DNA Analysis Research Articles

Forensic species identification: practical guide for animal and plant DNA analysis

The importance of non-human DNA in the forensic field has increased greatly in recent years, together with the type of applications. The molecular species identification of animal and botanical material may be crucial both for wildlife trafficking and crime scene investigation. However, especially for forensic botany, several challenges slow down the implementation of the discipline in the routine.Although the importance of molecular analysis of animal origin samples is widely recognized and the same value is acknowledged to the botanical counterpart, the latter does not find the same degree of application.The availability of molecular methods, especially useful in cases where the material is fragmented, scarce or spoiled preventing the morphological identification, is not well known. This work is intended to reaffirm the relevance of non-human forensic genetics (NHFG), highlighting differences, benefits and pitfalls of the current most common molecular analysis workflow for animal and botanical samples, giving a practical guide. A flowchart describing the analysis paths, divided in three major working areas (inspection and sampling, molecular analysis, data processing and interpretation), is provided. More real casework examples of the utility of non-human evidence in forensic investigations should be shared by the scientific community, especially for plants. Moreover, concrete efforts to encourage initiatives in order to promote quality and standardization in the NHFG field are also needed.

Genetic diversity and DNA fingerprinting of rice varieties of Manipur using microsatellite markers

Rice variety differentiation was based on morphological descriptors (DUS) before the advent of genomic and proteomic technology. DNA fingerprinting is becoming an important molecular marker approach because of its wide application in varietal protection, classification, and conflict settlement. In the present study, genetic diversity and variability among the 11 rice varieties of Manipur at the molecular level was studied using 42 SSR markers. The genotypic data of the SSR markers revealed four sub-populations. Cluster analysis indicated the close similarity between RC Maniphou 12 and RC Maniphou 15. To create a DNA fingerprinting database for 11 rice varieties, three separate multiplex PCR systems using different combinations of 11 polymorphic SSR markers based on amplicon size were successfully developed . These were able to distinguish the distinctness of all the eleven released rice varieties.

H-151, a Selective STING Inhibitor, Has Potential as a Treatment for Neovascular Age-Related Macular Degeneration.

The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) stimulator of interferon gene (STING) pathway is a crucial cascade in the inflammatory response initiated by the recognition of cytosolic double-stranded DNA (dsDNA). The aim of this study was to evaluate the effect of STING inhibitor in murine choroidal neovascularization (CNV). To investigate whether the cGAS-STING pathway is activated during CNV, CNV was induced using laser photocoagulation in male C57BL/6J mice. The expression of change of cGAS and STING during CNV development was confirmed by Western-blotting. H-151, a potent STING palmitoylation antagonist, was used as a STING inhibitor. H-151 was administered intravitreally immediately after laser induction. To confirm the role of the cGAS-STING pathway in CNV formation, we evaluated CNV size and performed fundus fluorescein angiography. The expression levels of cGAS and STING were significantly upregulated in the RPE-choroid complex after CNV induction, and dsDNA merged with cGAS was observed in CNV lesions. Intravitreal administration of H-151 suppressed CNV development and fluorescent leakage from neovessels. In CNV lesions, the high expression of STING and cGAS was observed in infiltrating F4/80+ macrophages. H-151 administration attenuated downstream signals of the cGAS-STING pathway, including the phosphorylation of nuclear factor-κB, and downregulated the expression of interleukin 1β. These findings support that the inhibition of cGAS-STING pathway treats abnormal ocular angiogenesis.

Comparison of CRISPR typing and conventional molecular methods for distinguishing Laribacter hongkongensis isolates from fish, frogs and humans

High-resolution and efficient typing for Laribacter hongkongensis (L. hongkongensis) is essential for epidemiological investigation of such emerging foodborne pathogens. Clustered regularly interspaced short palindromic repeats (CRISPR) typing is an innovative molecular method that shows great promise for L. hongkongensis typing. Here, we explored the CRISPR typing method by combining CRISPR1 and CRISPR2 loci to characterize a collection of 109 L. hongkongensis isolates from humans and animals and compared it to current molecular methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The results showed that all three methods have high discriminatory power (diversity index was 0.9902 for PFGE, 0.9663 for CRISPR and 0.9562 for MLST); strong congruence was observed between them (Rand index was 0.969 between CRISPR and PFGE, 0.953 between CRISPR and MLST, 0.958 between PFGE and MLST). CRISPR typing could well distinguish the isolates in the same STs or PFGE profiles, and the genetic information contained by the CRISPR array is useful for deep phylogenetic typing. We demonstrate that rapid CRISPR typing is a practical genetic fingerprinting tool with high resolution, comparable ease of use and lower cost, ability to track the source of various groups of L. hongkongensis strains and indication of genetic characteristics.

Dependence of post-segregational killing mediated by Type II restriction-modification systems on the lifetime of restriction endonuclease effective activity.

Plasmid-borne Type II restriction-modification (RM) systems mediate post-segregational killing (PSK). PSK is thought to be caused by the dilution of restriction and modification enzymes during cell division, resulting in accumulation of unmethylated DNA recognition sites and their cleavage by restriction endonucleases. PSK is the likely reason for stabilization of plasmids carrying RM systems in the absence of selection for plasmid maintenance. In this study, we developed a CRISPR interference-based method to eliminate RM-carrying plasmids and study PSK-related phenomena with minimal perturbation to the Escherichia coli host. Plasmids carrying the EcoRV, Eco29kI, and EcoRI RM systems were highly stable, and their loss resulted in SOS response and PSK. In contrast, plasmids carrying the Esp1396I system were poorly stabilized; their loss led to a temporary cessation of growth, followed by full recovery. We demonstrate that this unusual behavior is due to a limited lifetime of the Esp1396I restriction endonuclease activity, which, upon Esp1396I plasmid loss, disappears approximately after two cycles of cell division, i.e., before unmethylated sites appear in significant numbers. Our results indicate that whenever PSK induced by a loss of RM systems, and, possibly, other toxin-antitoxin systems, is considered, the lifetimes of individual system components and the growth rate of host cells shall be taken in account. Mathematical modeling shows, that unlike the situation with classical toxin-antitoxin systems, RM system-mediated PSK is possible when the lifetimes of restriction endonuclease and methyltransferase activities are similar, as long as the toxic restriction endonuclease activity persists for more than two chromosome replication cycles.IMPORTANCEIt is widely accepted that many Type II restriction-modification (RM) systems mediate post-segregational killing (PSK) if plasmids that encode them are lost. In this study, we harnessed an inducible CRISPR-Cas system to remove RM plasmids from Escherichia coli cells to study PSK while minimally perturbing cell physiology. We demonstrate that PSK depends on restriction endonuclease activity lifetime and is not observed when it is less than two replication cycles. We present a mathematical model that explains experimental data and shows that unlike the case of toxin-antitoxin-mediated PSK, the loss of an RM system induced PSK even when the RM enzymes have identical lifetimes.

Identification of orb-web spider species and their food source through environmental DNA analysis

Identification of orb-web spider species and their food source through environmental DNA analysis

Analysis of the privacy-performance tradeoff of reference testing in Forensic Investigative Genetic Genealogy.

During an investigation using Forensic Investigative Genetic Genealogy, which is a novel approach for solving violent crimes and identifying human remains, reference testing-when law enforcement requests a DNA sample from a person in a partially constructed family tree-is sometimes used when an investigation has stalled. Because the people considered for a reference test have not opted in to allow law enforcement to use their DNA profile in this way, reference testing is viewed by many as an invasion of privacy and by some as unethical. We generalize an existing mathematical optimization model of the genealogy process by incorporating the option of reference testing. Using simulated versions of 17 DNA Doe Project cases, we find that reference testing can solve cases more quickly (although many reference tests are required to substantially hasten the investigative process), but only rarely (<1%) solves cases that cannot otherwise be solved. Through a mixture of mathematical and computational analysis, we find that the most desirable people to test are at the bottom of a path descending from an ancestral couple that is most likely to be related to the target. We also characterize the rare cases where reference testing is necessary for solving the case: when there is only one descending path from an ancestral couple, which precludes the possibility of identifying an intersection (e.g., marriage) between two descendants of two different ancestral couples.

Establishing Silphids in the invertebrate DNA toolbox: a proof of concept.

Environmental DNA (eDNA) analyses are an increasingly popular tool for assessing biodiversity. eDNA sampling that uses invertebrates, or invertebrate DNA (iDNA), has become a more common method in mammal biodiversity studies where biodiversity is assessed via diet analysis of different coprophagous or hematophagous invertebrates. The carrion feeding family of beetles (Silphidae: Coleoptera, Latreille (1807)), have not yet been established as a viable iDNA source in primary scientific literature, yet could be useful indicators for tracking biodiversity in forested ecosystems. Silphids find carcasses of varying size for both food and reproduction, with some species having host preference for small mammals; therefore, iDNA Silphid studies could potentially target small mammal communities. To establish the first valid use of iDNA methods to detect Silphid diets, we conducted a study with the objective of testing the validity of iDNA methods applied to Silphids using both Sanger sequencing and high throughput Illumina sequencing. Beetles were collected using inexpensive pitfall traps in Alberta, Michigan in 2019 and 2022. We successfully sequenced diet DNA and environmental DNA from externally swabbed Silphid samples and diet DNA from gut dissections, confirming their potential as an iDNA tool in mammalian studies. Our results demonstrate the usefulness of Silphids for iDNA research where we detected species from the genera Anaxyrus, Blarina, Procyon, Condylura, Peromyscus, Canis, and Bos. Our results highlight the potential for Silphid iDNA to be used in future wildlife surveys.

Early Non-Invasive Prenatal Testing at 6-9 Weeks of Gestation.

Non-invasive prenatal testing (NIPT) is usually performed beyond 10 weeks of gestation, because earlier in pregnancy, the fetal fraction is low, resulting in failure to obtain reliable results. This study aimed to evaluate the clinical performance of NIPT earlier in pregnancy using a method for cell-free DNA (cfDNA) analysis that eliminates the need for polymerase chain reaction (PCR), DNA sequencing, or microarrays (Vanadis® system, PerkinElmer, Waltham, MA, USA). Cell-free DNA was extracted from the maternal plasma of 30 singleton pregnancies at 6-9 weeks of gestation (group 1) and at 11-14 weeks of gestation of the same patients (group 2). The mean crown-rump length (CRL) and gestational age in group A was 16.12 mm and that in group B was 61.45 mm. In group A, results were obtained in all, but one, cases (97%). From the remaining pregnancies, one miscarried at 8 weeks and, therefore, the follow-up NIPT at 12 weeks could not be performed. The fetal sex was diagnosed correctly in the 28 cases that had a successful early test, and the results were in accordance with the examination at 12 weeks. There were no cases of aneuploidies and disomy was diagnosed correctly in all. The "Vanadis" prenatal NIPT assay can successfully be used early during the first trimester at 6-9 weeks of gestation (early NIPT) to identify the fetal sex. Further studies are needed to explore the diagnostic potential for aneuploidies.

The Role of Molecular Investigations in Estimating the Time since Deposition (TSD) of Bloodstains: A Systematic Review of the Literature.

At many crime scenes, investigators are able to trace and find traces of blood. For many years, it was believed that such traces could only be subjected to genetic investigations, such as those aimed at comparing DNA profiling with a suspect to verify his identity, and that it was therefore not possible to backdate the traces. In recent years, various works have used experimental models to investigate the possibility of identifying markers and methodologies for estimating the time since deposition (TSD) of bloodstains. Despite the results, these methods are still not part of standard procedures, and there is no univocal analysis methodology. In this work we carried out a systematic literature review of all the papers published in the last ten years on this topic, comparing the experimental models created. This review demonstrates the potential that different molecular approaches, such as transcriptomics, metabolomics, proteomics, and spectrometry, can have in the analysis of TSD, with notable sensitivity and specificity. This paper also analyzes the intrinsic and extrinsic limits of these models and emphasizes the need to continue research work on this topic, considering the importance that this parameter can assume in forensic investigations against a suspect.

Unravelling the identity of lake ecotype of brown trout (Salmo trutta) in Slovenia: genetic insights and conservation implications

Abstract The genetic identity and conservation status of lake ecotype of brown trout (Salmo trutta) populations in Slovenia’s Alpine lakes Bled and Bohinj have been subjected to changes due to historical stocking practices. We aimed to evaluate the genetic differentiation and origins of these populations to advise for correct conservation strategies. Using microsatellite and mitochondrial DNA analysis on 365 trout specimens, we explored the genetic diversity and structure of these populations, comparing them with potential originators. Results revealed distinct genetic profiles between Bled and Bohinj lake trout, with Bohinj lake trout closely aligned with the native Danubian lineage and Bled lake trout showing a hybrid composition, significantly influenced by hatchery-introduced Atlantic lineage strains. The genetic distinction between these populations underscores the complexity of their conservation, showcasing the impact of human-mediated introductions on natural populations. The urgent need for conservation measures to preserve the Bohinj population’s native genetic pool is highlighted, underlining the must to prevent the transfer of individuals from Lake Bled or elsewhere and to reconsider stocking practises in order to prevent further genetic dilution of these iconic European lake dwellers.

The Role of Biomarkers in HPV-Positive Head and Neck Squamous Cell Carcinoma: Towards Precision Medicine.

Head and neck cancer (HNC) represents a significant global health challenge, with squamous cell carcinomas (SCCs) accounting for approximately 90% of all HNC cases. These malignancies, collectively referred to as head and neck squamous cell carcinoma (HNSCC), originate from the mucosal epithelium lining the larynx, pharynx, and oral cavity. The primary risk factors associated with HNSCC in economically disadvantaged nations have been chronic alcohol consumption and tobacco use. However, in more affluent countries, the landscape of HNSCC has shifted with the identification of human papillomavirus (HPV) infection, particularly HPV-16, as a major risk factor, especially among nonsmokers. Understanding the evolving risk factors and the distinct biological behaviors of HPV-positive and HPV-negative HNSCC is critical for developing targeted treatment strategies and improving patient outcomes in this complex and diverse group of cancers. Accurate diagnosis of HPV-positive HNSCC is essential for developing a comprehensive model that integrates the molecular characteristics, immune microenvironment, and clinical outcomes. The aim of this comprehensive review was to summarize the current knowledge and advances in the identification of DNA, RNA, and protein biomarkers in bodily fluids and tissues that have introduced new possibilities for minimally or non-invasive cancer diagnosis, monitoring, and assessment of therapeutic responses.

An innovative approach for low input forensic DNA sample analysis using the GlobalFiler™ IQC PCR amplification Kit on the Magelia® platform

Short Tandem Repeat (STR) markers have been the gold standard for human identification testing in the forensic field for the last few decades. The GlobalFiler™ IQC PCR amplification Kit has shown sensitivity, high power of discrimination and is therefore widely used. Samples with limited DNA quantities remain a significant hurdle for streamlined human forensic identification. Reaction volume reduction in a closed system paired with automation can provide solutions to secure DNA profiles when routine methods fall short. We automated and optimized the GlobalFilerTM IQC PCR Amplification Kit on the Magelia®, a closed molecular biology platform, to test whether reaction volume reduction in a confined automated system would improve signal and sensitivity. We evaluated the platform’s performance using reference and real casework samples (blood, cigarette butt, saliva and touch DNA) in the context of a 5-fold volume reduction when compared to the routine protocol. This strategy showed distinct advantages over standard treatment, notably increased signal for lower DNA inputs. Importantly, negative casework samples through routine treatment yielded “usable” DNA profiles after amplification using this strategy. This novel approach represents a first proof of concept for a method enabling users to treat limited samples, or to partition routine samples for multiple analyses.

Body-wide chimerism and mosaicism are predominant causes of naturally occurring ABO discrepancies.

Routine ABO blood group typing of apparently healthy individuals sporadically uncovers unexplained mixed-field reactions. Such blood group discrepancies can either result from a haematopoiesis-confined or body-wide dispersed chimerism or mosaicism. Taking the distinct clinical consequences of these four different possibilities into account, we explored the responsible cause in nine affected individuals. Genotype analyses revealed that more than three-quarters were chimaeras (two same-sex females, four same-sex males, one sex-mismatched male), while two were mosaics. Short tandem repeat analyses of buccal swab, hair root and nail DNA suggested a body-wide involvement in all instances. Moreover, genome-wide array analyses unveiled that in both mosaic cases the causative genetic defect was a unique copy-neutral loss of heterozygosity encompassing the entire long arm of chromosome 9. The practical transfusion- or transplantation-associated consequences of such incidental discoveries are well known and therefore easily manageable. Far less appreciated is the fact that such findings also call attention to potential problems that directly ensue from their specific genetic make-up. In case of chimerism, these are the appearance of seemingly implausible family relationships and pitfalls in forensic testing. In case of mosaicism, they concern with the necessity to delineate innocuous pre-existent or age-related from disease-predisposing and disease-indicating cell clones.

Paying Tribute: Returning to the Story of the “Qur‘ān of ‘Uthmān

Objectives: The Qur’an occupies a central position in a religious and philosophical system that has had a significant and emblematic impact on human history, influencing Arabic studies worldwide. Such is particularly true for Russian Arabic studies, where the '‘Uthmanic Qur’an', with copies in Saint Petersburg and Uzbekistan, has played a crucial role. This study aims to honour our teachers and the Arab community of Qashqadarya, who continue to preserve their language and culture. Methodology: Synthetic, Comparative. Findings: The history of these manuscripts spans over twelve centuries, providing rich material linked to the fate of human lineages, nations, cities, peoples, and Islamic civilisation since its emergence in the Arabian Peninsula in the 7th century until the triumph of Islam. This Islam endured against the communist regime in the Islamic republics of the Soviet Union. The Russian School of Arabic Studies survived the 1917 Revolution due to its concentration on research areas that were of particular importance at the time. These included the study of Arabic sources on the history of the USSR territories and the living Arabic dialects used in the Central Asian republics. Such led to the publication of significant works on the language, ethnography, and history of the Arabs of Qashqadarya, whose ancestors brought the '‘Uthmanic Qur’an' from the Arabian Peninsula via the Silk Road to their land in Uzbekistan. Originality: Today, advanced research techniques must be used to study and analyse the '‘Uthmanic Qur’an' manuscript and reconstruct the origin, culture, and dialects of the Arab population in Qashqadarya, Uzbekistan. These include DNA analysis and modern linguistic methods based on big data and artificial intelligence technology.

DORA: an interactive map for the visualization and analysis of ancient human DNA and associated data.

The ability to sequence ancient genomes has revolutionized the way we study evolutionary history by providing access to the most important aspect of evolution-time. Until recently, studying human demography, ecology, biology, and history using population genomic inference relied on contemporary genomic datasets. Over the past decade, the availability of human ancient DNA (aDNA) has increased rapidly, almost doubling every year, opening the way for spatiotemporal studies of ancient human populations. However, the multidimensionality of aDNA, with genotypes having temporal, spatial and genomic coordinates, and integrating multiple sources of data, poses a challenge for developing meta-analyses pipelines. To address this challenge, we developed a publicly-available interactive tool, DORA, which integrates multiple data types, genomic and non-genomic, in a unified interface. This web-based tool enables browsing sample metadata alongside additional layers of information, such as population structure, climatic data, and unpublished samples. Users can perform analyses on genotypes of these samples, or export sample subsets for external analyses. DORA integrates analyses and visualizations in a single intuitive interface, resolving the technical issues of combining datasets from different sources and formats, and allowing researchers to focus on the scientific questions that can be addressed through analysis of aDNA datasets.

A systematic approach to the analysis of illicit drugs for DNA with an overview of the problems encountered

Due to the restricted nature of illicit drugs, it is difficult to conduct research surrounding the analysis of this drug material for any potential DNA in sufficient quantities acceptable for high numbers of replicates. Therefore, the current research available in peer reviewed journals thus far regarding analysing illicit drugs for DNA has been performed under varying experimental conditions, often using surrogate chemicals in place of illicit drugs. The data presented within this study originated from the analysis of genuine illicit drugs prepared both in controlled environments and those seized at the Australian border (and therefore from an uncontrolled environment) to determine if DNA can be obtained from this type of material. This study has been separated into three main parts (total n=114 samples): firstly, methamphetamine synthesised within a controlled environment was spiked with both saliva and trace DNA to determine the yield following DNA extraction; secondly, methamphetamine also synthesised in a controlled environment but on a larger scale was tested for the amount of DNA added incidentally throughout the synthesis, including the additional steps of recrystallising, homogenising and “cutting” the drug material to simulate preparation for distribution; and thirdly, the detection of human DNA within samples of cocaine and heroin seized at the Australian border. The DNA Fast Flow Microcon Device was utilised to concentrate all replicates from the same source into one combined extract to improve the DNA profiles for the samples where no DNA spiking occurred. Full STR profiles were successfully obtained from drug samples spiked with both saliva and trace DNA. Methamphetamine was present in the final DNA extracts and caused incompatibilities with the quantification of DNA using Qubit. The yields of DNA from drugs not spiked with DNA sources were much lower, resulting in 36 % of samples yielding alleles where all others did not. These results were not unexpected given these were realistic drug samples where the history of the drug material was unknown. This is the first study to obtain DNA profiles from genuine illicit drug material in both controlled and uncontrolled environments and indicates that the analysis of illicit drugs for DNA is an avenue worth pursuing to provide information which can in turn assist with disrupting the supply of these drugs. Given that DNA profiling is carried out worldwide using essentially the same systems as described within this study, the potential for impact is on a national and international scale.

A DNA profiling system for conservation management of Kamchatka brown bear (Ursus arctos piscator); population data and system performance from 16 autosomal STRs

Despite the high density of brown bears (Ursus arctos piscator) on the Kamchatka peninsula their genetic variation has not been studied by STR analysis. Our aim was, therefore, to provide population data from the Kamchatka brown bear population applying a validated DNA profiling system. Twelve dinucleotide STRs commonly used in Western-European (WE) populations and four additional ones (G10C, G10J, G10O, G10X), were included. Template input ≥ 0.2 ng was successfully amplified. Measurements of precision, stutter and heterozygous balance showed that markers could be reliably genotyped applying the thresholds used for genotyping WE brown bears. However, locus G10X revealed an ancient allele-specific polymorphism that led to suboptimal amplification of all 174 bp alleles (Kamchatka and WE). Allele frequency estimates and forensic genetic parameters were obtained from 115 individuals successfully identified by genotyping 434 hair samples. All markers met the Hardy-Weinberg and linkage equilibrium expectations, and the power of discrimination ranged from 0.667 to 0.962. The total average probability of identity from the 15 STRs was 1.4 ×10−14 (FST = 0.05) while the total average probability of sibling identity was 6.0 ×10−6. Relationship tests revealed several parent-cub and full sibling pairs demonstrating that the marker set would be valuable for the study of family structures. The population data is the first of its kind from the Kamchatka brown bear population. Population pairwise FST`s revealed moderate genetic differentiation that mirrored the geographic distances to WE populations. The DNA profiling system, providing individual-specific profiles from non-invasive samples, will be useful for future monitoring and conservation purposes

O-268 Re-considering the endometrial microbiome: a comparison of DNA- and RNA-based sequencing techniques within the same cohort

Abstract Study question Does the analysis of the endometrial microbiome provide the same information when using DNA or RNA sequencing-based techniques? Summary answer DNA vs. RNA-based microbiome analysis techniques demonstrate significant microbial compositional differences, meaning that the previous endometrial 16S rRNA gene-based microbiome analysis results might be overestimated. What is known already In recent years, high-throughput sequencing technologies have revolutionised the field of reproductive microbiome research. Our understanding of the composition of endometrial microbiome predominantly relies on DNA-based 16S rRNA gene profiling method. Regardless of its effectiveness and low cost, its underestimation of microbial diversity, abundance, or functionality and lack of detection precision on species level have been highlighted. The meta-transcriptome analysis, RNA-based method, overcomes these limitations and identifies functional genes that are actively expressed by the alive microbes. This study aims to elucidate for the first time the endometrial microbiome using a dual approach of 16S rRNA gene-method and meta-transcriptomic analyses. Study design, size, duration In total forty-seven women with infertility at ages 27-42 years old were enrolled in the present study. Paired endometrial samples, endometrial brushing and Pipelle biopsy were collected from each participant at the mid-secretory menstrual phase (LH + 7/9). Participants/materials, setting, methods Endometrial samples were collected using Tao Brush for 16S rRNA analysis (DNA analysis) and Pipelle endometrial curette for meta-transcriptomic analysis (total RNA analysis). QIAamp UCP Pathogen Mini Kit was used for DNA extraction and 16S rRNA gene V3-V4 regions were sequenced on MiSeq. RNA was extracted using miRNeasy Micro kit, with rRNA removal by RiboZero kit and Libraries were generated using Stranded Total RNA Prep. Taxonomy was assigned by using Kraken2 (v2.2.1), Bracken (v2.7). Main results and the role of chance To the best of our knowledge, this is the first study to compare endometrial microbial identification using both metagenomics (16S rRNA sequencing) and meta-transcriptomics (meta-RNA sequencing) within the same cohort. Analysis of the composition of the microorganisms by bacterial 16S rRNA gene sequencing revealed that the most abundant bacterial genus in the endometrium was Lactobacillus. In the meta-transcriptome analysis, no microorganisms belonging to the Lactobacillus genus were detected in high abundance. This indicates that the relative abundance of this genus at the DNA level does not necessarily imply microbial activity or the presence of viable Lactobacillus in the endometrium. The meta-transcriptomics analysis detected microbial taxa such as Staphylococcus, Bacillus, Streptococcus, and Burkholderia as the most dominant. The results suggest that DNA-based detection of microorganisms in the endometrium may be overestimated due to the presence of naked DNA sequences or microorganisms from the vagina and cervix. Furthermore, RNA-based analysis demonstrates a population of active microorganisms different from the image shown by DNA analysis. Altogether, our study results indicate that the uterine microenvironment, as previously identified through DNA sequencing, may partly reflect the vaginocervical microenvironment, with the uterine tissue being a low biomass site not dominated by lactobacilli. Limitations, reasons for caution The endometrial samples were paired; however, DNA was analysed in the endometrial brushing sample while RNA in the tissue biopsy, which might result in some differences in microbial composition. Wider implications of the findings Contrary to the general belief of the Lactobacillus dominance in the human endometrium, our study suggests that the endometrial microenvironment may be harbouring DNA fragments and/or cells of lactobacilli originating from the lower reproductive tract. Our study results call out to re-consider/re-analyse the endometrial microbiome in health and disease. Trial registration number not applicable

P-039 Parthenogenetic chimeras may be the cause of the development of a case of 46, XX/46, XY chimeras complicated by severe oligozoospermia

Abstract Study question How did the 46,XX/46,XY chimeric case of severe oligozoospermia arise? Summary answer DNA analysis suggested the possibility of monogenetic chimerism as a mechanism for the development of this case. What is known already Sex-chromosome discordant chimerism in humans(XX/XY chimerism)is a rare chromosomal abnormality. Although the first case was described in 1962, its incidence is still unknown. The XX/XY chimera manifests variable genital phenotypes. The XX/XY chimera is classified into subtypes; tetragametic chimeras, parthenogenetic chimeras, androgenetic chimeras and sesquizygotic twinning chimeras. Genotyping of XX/XY chimeras is important no only to clarify its developmental mechanism but also for diagnosis and treatment. As a result, patients occasionally present with infertility. We present a case of a parthenogenetic chimera with a karyotype of XX/XY in relation to infertility. Study design, size, duration case research. We researched from July 2023 to December 2023. Participants/materials, setting, methods A 38-year-old man diagnosed with azoospermia by his previous physician presented to our clinic with primary infertility for 1 year and 6 months. At 28 years old, he presented with left testicular inflammation and subsequent atrophy. He has no sexual problems. He has an older sister and a younger sister, both married with children. Main results and the role of chance Physical findings: BH 163 cm, BW 53 kg, BMI 19.95, external genitalia: male. Semen analysis: severe oligozoospermia: mostly immobile sperm with some sperm moving slightly in situ. Morphology (Kurger strict criteria): no abnormal sperm morphology. Left testicular hypoplasia: USG: 12 ml right testis, 1 ml left, no other problems. Abdominal MRI: no abnormalities in the kidney to the bladder. No uterus or ovaries. Prostate and seminal vesicles normal; AMH 3.34ng/ml (0.77-14.5) Testosterone 6.55ng/ml (1.95-7.63), DHEA-S 199 μg/dl(106-464),LH 6.2, FSH 7.5, E2 32.5, PRL 12.43, blood type A, Rh+, RH-Hr:CcDee, HLA-A, B, C, DR: all undetermined, Y chromosome SRY (FISH): SRY signal numbers 0 and 1 were observed. Chromosome analysis (G band): 46, XX[22]/46, XY[8]. SNP microarray chromosome examination: presumptive parthenogenic chimerism Treatment: His wife underwent oocyte pick up and intracytoplasmic sperm injection(ICSI) was performed using his frozen sperm. One blastocyst was frozen, but one frozen-thawed blastocyst transfer during an HRT cycle failed to result in pregnancy. The use of assisted reproductive technology(ART) is currently ongoing. Limitations, reasons for caution DNA analysis of both parents was necessary to determine the cause of the problem, but the patient refused to have her parents tested. Wider implications of the findings By accumulating and studying a large number of cases like this one, it may be possible to correctly treat patients with 46, XX/46, XY chimeric infertility. Trial registration number not applicable