, 6, 14 and 20 in Norwegian Red cattle.

Mastitis is the most frequent and costly disease in dairy production and solutions leading to a reduction in the incidence of mastitis are highly demanded. Here a genome-wide association study was performed to identify polymorphisms affecting susceptibility to mastitis. Genotypes for 17 349 SNPs distributed across the 29 bovine autosomal chromosomes from a total of 2589 sires with 1 389 776 daughters with records on clinical mastitis were included in the analysis. Records of occurrence of clinical mastitis were divided into seven time periods in the first three lactations in order to identify quantitative trait loci affecting mastitis susceptibility in particular phases of lactation. The most convincing results from the association mapping were followed up and validated by a combined linkage disequilibrium and linkage analysis. The study revealed quantitative trait loci affecting occurrence of clinical mastitis in the periparturient period on chromosomes 2, 6 and 20 and a quantitative trait locus affecting occurrence of clinical mastitis in late lactation on chromosome 14. None of the quantitative trait loci for clinical mastitis detected in the study seemed to affect lactation average of somatic cell score. The SNPs highly associated with clinical mastitis lie near both the gene encoding interleukin 8 on chromosome 6 and the genes encoding the two interleukin 8 receptors on chromosome 2.

Genome-wide association mapping in Norwegian Red cattle identifies quantitative trait loci for fertility and milk production on BTA12.

Reproductive performance is a critical trait in dairy cattle. Poor reproductive performance leads to prolonged calving intervals, higher culling rates and extra expenses related to multiple inseminations, veterinary treatments and replacements. Genetic gain for improved reproduction through traditional selection is often slow because of low heritability and negative correlations with production traits. Detection of DNA markers associated with improved reproductive performance through genome-wide association studies could lead to genetic gain that is more balanced between fertility and production. Norwegian Red cattle are well suited for such studies, as very large numbers of detailed reproduction records are available. We conducted a genome-wide association study for non-return rate, fertility treatments and retained placenta using almost 1 million records on these traits and 17 343 genome-wide single-nucleotide polymorphisms. Genotyping costs were minimized by genotyping the sires of the cows recorded and by using daughter averages as phenotypes. The genotyped sires were assigned to either a discovery or a validation population. Associations were only considered to be validated if they were significant in both groups. Strong associations were found and validated on chromosomes 1, 5, 8, 9, 11 and 12. Several of these were highly supported by findings in other studies. The most important result was an association for non-return rate in heifers in a region of BTA12 where several associations for milk production traits have previously been found. Subsequent fine-mapping verified the presence of a quantitative trait loci (QTL) having opposing effects on non-return rate and milk production at 18 Mb. The other reproduction QTL did not have pleiotropic effects on milk production, and these are therefore of considerable interest for use in marker-assisted selection.

Revealing genetic relationships between compounds affecting boar taint and reproduction in pigs.

Boar taint is characterized by an unpleasant taste or odor in intact male pigs and is primarily attributed to increased concentrations of androstenone and skatole and to a lesser extent by increased indole. The boar taint compounds skatole and indole are produced by gut bacteria, metabolized in the liver, and stored in the fat tissue. Androstenone, on the other hand, is synthesized in the testis along with testosterone and estrogens, which are known to be important factors affecting fertility. The main goal of this study was to investigate the relationship between genetic factors involved in the primary boar taint compounds in an attempt to discover ways to reduce boar taint without decreasing fertility-related compounds. Heritabilities and genetic correlations between traits were estimated for compounds related to boar taint (androstenone, skatole, indole) and reproduction (testosterone, 17ß-estradiol, and estrone sulfate). Heritabilities in the range of 0.47 to 0.67 were detected for androstenone concentrations in both fat and plasma, whereas those for skatole and indole were slightly less (0.27 to 0.41). The genetic correlations between androstenone in plasma and fat were extremely high (0.91 to 0.98) in Duroc and Landrace. In addition, genetic correlations between androstenone (both plasma and fat) and the other sex steroids (estrone sulfate, 17ß-estradiol, and testosterone) were very high, in the range of 0.80 to 0.95. Furthermore, a genome-wide association study (GWA) and a combined linkage disequilibrium and linkage analysis (LDLA) were conducted on 1,533 purebred Landrace and 1,027 purebred Duroc to find genome regions involved in genetic control of the boar taint compounds androstenone, skatole, and indole, and sex hormones related to fertility traits. Up to 3,297 informative SNP markers were included for both breeds, including SNP from several boar taint candidate genes. From the GWA study, we found that altogether 27 regions were significant at a genome-wide level (P < 0.05) and an additional 7 regions were significant at a chromosomal level. From the LDLA study, 7 regions were significant on a genome-wide level and an additional 7 regions were significant at a chromosomal level. The most convincing associations were obtained in 6 regions affecting skatole and indole in fat on chromosomes 1, 2, 3, 7, 13, and 14, 1 region on chromosome 6 affecting androstenone in plasma only, and 5 regions on chromosomes 3, 4, 13, and 15 affecting androstenone, testosterone, and estrogens.