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    Sperm Syringe: 3D Sorting Platform for Assisted Reproduction (Adv. Mater. Technol. 9/2022)
    Yazdan Parast, F ; O'Bryan, MK ; Nosrati, R (Wiley, 2022-09)
    In article number 2101291, Reza Nosrati and co-workers develop a scalable 3D sorting platform for one-step semen purification and high-quality sperm selection. The Sperm Syringe selects sperm with over 65% improvement in both DNA integrity and morphology, considerably outperforming the current best clinical practices for sperm selection in assisted reproduction.
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    Actionable secondary findings following exome sequencing of 836 non-obstructive azoospermia cases and their value in patient management
    Kasak, L ; Lillepea, K ; Nagirnaja, L ; Aston, K ; Schlegel, PN ; Goncalves, J ; Carvalho, F ; Moreno-Mendoza, D ; Almstrup, K ; Eisenberg, ML ; Jarvi, KA ; O'Bryan, MK ; Lopes, AM ; Conrad, DF ; Punab, M ; Laan, M (OXFORD UNIV PRESS, 2022-06-30)
    STUDY QUESTION: What is the load, distribution and added clinical value of secondary findings (SFs) identified in exome sequencing (ES) of patients with non-obstructive azoospermia (NOA)? SUMMARY ANSWER: One in 28 NOA cases carried an identifiable, medically actionable SF. WHAT IS KNOWN ALREADY: In addition to molecular diagnostics, ES allows assessment of clinically actionable disease-related gene variants that are not connected to the patient's primary diagnosis, but the knowledge of which may allow the prevention, delay or amelioration of late-onset monogenic conditions. Data on SFs in specific clinical patient groups, including reproductive failure, are currently limited. STUDY DESIGN, SIZE, DURATION: The study group was a retrospective cohort of patients with NOA recruited in 10 clinics across six countries and formed in the framework of the international GEMINI (The GEnetics of Male INfertility Initiative) study. PARTICIPANTS/MATERIALS, SETTING, METHODS: ES data of 836 patients with NOA were exploited to analyze SFs in 85 genes recommended by the American College of Medical Genetics and Genomics (ACMG), Geisinger's MyCode, and Clinical Genome Resource. The identified 6374 exonic variants were annotated with ANNOVAR and filtered for allele frequency, retaining 1381 rare or novel missense and loss-of-function variants. After automatic assessment of pathogenicity with ClinVar and InterVar, 87 variants were manually curated. The final list of confident disease-causing SFs was communicated to the corresponding GEMINI centers. When patient consent had been given, available family health history and non-andrological medical data were retrospectively assessed. MAIN RESULTS AND THE ROLE OF CHANCE: We found a 3.6% total frequency of SFs, 3.3% from the 59 ACMG SF v2.0 genes. One in 70 patients carried SFs in genes linked to familial cancer syndromes, whereas 1 in 60 cases was predisposed to congenital heart disease or other cardiovascular conditions. Retrospective assessment confirmed clinico-molecular diagnoses in several cases. Notably, 37% (11/30) of patients with SFs carried variants in genes linked to male infertility in mice, suggesting that some SFs may have a co-contributing role in spermatogenic impairment. Further studies are needed to determine whether these observations represent chance findings or the profile of SFs in NOA patients is indeed different from the general population. LIMITATIONS, REASONS FOR CAUTION: One limitation of our cohort was the low proportion of non-Caucasian ethnicities (9%). Additionally, as comprehensive clinical data were not available retrospectively for all men with SFs, we were not able to confirm a clinico-molecular diagnosis and assess the penetrance of the specific variants. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, this study analyzed medically actionable SFs in men with spermatogenic failure. With the evolving process to incorporate ES into routine andrology practice for molecular diagnostic purposes, additional assessment of SFs can inform about future significant health concerns for infertility patients. Timely detection of SFs and respective genetic counseling will broaden options for disease prevention and early treatment, as well as inform choices and opportunities regarding family planning. A notable fraction of SFs was detected in genes implicated in maintaining genome integrity, essential in both mitosis and meiosis. Thus, potential genetic pleiotropy may exist between certain adult-onset monogenic diseases and NOA. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Estonian Research Council grants IUT34-12 and PRG1021 (M.L. and M.P.); National Institutes of Health of the United States of America grant R01HD078641 (D.F.C., K.I.A. and P.N.S.); National Institutes of Health of the United States of America grant P50HD096723 (D.F.C. and P.N.S.); National Health and Medical Research Council of Australia grant APP1120356 (M.K.O'B., D.F.C. and K.I.A.); Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação grant POCI-01-0145-FEDER-007274 (A.M.L., F.C. and J.G.) and FCT: IF/01262/2014 (A.M.L.). J.G. was partially funded by FCT/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), through the Centre for Toxicogenomics and Human Health-ToxOmics (grants UID/BIM/00009/2016 and UIDB/00009/2020). M.L.E. is a consultant for, and holds stock in, Roman, Sandstone, Dadi, Hannah, Underdog and has received funding from NIH/NICHD. Co-authors L.K., K.L., L.N., K.I.A., P.N.S., J.G., F.C., D.M.-M., K.A., K.A.J., M.K.O'B., A.M.L., D.F.C., M.P. and M.L. declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.
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    Impact of Chronic Multi-Generational Exposure to an Environmentally Relevant Atrazine Concentration on Testicular Development and Function in Mice
    Kolaitis, ND ; Finger, BJ ; Merriner, DJ ; Nguyen, J ; Houston, BJ ; O'Bryan, MK ; Stringer, JM ; Zerafa, N ; Nguyen, N ; Hutt, KJ ; Tarulli, GA ; Green, MP (MDPI, 2023-02)
    A common herbicide, atrazine, is associated with poor health. Atrazine acts as an endocrine disruptor at supra-environmental levels. Little research, however, has been conducted regarding chronic exposure to environmental atrazine concentrations across generations. This study utilized comprehensive endpoint measures to investigate the effects of chronic exposure to a conservative atrazine concentration (0.02 ng/mL), measured in Australian waterways, on male mice fertility across two generations. Mice were exposed through the maternal line, from the pre-conception period and through the F1 and F2 generations until three or six months of age. Atrazine did not impact sperm function, testicular morphology nor germ cell parameters but did alter the expression of steroidogenic genes in the F1, down-regulating the expression of Cyp17a1 (Cytochrome P450 family 17, subfamily A member 1; p = 0.0008) and Ddx4 (DEAD-box helicase 4; p = 0.007), and up-regulating the expression of Star (Steroidogenic acute regulatory protein; p = 0.017). In the F2, atrazine induced up-regulation in the expression of Star (p = 0.016). The current study demonstrates that chronic exposure to an environmentally relevant atrazine concentration perturbs testicular steroid-associated gene expression that varies across generations. Future studies through the paternal and combined parental lineages should be undertaken to further elucidate the multigenerational effects of atrazine on male fertility.
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    Diverse monogenic subforms of human spermatogenic failure
    Nagirnaja, L ; Lopes, AM ; Charng, W-L ; Miller, B ; Stakaitis, R ; Golubickaite, I ; Stendahl, A ; Luan, T ; Friedrich, C ; Mahyari, E ; Fadial, E ; Kasak, L ; Vigh-Conrad, K ; Oud, MS ; Xavier, MJ ; Cheers, SR ; James, ER ; Guo, J ; Jenkins, TG ; Riera-Escamilla, A ; Barros, A ; Carvalho, F ; Fernandes, S ; Goncalves, J ; Gurnett, CA ; Jorgensen, N ; Jezek, D ; Jungheim, ES ; Kliesch, S ; McLachlan, R ; Omurtag, KR ; Pilatz, A ; Sandlow, J ; Smith, J ; Eisenberg, ML ; Hotaling, JM ; Jarvi, KA ; Punab, M ; Rajpert-De Meyts, E ; Carrell, DT ; Krausz, C ; Laan, M ; O'Bryan, MK ; Schlegel, PN ; Tuettelmann, F ; Veltman, JA ; Almstrup, K ; Aston, K ; Conrad, DF (NATURE PORTFOLIO, 2022-12-26)
    Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human "knockouts", and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification.
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    Sperm Syringe: 3D Sorting Platform for Assisted Reproduction
    Parast, FY ; O'Bryan, MK ; Nosrati, R (WILEY, 2022-09)
    Abstract Selection of high‐quality sperm is crucial to assisted reproduction. However, conventional clinical methods for sperm selection are manual and prone to operator errors. This article presents ‘sperm syringe', a scalable technology that mimics the highly parallelized 3D selection process in vivo via a 3D network of 560 microchannels to select high‐quality sperm. Sperm syringe retrieves more than 41% of healthy sperm from the initial sperm sample in under 15 min, providing a sufficient volume (≈500 µL) and number (1,600,000) of high‐quality sperm for fertility treatments. Experiments with bull and human sperm indicate 65% improvement in selected sperm morphology and DNA integrity, considerably outperforming the current best clinical practices. This approach enables the selection of a subpopulation of high‐quality human sperm from an oligozoospermia sample with DNA integrity below the reference value for fertility. The fabrication method of the device is also simple and scalable, representing a commercially viable technology for translation and clinical adoption. Sperm syringe provides a promising opportunity for clinics to use less invasive assisted reproductive technologies (ARTs) over intracytoplasmic sperm injection, thus reducing the associated clinical workload and optimizing long‐term health outcomes for ART‐conceived children.
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    Activation of the viral sensor oligoadenylate synthetase 2 (Oas2) prevents pregnancy-driven mammary cancer metastases
    Ho, W-HJ ; Law, AMK ; Masle-Farquhar, E ; Castillo, LE ; Mawson, A ; O'Bryan, MK ; Goodnow, CC ; Gallego-Ortega, D ; Oakes, SR ; Ormandy, CJ (BMC, 2022-05-03)
    BACKGROUND: The interferon response can influence the primary and metastatic activity of breast cancers and can interact with checkpoint immunotherapy to modulate its effects. Using N-ethyl-N-nitrosourea mutagenesis, we found a mouse with an activating mutation in oligoadenylate synthetase 2 (Oas2), a sensor of viral double stranded RNA, that resulted in an interferon response and prevented lactation in otherwise healthy mice. METHODS: To determine if sole activation of Oas2 could alter the course of mammary cancer, we combined the Oas2 mutation with the MMTV-PyMT oncogene model of breast cancer and examined disease progression and the effects of checkpoint immunotherapy using Kaplan-Meier survival analysis with immunohistochemistry and flow cytometry. RESULTS: Oas2 mutation prevented pregnancy from increasing metastases to lung. Checkpoint immunotherapy with antibodies against programmed death-ligand 1 was more effective when the Oas2 mutation was present. CONCLUSIONS: These data establish OAS2 as a therapeutic target for agents designed to reduce metastases and increase the effectiveness of checkpoint immunotherapy.
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    High-Frequency Ultrasound Boosts Bull and Human Sperm Motility
    Gai, J ; Dervisevic, E ; Devendran, C ; Cadarso, VJ ; O'Bryan, MK ; Nosrati, R ; Neild, A (WILEY, 2022-04)
    Sperm motility is a significant predictor of male fertility potential and is directly linked to fertilization success in both natural and some forms of assisted reproduction. Sperm motility can be impaired by both genetic and environmental factors, with asthenozoospermia being a common clinical presentation. Moreover, in the setting of assisted reproductive technology clinics, there is a distinct absence of effective and noninvasive technology to increase sperm motility without detriment to the sperm cells. Here, a new method is presented to boost sperm motility by increasing the intracellular rate of metabolic activity using high frequency ultrasound. An increase of 34% in curvilinear velocity (VCL), 10% in linearity, and 32% in the number of motile sperm cells is shown by rendering immotile sperm motile, after just 20 s exposure. A similar effect with an increase of 15% in VCL treating human sperm with the same setting is also identified. This cell level mechanotherapy approach causes no significant change in cell viability or DNA fragmentation index, and, as such, has the potential to be applied to encourage natural fertilization or less invasive treatment choices such as in vitro fertilization rather than intracytoplasmic injection.
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    CRISPs Function to Boost Sperm Power Output and Motility
    Gaikwad, AS ; Nandagiri, A ; Potter, DL ; Nosrati, R ; O'Connor, AE ; Jadhav, S ; Soria, J ; Prabhakar, R ; O'Bryan, MK (FRONTIERS MEDIA SA, 2021-08-05)
    Fertilization requires sperm to travel long distances through the complex environment of the female reproductive tract. Despite the strong association between poor motility and infertility, the kinetics of sperm tail movement and the role individual proteins play in this process is poorly understood. Here, we use a high spatiotemporal sperm imaging system and an analysis protocol to define the role of CRISPs in the mechanobiology of sperm function. Each of CRISP1, CRISP2, and CRISP4 is required to optimize sperm flagellum waveform. Each plays an autonomous role in defining beat frequency, flexibility, and power dissipation. We thus posit that the expansion of the CRISP family from one member in basal vertebrates, to three in most mammals, and four in numerous rodents, represents an example of neofunctionalization wherein proteins with a common core function, boosting power output, have evolved to optimize different aspects of sperm tail performance.
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    Correction: HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse.
    Lim, SL ; Qu, ZP ; Kortschak, RD ; Lawrence, DM ; Geoghegan, J ; Hempfling, A-L ; Bergmann, M ; Goodnow, CC ; Ormandy, CJ ; Wong, L ; Mann, J ; Scott, HS ; Jamsai, D ; Adelson, DL ; O'Bryan, MK (Public Library of Science (PLoS), 2015-12)
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    Validation and application of a novel integrated genetic screening method to a cohort of 1,112 men with idiopathic azoospermia or severe oligozoospermia
    Oud, MS ; Ramos, L ; O'Bryan, MK ; McLachlan, RI ; Okutman, O ; Viville, S ; de Vries, PF ; Smeets, DFCM ; Lugtenberg, D ; Hehir-Kwa, JY ; Gilissen, C ; van de Vorst, M ; Vissers, LELM ; Hoischen, A ; Meijerink, AM ; Fleischer, K ; Veltman, JA ; Noordam, MJ (WILEY-HINDAWI, 2017-11)
    Microdeletions of the Y chromosome (YCMs), Klinefelter syndrome (47,XXY), and CFTR mutations are known genetic causes of severe male infertility, but the majority of cases remain idiopathic. Here, we describe a novel method using single molecule Molecular Inversion Probes (smMIPs), to screen infertile men for mutations and copy number variations affecting known disease genes. We designed a set of 4,525 smMIPs targeting the coding regions of causal (n = 6) and candidate (n = 101) male infertility genes. After extensive validation, we screened 1,112 idiopathic infertile men with non-obstructive azoospermia or severe oligozoospermia. In addition to five chromosome YCMs and six other sex chromosomal anomalies, we identified five patients with rare recessive mutations in CFTR as well as a patient with a rare heterozygous frameshift mutation in SYCP3 that may be of clinical relevance. This results in a genetic diagnosis in 11-17 patients (1%-1.5%), a yield that may increase significantly when more genes are confidently linked to male infertility. In conclusion, we developed a flexible and scalable method to reliably detect genetic causes of male infertility. The assay consolidates the detection of different types of genetic variation while increasing the diagnostic yield and detection precision at the same or lower price compared with currently used methods.