Mycoplasma species (spp.) are bacteria that are difficult to detect. Currently, the polymerase chain reaction (PCR) is considered the most effective diagnostic tool to detect these microorganisms in both human and veterinary medicine. There are 13 known species of human Mycoplasma and 15 species of canine Mycoplasma. Owing to the difficulties in identifying the individual species of Mycoplasma, there is a lack of information regarding which species are saprophytic and which are pathogenic. The prevalence of the individual species is also unknown. In addition, in both humans and dogs, the results of some studies on the impact of Mycoplasma are conflicting. The presence of Mycoplasma spp. on the epithelium of reproductive tract is often associated with infertility, although they are also detected in healthy individuals. The occurrence of Mycoplasma spp. is more common in dogs (even 89%) than in humans (1.3%-4%). This is probably because the pH of a dog's genital is more conducive to the growth of Mycoplasma spp. than that of humans. Phylogenetically, human and canine Mycoplasma are related, and majority of them belong to the same taxonomic group. Furthermore, 40% of canine Mycoplasma spp. are placed in common clusters with those of human. This suggests that species from the same cluster can play a similar role in the canine and human reproductive tracts. This review summarizes the current state of knowledge about the impact of Mycoplasma on canine and human male fertility as well as the prospects of further development in this field.
Humans ; Dogs ; Male ; Animals ; Mycoplasma/genetics* ; Infertility ; Semen Analysis ; Polymerase Chain Reaction/methods* ; Prevalence ; Semen/chemistry*

This study aimed to analyze aflatoxins content and fungal community distribution in the harvesting and processing of Platycladi Semen, and explore the key link that affects aflatoxins contamination. The related Platycladi Semen samples of different maturity periods(cone non-rupture period, early rupture, and complete rupture period) and different processing periods(before drying, during 2-d drying, during 7-d drying, before and after seed scale removal, before and after peeling, 1 d after color sorting, and 7 d after color sorting) were collected for identifying the fungal community composition on sample surface by ITS amplicon sequencing. Then the content of aflatoxins B_1, B_2, G_1 and G_2 was determined by HPLC-MS/MS. The results showed that during the harvesting of Platycladi Semen from cone non-rupture to complete rupture, aflatoxins were only detected in the seed scale and seed coat, with aflatoxin G_2 in the seed scale and aflatoxin B_1 in the seed coat. During the drying, with the prolongation of drying time, aflatoxins B_1 and G_2 were detected simultaneously in the seed scale, aflatoxin B_1 in the seed coat, and low-content aflatoxin B_1 in the seed kernel. During subsequent processing, the aflatoxin content in seed kernel during subsequent processing was slighted increased. As demonstrated by fungal detection, Aspergillus flavus was not present during the harvesting of Platycladi Semen, but present during the drying and processing. Its content in the seed coat during the drying process was relatively higher. In short, Platycladi Semen should be harvested as soon as possible after it becomes fully mature. Drying process is the key link of preventing aflatoxin contamination. It is advised to build a sunlight room or adopt similar settings, standardize the operations in other processes, and keep the surrounding environment clean to minimize aflatoxin contamination.
Aflatoxins/analysis* ; Aspergillus flavus ; Food Contamination/prevention & control* ; Mycobiome ; Semen/chemistry* ; Tandem Mass Spectrometry

OBJECTIVES: To establish a system for simultaneous detection of miR-888 and miR-891a by droplet digital PCR (ddPCR), and to evaluate its application value in semen identification. METHODS: The hydrolysis probes with different fluorescence modified reporter groups were designed to realize the detection of miR-888 and miR-891a by duplex ddPCR. A total of 75 samples of 5 body fluids (including peripheral blood, menstrual blood, semen, saliva and vaginal secretion) were detected. The difference analysis was conducted by Mann-Whitney U test. The semen differentiation ability of miR-888 and miR-891a was evaluated by ROC curve analysis and the optimal cut-off value was obtained. RESULTS: There was no significant difference between the dual-plex assay and the single assay in this system. The detection sensitivity was up to 0.1 ng total RNA, and the intra- and inter-batch coefficients of variation were less than 15%. The expression levels of miR-888 and miR-891a detected by duplex ddPCR in semen were both higher than those in other body fluids. ROC curve analysis showed that the AUC of miR-888 was 0.976, the optimal cut-off value was 2.250 copies/μL, and the discrimination accuracy was 97.33%; the AUC of miR-891a was 1.000, the optimal cut-off value was 1.100 copies/μL, and the discrimination accuracy was 100%. CONCLUSIONS In this study, a method for detection of miR-888 and miR-891a by duplex ddPCR was successfully established. The system has good stability and repeatability and can be used for semen identification. Both miR-888 and miR-891a have high ability to identify semen, and the discrimination accuracy of miR-891a is higher.
Female ; Humans ; Body Fluids/chemistry* ; MicroRNAs/analysis* ; Real-Time Polymerase Chain Reaction/methods* ; Saliva/chemistry* ; Semen/chemistry* ; Male

In forensic physical evidence identification, the accurate identification of the individual origin and their body fluid composition of the biological samples obtained from the crime scene play a critical role in determining the nature of a crime. In recent years, RNA profiling has become one of the fastest developing methods for body fluids identification. Due to the characteristics of tissue or body fluid specific expression, various types of RNA markers have been proven to be promising candidate markers for body fluids identification in previous studies. This review summarizes the research progress of RNA markers in body fluids identification, including the RNA markers that have been effectively verified in current research and their advantages and disadvantages. Meanwhile, this review prospects the application of RNA markers in forensic medicine.
Forensic Medicine/methods* ; Body Fluids/chemistry* ; RNA/analysis* ; Feces ; Forensic Genetics ; Semen/chemistry* ; Saliva/chemistry*

This study adopted headspace-gas chromatography-mass spectrometry(HS-GC-MS) and electronic nose to detect volatile components from Myristicae Semen samples with varying degrees of mildew, aiming at rapidly identifying odor changes and substance basis of Myristicae Semen mildew. The experimental data were analyzed by electronic nose and principal component analysis(PCA). The results showed that Myristicae Semen samples were divided into the following three categories by electronic nose and PCA: mildew-free samples, slightly mildewy samples, and mildewy samples. Myristicae Semen samples with different degrees of mildew greatly varied in volatile components. The volatile components in the samples were qualitatively and quantitatively detected by HS-GC-MS, and 59 compounds were obtained. There were significant differences in the composition and content in Myristicae Semen samples with different degrees of mildew. The PCA results were the same as those by electronic nose. Among them, 3-crene, D-limonene, and other terpenes were important indicators for the identification of mildew. Bicyclo[3.1.0]hexane, 4-methylene-1-(1-methylethyl)-, terpinen-4-ol, and other alcohols were key substances to distinguish the degree of mildew. In the later stage of mildew, Myristicae Semen produced a small amount of hydroxyl and aldehyde compounds such as acetaldehyde, 2-methyl-propionaldehyde, 2-methyl-butyraldehyde, and formic acid, which were deduced as the material basis of the mildew. The results are expected to provide a basis for the rapid identification of Myristicae Semen with different degrees of mildew, odor changes, and the substance basis of mildew.
Electronic Nose ; Gas Chromatography-Mass Spectrometry ; Odorants/analysis* ; Semen/chemistry* ; Solid Phase Microextraction ; Volatile Organic Compounds/analysis*

In the process of harvesting, production and processing, storage, and transportation, the traditional Chinese medicine Platycladi Semen is prone to mildew due to its own and environmental factors, which can nourish the production of toxic or pathogenic fungi, and even produce mycotoxins, which affects the safety of clinical medication. The 2020 edition of Chinese Pharmacopoeia limits the highest standard of aflatoxin content in Platycladi Semen. However, there are few studies on the fungal contamination of Platycladi Semen, and it is difficult to prevent and control it in a targeted manner. Therefore, based on the Illumina NovaSeq6000 platform, this article uses ITS sequence amplicon technology to analyze the distribution and diversity of fungi in 27 batches of commercially available Platycladi Semen in the Chinese market. A total of 10 phyla, 35 classes, 93 orders, 193 families, 336 genera, and 372 species of fungi were identified in China. Among them, Aspergillus, Alternaria spp. were dominant, 20 batches of samples were detected for A. flavus, 10 batches of samples were detected for A. nidulans, and all samples were detected for potential pathogenic fungi such as A. fumigatus and A. niger. According to diversity analysis, the diversity of the fungal communities in the samples from Gansu province was high, the samples in Shandong province contain the largest number of fungal species, and the samples in Guangxi province had the lo-west diversity and the least number of species. In most samples, pathogenic fungi such as A. fumigatus, A. niger, A. flavus, A. parasiticus were detected in varying degrees. This study systematically investigated the fungal contamination of Platycladi Semen from the markets in the last link of the its industrial chain, and clarified the distribution of Platycladi Semen fungi, especially toxin-producing fungi, and provided theoretical basis for the targeted prevention and control of fungal contamination in Platycladi Semen.
Aflatoxins ; China ; Fungi/genetics* ; Humans ; Mycobiome ; Mycotoxins/analysis* ; Semen/chemistry*

Based on the systematic retrieval and the reported components of Sojae Semen Nigrum and Sojae Semen Praeparatum, this study conducted in-depth analysis of conversion of components in the fermentation process, and discussed types and possible mec-hanisms of conversion of chemical components, so as to provide the basis for studying technology, medicinal ingredients and quality standards. According to the analysis, there is a certain degree of conversion of nutrients(like protein, sugar, lipid), bioactive substances(like isoflavones, saponins, γ-aminobutyric acid) and other substances(like nucleosides, melanoids, biamines, etc) in the process of fermentation.
Chromatography, High Pressure Liquid ; Fermentation ; Isoflavones/analysis* ; Semen/chemistry* ; Soybeans

A highly sensitive monoclonal antibody against aflatoxin B_1(AFB_1) was prepared and an indirect competition enzyme-linked immunosorbent assay(ic-ELISA) was established based on the antibody which was used for high-throughput and rapid screening of AFB_1 contamination in Chinese herbal medicines to ensure the safety of medication. In this study, the structure of AFB_1 was modified by improved oxime method, and the carrier protein was coupled by EDC-NHS method to obtain the complete antigen of AFB_1, which was more convenient and environmental friendly. The Balb/c female mice were immunized using increasing the immunization dose and various ways of injection, and finally the AFB_1 monoclonal antibody was prepared. The AFB_1 monoclonal antibody belongs to IgG_(2 b) immunoglobulin by identifying its immunological characteristics, and its sensitivity(IC_(50)) can reach 0.15 μg·L~(-1), and the affi-nity is 2.81×10~8 L·mol~(-1). The cross-reaction rates of AFB_2, AFG_1, and AFG_2 were 35.07%, 8.75%, and 1.15%, respectively, and there was almost no cross-reactivity with other mycotoxins. Based on the high sensitivity and specificity of the antibody, an ic-ELISA method was established and applied to the determination of AFB_1 contamination in Ziziphi Spinosae Semen. According to the matrix matching standard curve, the linear concentration range for AFB_1 was 0.05-0.58 μg·L~(-1)(R~2=0.992), the recoveries were 88.00%-119.0%, and the detection limit was 1.69 μg·kg~(-1). The AFB_1 in 33 batches of Ziziphi Spinosae Semen samples was determined by ic-ELISA, and the contamination level was 3.62-206.58 μg·kg~(-1). The linear correlation coefficient between the detection results of ic-ELISA and UHPLC-MS/MS was 0.996, and there were no false positive and false negative cases. It indicates that the established ic-ELISA is accurate and reliable, and could provide a simple and effective technique for fast screening of AFB_1 contamination in Ziziphi Spinosae Semen, and also could be considered as the reference for the detection and monitoring of AFB_1 contamination in other Chinese herbal medicines.
Aflatoxin B1 ; analysis ; Animals ; Antibodies, Monoclonal ; Drug Contamination ; Enzyme-Linked Immunosorbent Assay ; Female ; Mice ; Semen ; chemistry ; Tandem Mass Spectrometry

Lack of standardized, reproducible protocols and reference values is among the challenges faced when using new or upgraded versions of instruments in reproductive laboratories and flow cytometry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay combined with flow cytometry routinely used for diagnostic measurement of sperm DNA fragmentation (SDF) is a unique example. Any change in the setting of the standard instrument, including upgrades of hardware or software, can lead to different results and may affect clinicians' decision for treatment. Therefore, we compared TUNEL results of SDF obtained from a standard (C6) flow cytometer with a newer version of the same instrument (C6 Plus) and examined the cutoff, sensitivity, and specificity without calibration (adjustment) and after adjustment. Identical sperm preparation and matched acquisition settings were used to examine the performance of two flow cytometers. The strength of agreement of the results between the two observers was also assessed. After adjustment of the settings, overall concordance became high and the two cytometers showed 100% positive and negative predictive value with 100% area under the curve. The overall correlation coefficient observed between C6 and C6 Plus was highly significant (P < 0.0001; r = 0.992; 95% confidence interval [CI]: 0.982-0.997). After adjustment, the two cytometers showed very high precision of 98% and accuracy of >99%. The interobserver agreement on C6 flow cytometer for the two observers was 0.801 ± 0.062 and 0.746 ± 0.044 for C6 Plus. We demonstrated a strong agreement between the samples tested on the two flow cytometers after calibration and established the robustness of both instruments.
Adult ; Calibration ; DNA Fragmentation ; Flow Cytometry/instrumentation* ; Humans ; In Situ Nick-End Labeling ; Male ; Observer Variation ; Reference Values ; Reproducibility of Results ; Semen Analysis/methods* ; Sensitivity and Specificity ; Spermatozoa/chemistry*

Seminal plasma is a rich source of proteins and serves as an ideal sample for proteomic analysis of male infertility. In varicocele-associated infertility, the contributory role of seminal plasma proteins specific to unilateral and bilateral varicocele is not clear. Furthermore, there is a lack of specific protein biomarker to differentiate bilateral from unilateral varicocele. The main objective is to identify the differentially regulated molecular and cellular pathways in bilateral varicocele. Furthermore, we intend to identify seminal plasma biomarkers to differentiate bilateral and unilateral varicocele patients in comparison with fertile healthy men. Global proteomic analysis of seminal plasma proteins has identified the functionality of differentially expressed proteins (DEPs) in varicocele patients. Bioinformatic analysis has revealed response to reactive oxygen species and oxidative stress, and tissue homeostasis as top process pathways that are affected in bilateral varicocele patients compared to fertile healthy men. In comparison with unilateral varicocele patients, inflammatory response pathways were dysregulated, especially interleukin 6 (IL-6) signaling and Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathways, in bilateral varicocele patients, owing to the involvement of underexpressed DEPs. Key DEPs associated with oxidative stress (peroxiredoxin 2; PRDX2), DNA fragmentation (fatty acid synthase; FASN), and inflammatory response (fibronectin 1; FN1) validated by western blot analysis revealed differential expression of these proteins in unilateral and bilateral varicocele groups. Altered expression of DEPs and its association with key processes show that the seminal plasma homeostasis is compromised in bilateral varicocele patients. Furthermore, we propose PRDX2, FASN, and FN1 as potential noninvasive seminal plasma markers for the differentiation of unilateral and bilateral varicocele patients.
Biomarkers/analysis* ; Blotting, Western ; Humans ; Inflammation/metabolism* ; Male ; Metabolic Networks and Pathways ; Oxidative Stress ; Proteins/analysis* ; Proteomics ; Semen/chemistry* ; Varicocele/metabolism* ; Young Adult