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Metabolomic fingerprinting of pig seminal plasma identifies in vivo fertility biomarkers

Journal of Animal Science and Biotechnology 2022年第2期13卷 473-487页

作者： Yentel Mateo-Otero Pol Fernández-López Ariadna Delgado-Bermúdez Pau Nolis Jordi Roca Jordi Miró Isabel Barranco Marc Yeste Biotechnology of Animal and Human Reproduction(TechnoSperm) Institute of Food and Agricultural TechnologyUniversity of GironaES-17003 GironaSpain Department of Biology Unit of Cell BiologyFaculty of SciencesUniversity of GironaES-17003 GironaSpain Centre d’Estudis Avançats de Blanes(CEAB) Spanish Research Council(CSIC)ES-17300 GironaSpain Magnetic Nuclear Resonance Facility Autonomous University of BarcelonaBellaterraES-08193 Cerdanyola del VallèsSpain Department of Animal Medicine and Surgery Faculty of Veterinary MedicineUniversity of MurciaES-30100 MurciaSpain Equine Reproduction Service Department of Animal Medicine and SurgeryFaculty of Veterinary MedicineAutonomous University of BarcelonaBellaterraES-08193 Cerdanyola del VallèsSpain Department of Veterinary Medical Sciences University of BolognaIT-40064 Ozzano dell’EmiliaBolognaItaly

Background:Metabolomic approaches,which include the study of low molecular weight molecules,are an emerging-omics technology useful for identification of biomarkers.In this field,nuclear magnetic resonance(NMR)spectroscopy has already been used to uncover(in)fertility biomarkers in the seminal plasma(SP)of several mammalian species.However,NMR studies profiling the porcine SP metabolome to uncover in vivo fertility biomarkers are yet to be carried out.Thus,this study aimed to evaluate the putative relationship between SPmetabolites and in vivo fertility outcomes.To this end,24 entire ejaculates(three ejaculates per boar)were collected from artificial insemination(AI)-boars throughout a year(one ejaculate every 4 months).Immediately after collection,ejaculates were centrifuged to obtain SP-samples,which were stored for subsequent metabolomic analysis by NMR spectroscopy.Fertility outcomes from 1525 inseminations were recorded over a year,including farrowing rate,litter size,stillbirths per litter and the duration of pregnancy.Results:A total of 24 metabolites were identified and quantified in all SP-samples.Receiver operating characteristic(ROC)curve analysis showed that lactate levels in SP had discriminative capacity for farrowing rate(area under the curve[AUC]=0.764)while carnitine(AUC=0.847),hypotaurine(AUC=0.819),sn-glycero-3-phosphocholine(AUC=0.833),glutamate(AUC=0.799)and glucose(AUC=0.750)showed it for litter size.Similarly,citrate(AUC=0.743),creatine(AUC=0.812),phenylalanine(AUC=0.750),tyrosine(AUC=0.753)and malonate(AUC=0.868)levels had discriminative capacity for stillbirths per litter;and malonate(AUC=0.767)and fumarate(AUC=0.868)levels for gestation length.Conclusions:The assessment of selected SP-metabolites in ejaculates through NMR spectroscopy could be considered as a promising non-invasive tool to predict in vivo fertility outcomes in pigs.Moreover,supplementing AI-doses with specific metabolites should also be envisaged as a way to improve their fertility potential.

关键词： Artificial insemination in vivo fertility Metabolomics NMR Pregnancy outcomes seminal plasma

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Oxytocin in pig seminal plasma is positively related with in vivo fertility of inseminated sows

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Journal of Animal Science and Biotechnology 2022年第2期13卷 430-440页

作者： Lorena Padilla Marina López-Arjona Silvia Martinez-Subiela Heriberto Rodriguez-Martinez Jordi Roca Isabel Barranco Department of Medicine and Animal Surgery Faculty of Veterinary MedicineUniversity of MurciaE-30100 MurciaSpain Department of Biomedical&Clinical Sciences(BKV) BKH/Obstetrics&GynaecologyFaculty of Medicine and Health SciencesLinköping UniversitySE-58185 LinköpingSweden Department of Veterinary Medical Sciences University of BolognaIT-40064 Ozzano dell’EmiliaBolognaItaly

Background:Identification of relevant in vivo biomarkers for fertility remains a challenge for the livestock industry.Concentrations of the small peptide hormone oxytocin(OXT),involved in male reproductive function and present in the seminal plasma(SP)of several species could be a robust one.This study characterized concentrations of SPOXT in ejaculates from boars used in artificial insemination(AI)programs aiming to evaluate its relationship with sperm quality variables and in vivo fertility of their liquid-stored AI-semen.seminal OXT concentrations(ng/mL)were measured in 169 ejaculates from 61 boars of the Duroc,Pietrain,Landrace and Large White breeds using a direct competitive immunoassay test based on AlphaLISA®technology.Ejaculate(ejaculate volume,sperm concentration,total sperm count)and sperm parameters(motility,viability,intracellular generation of reactive oxygen species,plasma membrane fluidity)were assessed at 0 h and 72 h in AI-semen samples stored at 17℃.In vivo fertility included only 18 Large White and Landrace boars whose AI-semen was used to inseminated>100 sows and evaluated both farrowing rate and litter size of 3,167 sows.Results:The results showed that SP-OXT differed between boars and between ejaculates within boar(P<0.05)but not between breeds(Duroc,Pietrain,Landrace and Large White).Ejaculates with higher SP-OXT concentration/mL(hierarchically grouped;P<0.001)had larger volume and came from younger boars(P<0.05).Ejaculates of boars showing positive farrowing rate deviation exhibited higher(P<0.05)SP-OXT concentration/mL than those with negative farrowing rate deviation.Conclusion:The SP concentrations of OXT are boar,ejaculate and age dependent,and positively related with ejaculate volume and farrowing rates of liquid-stored semen AI-doses.

关键词： Artificial insemination Ejaculate Fertility Oxytocin Pig Semen quality seminal plasma

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seminal plasma anti-Müllerian hormone level correlates with semen parameters but does not predict success of testicular sperm extraction （TESE）

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Asian Journal of Andrology 2007年第2期9卷 265-270页

作者： Taymour Mostafa Medhat K. Amer Guirgis Abdel-Malak Taha Abdel Nsser Wael Zohdy Shedeed Ashour Dina El-Gayar Hosam H. Awad Andrology ＆ Sexology Department Faculty of Medicine Cairo University Cairo 11553 Egypt Adam International Clinic Mohandesseen Giza 12411 Egypt Artificial Insemination and Embryo Transfer Department Reproductive Research Institute Alharam Giza 12111 Egypt Chemical Pathology Department Faculty of Medicine Cairo University Cairo 11553 Egypt

Aim： To assess seminal plasma anti-Müllerian hormone （AMH） level relationships in fertile and infertile males. Methods： Eighty-four male cases were studied and divided into four groups： fertile normozoospermia （n = 16）, oligoastheno- teratozoospermia （n = 15）, obstructive azoospermia （OA）（n = 13） and non-obstructive azoospermia （NOA）（n = 40）. Conventional semen analysis was done for all cases. Testicular biopsy was done with histopathology and fresh tissue examination for testicular sperm extraction （TESE） in NOA cases. NOA group was subdivided according to TESE results into unsuccessful TESE （n = 19） and successful TESE （n = 21）. seminal plasma AMH was estimated by enzyme linked immunosorbent assay （ELISA） and serum follicular stimulating hormone （FSH） was estimated in NOA cases only by radioimmunoassay （RIA）. Results： Mean seminal AMH was significantly higher in fertile group than in oligoasthenoteratozoospermia with significance （41.5±10.9 pmol/L vs. 30.5±10.3 pmol/L, P 〈 0.05）. seminal AMH was not detected in any OA patients. seminal AMH wascorrelated positively with testicular volume （r = 0.329, P = 0.005）, sperm count （r = 0.483, P = 0.007）, sperm motility percent （r = 0.419, P = 0.021） and negatively with sperm abnormal forms percent （r = -0.413, p = 0.023）. Nonsignificant correlation was evident with age （r = -0.155, P = 0.414） and plasma FSH （ r = -0.014, P = 0.943）. In NOA cases, seminal AMH was detectable in 23/40 cases, 14 of them were successful TESE （57.5%） and was undetectable in 17/40 cases, 10 of them were unsuccessful TESE （58.2%）. Conclusion： seminal plasma AMH is an absolute testicular marker being absent in all OA cases. However, seminal AMH has a poor predictability for successful testicular sperm retrieval in NOA cases.

关键词： seminal plasma anti-Müllerian hormone spermatogenesis azoospermia testicular sperm extraction

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seminal plasma miR-192a： a biomarker predicting successful resolution of nonobstructive azoospermia following varicocele repair

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Asian Journal of Andrology 2018年第4期20卷 396-399页

作者： Er-Lei Zhi Guo-Qing Liang Peng Li Hui-Xing Chen Ru-Hui Tian Peng Xu Zheng Li Department of Andrology Center for Men's Health Department of ART Institute of Urologic Medical Center Shanghai General Hospital Shanghai Key Lab of Reproductive Medicine Shanghai Jiao Tong University 100 Haining Road Shanghai 200080 China Department of Andrology Seventh People's Hospital of Shangha University of TCM Shanghai 200000 China Oriental Jinghua Hospital of Shenyang Department of ART 156 Nanjing Road Shenyang 110005 China

This study was performed to investigate a potential marker for the presence of spermatozoa in the ejaculate following varicocelectomy in Chinese men with nonobstructive azoospermia and varicoceles. The micro-RNA （miR）-192a levels in seminal plasma and testicular tissue were evaluated by quantitative real-time polymerase chain reaction from 60 men with nonobstructive azoospermia and varicoceles （Group A： 27 men with spermatozoa found in the ejaculate after surgery; Group B： 33 men without spermatozoa found in the ejaculate after surgery） and 30 controls. The seminal plasma and testicular tissue miR-192a levels were higher in Group B than in Group A and the controls （P〈 0.001）, and there was no significant difference between Group A and the controls （P〉 0.05）. Apoptosis and proliferation assays with miR mimics and inhibitors showed that miR-192a induced GC-2 cell apoptosis through the activation of Caspase-3 protein. Thus, seminal plasma miR-192a appears to be a potential marker for successfully indicating spermatozoa in the ejaculate following microsurgical varicocelectomy in men with nonobstructive azoospermia and varicoceles. seminal plasma miR-192a may be a useful clinical marker for prescreening to determine which patients with nonobstructive azoospermia and varicoceles would benefit from varicocelectomy.

关键词： miR-192a nonobstructive azoospermia seminal plasma varicocelectomy

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Quick recovery and characterization of cell-free DNA in seminal plasma of normozoospermia and azoospermia： implications for non-invasive genetic utilities

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Asian Journal of Andrology 2009年第6期11卷 703-709页

作者： Hong-Gang Li Shi-Yun Huang Hui Zhou Ai-Hua Liao Cheng-Liang Xiong Family Planning Research Institute and Centre for Reproductive Medicine Tongji Medical College Huazhong Universityof Science and Technology Wuhan 430030 China

We established a quick and reliable method for recovering cell-free seminal DNA （cfsDNA）, by using the binding-washing-elution procedure on the DNA purification column. Low variations （below 15%） among the triplicate values of cfsDNA quantity verified the reproducibility of our cfsDNA recovery method. Similar cfsDNA yield and size distribution between seminal plasma acquired by filtration and centrifugation confirmed the presence of cfsDNA. To investigate the general characterization of cfsDNA, the quantitation and size distribution of cfsDNA from normozoospermic and azoospermic semen were analyzed by real-time PCR and electrophoresis, respectively. CfsDNA concentration in semen with normozoospermia （n = 11） was 1.34 ± 0.65 μg ·mL^-1, whereas a higher cfsDNA concentration was observed in azoospermia （2.56 ± 1.43 μg ·mL^-1, n = 9）. The continuous distribution of DNA fragments ranging from -1 kb to 15 kb and a spectrum of multiples of 180-bp fragments were observed in each normozoospermic and azoospermic sample. Distinct characteristic DNA ladder fragmentations in some azoospermic samples implicated that cfsDNA originate partly from apoptotic cells. CfsDNAs of 36 selected azoospermic patients with known information of Y chromosome microdeletion were subjected to the same microdeletion analysis by multiplex PCR and PCR amplification of sY114 （1 450 bp）. All multiplex PCR reactions with cfsDNA amplified successfully and provided the same result as leukocyte DNA. PCR amplification of sY114 gave a 1 450-bp amplicon as expected. Our data suggested the potential use of cfsDNA in search of biomarker or diagnostic procedures.

关键词： azoospermia cell-free DNA normozoospermia seminal plasma Y chromosome microdeletion

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Proteomic analysis of seminal plasma from asthenozoospermia patients reveals proteins that affect oxidative stress responses and semen quality

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Asian Journal of Andrology 2009年第4期11卷 484-491页

作者： Jun Wang Jian Wang Hua-Rong Zhang Hui-Juan Shi Duan Ma Hong-Xin Zhao Biaoyang Lin Run-Sheng Li Systems Biology Division Zhejiang-California Nanosystems Institute （ZCNI） Zhejiang University Zhejiang University Huajiachi Campus Hangzhou 310029 China Key Laboratory of Contraceptive Drugs and Devices of National population and family planning committee （NPFPC） Shanghai Institute of Planned Parenthood Research Shanghai 200030 China Key Laboratory of Molecular Medicine Ministry of Education Institute of Biomedical Sciences Shanghai Medical College Fudan University Shanghai 200030 China

Asthenozoospermia （AS） is a common cause of human male infertility. In one study, more than 80% of the samples from infertile men had reduced sperm motility. seminal plasma is a mixture of secretions from the testis, epididymis and several male accessory glands, including the prostate, seminal vesicles and Cowper＇s gland. Studies have shown that seminal plasma contains proteins that are important for sperm motility. To further explore the pathophysiological character of AS, we separated the seminal plasma proteins from AS patients and healthy donors using sodium dodecyl sulfate polyacrylamide gel electrophoresis and in-gel digestion, and then subjected the proteins to liquid chromatography-mass spectrometry （LC-MS/MS） analysis. A total of 741 proteins were identified in the seminal plasma, with a false discovery rate of 3.3%. Using spectral counting, we found that 45 proteins were threefold upregulated and 56 proteins were threefold downregulated in the AS group when compared with the control. Most of these proteins originated from the epididymis and prostate. This study identified a rich source of biomarker candidates for male infertility and indicates that functional abnormalities of the epididymis and prostate can contribute to AS. We identified D J-1--a protein that has been shown elsewhere to be involved in the control of oxidative stress （OS）-as a downregulated protein in AS seminal plasma. The levels of D J-1 in AS seminal plasma were about half of those in the control samples. In addition, the levels of reactive oxygen species were 3.3-fold higher in the AS samples than in the controls. Taken together, these data suggest that downregulation of DJ-1 is involved in OS in semen, and therefore affects the quality of the semen.

关键词： asthenozoospermia comparative proteomics DJ-1 seminal plasma

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Proteomic profile of seminal plasma in adolescents and adults with treated and untreated varicocele

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Asian Journal of Andrology 2016年第2期18卷 194-201,J0003页

作者： Mariana Camargo Paula Intasqui Ricardo Pimenta Bertolla Department of Surgery Division of Urology Human Reproduction Section Sao Paulo Federal University R. Embau 231 04039-060 Sao Paulo Brazil Sao Paulo Hospital Sao Paulo Brazil.

Varicocele, the most important treatable cause of male infertility, is present in 15% of adult males, 35% of men with primary infertility, and 80% of men with secondary infertility. On the other hand, 80% of these men will not present infertility. Therefore, there is a need to differentiate a varicocele that is exerting a deleterious effect that is treatable from a ＂silent＂ varicocele. Despite the growing evidence of the cellular effects of varicocele, its underlying molecular mechanisms are still eluding. Proteomics has become a promising area to determine the reproductive biology of semen as well as to improve diagnosis of male infertility. This review aims to discuss the state-of-art in seminal plasma proteomics in patients with varicocele to discuss the challenges in undertaking these studies, as well as the future outlook derived from the growing body of evidence on the seminal proteome.

关键词： male infertility proteomics seminal plasma varicocele varicocelectomy

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Assessment of seminal plasma laminin in fertile and infertile men

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Asian Journal of Andrology 2007年第1期9卷 63-67页

作者： Mohamed R. El-Dakhly Gamil A. Tawadrous Taymour Mostafa Mohamed M. F. Roaia Abdel R. M. El-Nashar Shedeed A. Shedeed Ihab I. Kamel Amal A. Aziz Yasser El-Mohtaseb Andrology Department Faculty of Medicine Cairo University Cairo 12311 Egypt Medical Biochemistry Department Faculty of Medicine Cairo University Cairo 12311 Egypt Clinical Pathology Department Faculty of Medicine Cairo University Cairo 12311 Egypt

Aim： To assess laminin levels in the seminal plasma of infertile and fertile men, and to analyze the correlation of laminin levels with sperm count, age, sperm motility and semen volume. Methods： One hundred and twenty-five recruited men were equally divided into five groups according to their sperm concentration and clinical examination： fertile normozoospermia, oligoasthenozoospermia, non-obstructive azoospermia （NOA）, obstructive azoospermia （OA） and congenital bilateral absent vas deferens （CBAVD）. The patients＇ medical history was investigated and patients underwent clinical examination, conventional semen analysis and estimation of seminal plasma laminin by radioimmunoassay. Results： seminal plasma laminin levels of successive groups were： 2.82 ± 0.62, 2.49 ± 0.44, 1.77 ± 0.56, 1.72 ± 0.76, 1.35 ± 0.63 U/mL, respectively. The fertile normozoospermic group showed the highest concentration compared to all infertile groups with significant differences compared to azoospermic groups （P 〈 0.05）. Testicular contribution was estimated to be approximately one-third of the seminal laminin. seminal plasma laminin demonstrated significant correlation with sperm concentration （r = 0.460, P 〈 0.001） and nonsignificant correlation with age （r = 0.021, P = 0.940）, sperm motility percentage （r = 0.142, P = 0.615） and semen volume （r = 0.035, P = 0.087）. Conelusion： seminal plasma laminin is derived mostly from prostatic and testicular portions and minimally from the seminal vesicle and vas deferens. Estimating seminal laminin alone is not conclusive in diagnosing different cases of male infertility.

关键词： male infertility semen seminal plasma testis basement membrane laminin azoospermia congenital bilateral absent vas deferens

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Beta-endorphin in serum and seminal plasma in infertile men

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Asian Journal of Andrology 2006年第6期8卷 709-712页

作者： Shawky El-Haggar Salah El-Ashmawy Ahmed Attia Taymour Mostafa M.M.Farid Roaiah Ashraf Fayez Sherif Ghazi Wael Zohdy Nagwa Roshdy Andrology Department Clinical Pathology Department Medical Biochemistry Department Faculty of MedicineCairo UniversityCairo 12311Egypt

Aim： To access beta-endorphin levels in serum as well as seminal plasma in different infertile male groups. Methods： Beta-endorphin was estimated in the serum and seminal plasma by enzyme-linked immunosorbent assay （ELISA） method in 80 infertile men equally divided into four groups： non-obstructive azoospermia （NOA）, obstructive azoospermia （OA）, congenital bilateral absent vas deferens （CBVAD） and asthenozoospermia. The results were compared to those of 20 normozoospermic proven fertile men. Results： There was a decrease in the mean levels of betaendorphin in the seminal plasma of all successive infertile groups （mean ± SD： NOA 51.30 ± 27.37, OA 51.88 ± 9.47, CBAVD 20.36 ± 13.39, asthenozoospermia 49.26 ± 12.49 pg/mL, respectively） compared to the normozoospermic fertile control （87.23 ± 29.55 pg/mL）. This relation was not present in mean serum level of beta-endorphin between four infertile groups （51.09 ± 14.71, 49.76 ± 12.4, 33.96 ± 7.2, 69.1 ± 16.57 pg/mL, respectively） and the fertile control group （49.26 ± 31.32 pg/mL）. The CBVAD group showed the lowest seminal plasma mean level of beta-endorphin. Testicular contribution of seminal beta-endorphin was estimated to be approximately 40%. seminal beta-endorphin showed significant correlation with the sperm concentration （r = 0.699, P = 0.0188） and nonsignificant correlation with its serum level （r = 0.375, P = 0.185） or with the sperm motility percentage （r = 0.470, P = 0.899）. Conclusion： The estimation of beta-endorphin alone is not conclusive to evaluate male reproduction as there are many other opiates acting at the hypothalamic pituitary gonadal axis.

关键词： azoospermia beta-endorphin male infertility opioid peptides semen seminal plasma

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Cytokines in Human seminal plasma and Their Effect on Male Fertility

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Journal of Reproduction and Contraception 2005年第2期16卷 107-126页

作者： Snje■ana ■idovec Sanja Vujisic Division of cellular immunology University Hospital Zagreb Croatia Clinical Laboratory for Human Reproduction Department of Obstetrics and Gynecology Medical School University of Zagreb Sveti Duh Hospital Zagreb Croatia

Cytokines are a heterogeneous group of peptides that play an important role in intercellular communication, regulation of innate and specific immunity, hematopoiesis, interaction between the immune system and neuroendocrine network as well as in reproduction and development. seminal plasma provides an immunological environ- ment for the semen and contains important biological response mediators. Numerous studies investigated the presence of various cytokines in the seminal plasma and tried to correlate cytokine levels with sperm quality and male fertility. However, the patho- physiological significance of seminal cytokines in sperm function is still not completely understood. The purpose of this review is to provide a brief summary of the extensive literature dealing with cytokines in the seminal plasma and to discuss the contribution of local cytokine immunity to male fertility.

关键词： seminal plasma cytokines fertility semen quality

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