36. Jahrestagung der Deutschen Gesellschaft für Andrologie

Köln, 14.–16. November 2024

Abstracts*

*Begutachtet und zusammengestellt vom wissenschaftlichen Komitee; alphabetisch geordnet nach Erstautor.

Freie Vorträge zu aktuellen Themen – Klinik

One family, two causes: DNAH17 and HIPK4 variants and male infertility

S. A. Koser1, A. S. Gaikwad1, I. Aprea2, C. Rieck2, C. Krallmann3, A. Kovacevic4, B. Stallmeyer1, J. Raidt2, H. Omran2, S. Kliesch3, H. Schorle4, C. Friedrich1, F. Tüttelmann1

1Centre of Medical Genetics, Institute of Reproductive ­Genetics, University of Münster, Münster; 2Department of General Paediatrics, University Hospital Münster, Münster; 3Centre of Reproductive Medicine and Andrology (CeRA), Department of Clinical and Surgical Andrology, University of Münster, Münster; 4Institute of Pathology, Department of Developmental Pathology, Medical Faculty, University of Bonn, Bonn, Germany

Introduction In male infertility, both clinical characteristics – especially semen parameters – and underlying causes are highly heterogeneous. In familial cases, however, a shared genetic cause represents the most likely aetiology.

Methods Exome sequencing was performed in four infertile brothers, their parents and their fertile brother. Andrological diagnostics including standard semen analysis, as well as immunofluorescence staining, transmission electron microscopy (TEM) and heterologous expression served for phenotypic analysis and variant validation.

Results In the four affected brothers, semen analysis revealed variable quantitative and qualitative spermatogenic failure that could be summarised as oligoterato- or oligoasthenoteratozoospermia (OAT). Surprisingly, exome sequencing did not identify a shared genetic cause. One brother carries bi-allelic DNAH17 variants causing an outer dynein arm defect leading to impaired sperm motility and morphological abnormalities of the sperm flagella. In contrast, his three brothers are homozygous for a variant in HIPK4, a novel candidate gene for male infertility expressed in spermatids and required for sperm head shaping. Concordantly, all three brothers had 100% sperm head defects with variable morphological abnormalities.

Conclusion In addition to novel variants in the established infertility gene DNAH17, we identified a homozygous HIPK4 variant likely causing familial infertility due to OAT. This is in line with published male Hipk4 knockout mice displaying an OAT phenotype with abnormal sperm heads and infertility. Our work raises awareness for the evaluation of family history in standard infertility diagnostics. It highlights the possibility of more than one genetic cause in one family and, thus, the utility of exome-based segregation analysis of a whole family.

Grants: This work was supported by the DFG Clinical Research Unit 326 ‚Male Germ Cells‘ (CRU326) and the Medical Faculty Münster’s ‚CareerS‘ programme.

Facetten des sexuellen Selbstbildes bei Männern nach Vasektomie

L. J. Schmalbrock1, C.Nöckel1, V. H. Meissner1, S. Schiele1, M. Jahnen1, H. Schulwitz1, A. Dinkel2, J. E. Gschwend1, K. Herkommer1

1Klinik und Poliklinik für Urologie, School of Medicine and Health, Klinikum rechts der Isar der Technischen Universität München, München; 2Klinik und Poliklinik für Psychosomatische Medizin und Psychotherapie, School of Medicine and Health, Klinikum rechts der Isar der Technischen Universität München, München, Deutschland

Einleitung Einige Männer befürchten negative Veränderungen ihres Sexuallebens nach einer Vasektomie, wobei psychologische Faktoren wie das sexuelle Selbstbild eine wesentliche Rolle spielen können. Ziel dieser Studie war es, Männer mit und ohne Vasektomie hinsichtlich ihres sexuellen Selbstbildes zu vergleichen.

Methoden Die Datenerhebung erfolgte im Rahmen der Bavarian Men’s Health- (BMH-) Study. Soziodemographische, lebensstilbezo­gene, gesundheitliche und psychologische Merkmale sowie das Sexualverhalten wurden mittels Fragebögen und Arztgespräch erfasst. Das sexuelle Selbstbild wurde anhand von vier Facetten analysiert: Körperbild, Verständnis von Maskulinität/Toughness, sexuelles Selbstwertgefühl und wahrgenommener Druck bezüglich sexueller Leistungsfähigkeit. Männer mit und ohne Vasektomie wurden hinsichtlich der genannten Parameter verglichen.

Ergebnisse Insgesamt wurden 5355 Männer (Alter: 50,4 ± 0,8 Jahre) eingeschlossen, 88,0 % waren in einer festen Partnerschaft und 72,9 % hatten Kinder. 63,3 % waren mindestens einmal im Monat sexuell aktiv, 61,4 % empfanden ihr Sexualleben als (sehr) wichtig und 45,4 % waren (sehr) zufrieden damit. 12,6 % berichteten von einer Vasektomie, die durchschnittlich 8,6 ± 5,8 Jahre zurücklag. Vasektomierte Männer waren häufiger sexuell aktiv, zufriedener mit ihrem Sexual­leben und empfanden dieses als wichtiger (alle p < 0,001). Zudem wiesen sie ein positiveres Körperbild (p < 0,001), moderneres Maskulinitätsverständnis (p < 0,01), höheres sexuelles Selbstwertgefühl (p < 0,001) und weniger Druck bezüglich sexueller Leistungsfähigkeit (p < 0,05) auf.

Schlussfolgerung Unsere Ergebnisse zeigen, dass Männer etwa acht Jahre nach einer Vasektomie ein positiveres Körperbild, ein höheres sexuelles Selbstwertgefühl, weniger Druck bezüglich sexueller Leistungsfähigkeit und moderneres Maskulinitätsverständnis haben. Diese Erkenntnisse können Männern, die eine Vasektomie in Erwägung ziehen, die Sorge vor negativen Auswirkungen auf ihr sexuelles Selbstbild nehmen.

Normozoospermie und Infertilität – ein frühzeitiger Indikator für relevante Erkrankungen?

S. Bier, A. Wolff, M. Zitzmann, S. Kliesch

Abteilung für klinische und operative Andrologie, Centrum für Reproduktionsmedizin und Andrologie, Universitäts­klinikum Münster, Münster, Deutschland

Einleitung Weltweit sind etwa 15 % der Paare von Infertilität betroffen, definiert als unerfüllter Kinderwunsch trotz eines Jahres regelmäßigen ungeschützten Geschlechtsverkehrs. In 50 % der Fälle sind männliche Faktoren für diese Infertilität verantwortlich. Aktuelle Studien haben gezeigt, dass männliche Infertilität mit einem erhöhten Risiko für Begleiterkrankungen einhergeht. Dennoch wird die Notwendigkeit einer andrologischen Untersuchung bei Männern mit Infertilität und Normozoospermie weiterhin diskutiert.

Material und Methoden In dieser retro­spektiven Studie wurden die Untersuchungs­ergebnisse von 997 Männern analysiert, die sich zwischen 2010 und 2020 mit Normozoospermie und Infertilität in unserem Zentrum vorstellten. Die Daten umfassten Anamnese, Hormon- und Blutuntersuchungen, Sonographie der Hoden sowie Ejakulatuntersuchungen. Diese Ergebnisse wurden mit denen gesunder Männer aus unserer FAMe-Studie [Laurentino et al. 2020] sowie von Vätern verglichen, die nach einer Vasektomie mit erneutem Kinderwunsch unser Zentrum aufsuchten.

Ergebnisse Patienten der Infertilitätsgruppe (IG) wiesen signifikant häufiger Fehlbildungen des Genitales (z. B. Hypospadie) (p = 0,024) und/oder eine erektile Dysfunktion (p < 0,001) auf als Männer der Kontrollgruppe (KG). Bei den Hormonuntersuchungen zeigte sich in der IG signifikant häufiger ein Hypogonadismus (p < 0,001), während Männer der KG signifikant häufiger einen kompensierten Hypogonadismus aufwiesen. In der IG wurden signifikant mehr Rundzellen (p < 0,001) und Leukozyten (p = 0,013) in der Ejakulatuntersuchung nachgewiesen als bei gesunden Männern der FAMe-Studie.

Zusammenfassung Männer mit Infertilität und Normozoospermie haben ein erhöhtes Risiko für relevante Vorerkrankungen. Die Ergebnisse unserer Studie unterstreichen die Notwendigkeit einer umfassenden medizinischen Betreuung dieses besonderen Patientenkollektivs, um langfristige negative Gesundheitsfolgen zu vermeiden.

Modifizierte Berechnung des International Index of Erectile Function (IIEF-6): Präzisere Erhebung der erektilen Funktion bei Männern mittleren Alters

R. D. Hübner1, F. Kirchhoff1, M. Kron2, S. Schiele1, H. Schulwitz1, V. H. Meissner1, M. Jahnen1, C. Peter1, J. E. Gschwend1, K. Herkommer1

1Klinik und Poliklinik für Urologie, Klinikum rechts der Isar der Technischen Universität München, München; 2Institut für Epidemiologie und Medizinische Biometrie, Universität Ulm, Ulm, Deutschland

Einleitung Die Erectile Function Domain des International Index of Erectile Function (IIEF-6) ist ein etablierter Fragebogen mit sechs Items zur Diagnostik der erektilen Dysfunktion (ED). Da sich jedoch drei Items auf den Geschlechtsverkehr (GV) in den letzten vier Wochen beziehen, werden Männer, die keinen GV hatten, nicht adäquat bewertet. Vickers et al. publizierten daher 2020 eine modifizierte Berechnung, die an Patienten nach radikaler Prostatektomie evaluiert wurde. Ziel unserer Arbeit ist es, diese Modifikation des IIEF-6 an einem bevölkerungsbasierten Kollektiv von Männern mittleren Alters zu untersuchen.

Methoden Im Rahmen der Bavarian Men‘s Health Study wurde die erektile Funktion (EF) mittels IIEF-6 und Erection Hardness Score (EHS) bei Männern im Alter von 50 Jahren zwischen 2020 und 2023 erhoben. Bei nicht stattgehabtem GV wurden zusätzlich die Gründe erfragt und eine Einteilung in drei Gruppen vorgenommen: (I) Männer mit GV, (II) Männer ohne GV aus persönlichem Grund (z. B. selbst kein Interesse), (III) Männer ohne GV aus externem Grund (z. B. kein passender Partner). In Gruppe III wurde der IIEF-6 modifiziert berechnet (doppelte Gewichtung der Fragen ohne GV-Bezug; Items 1, 5 und 15 des ursprünglichen IIEF-15).

Ergebnisse Im Gesamtkollektiv (n = 4924 Männer, Durchschnittsalter: 50,6 ± 0,7 Jahre) betrug die Prävalenz der ED 33,5 % bei klassischer Berechnung. Keinen GV in den letzten vier Wochen gaben 15,1 % an, hiervon fast 3/4 (74,2 %) aus einem externen Grund (Gruppe III). Durch die modifizierte Berechnung sank die Prävalenz der ED in Gruppe III von 100,0 % auf 44,3 %. Die Übereinstimmung der ED-Schweregrade zwischen IIEF-6 und EHS wurde von 2,7 % auf 73,4 % verbessert (vgl. Gruppe I: 97,2 %). Die Prävalenz der ED im Gesamtkollektiv sank durch die Modifikation auf 27,2 %.

Schlussfolgerung Eine adäquatere Bewertung der EF aller Männer kann durch die Anwendung der modifizierten Version des IIEF-6 erreicht werden.

Freie Vorträge zu aktuellen Themen – Grundlagenforschung

The epididymis: shaping the health of the next generation

D. A. Skerrett-Byrne1, N. A. Trigg2, J. E. Schjeken2, J. H. Martin2, S. M. Aljabali1, A. Tomar1, R. Teperino1, B. Nixon2

1Institute of Experimental Genetics, Helmholtz Zentrum München, München, Germany; 2Infertility and Reproduction Research Program, The University of Newcastle, Newcastle, Australia

Introduction The epididymis has long been of interest owing to its role in the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue, responsible for endowing sperm with important epi­genetic signatures (e.g. sncRNAs), that are ­delivered to the oocyte at conception and thereafter influence the lifetime health of offspring. This provides a mechanism by which our environment and lifestyle choices can rapidly alter the sperm epigenome and in turn, the phenotypic characteristics of the next generation. However, the molecular mechanisms by which the epididymis responds to paternal exposures remain in­adequately understood.

Methods To bridge this knowledge gap, we developed several stress models to study epididymal contributions to the sperm epigenome, tracing its impact through embryo development and placental function, utilising advanced multiomic approaches.

Results In one such model, whole-body hyperthermic stress in mice, designed to emulate a relatively modest heatwave event can elicit a rapid and marked effect on the sperm sncRNA landscape without compromising DNA integrity or sperm function. Accordingly, heat-stressed spermatozoa retained the ability to sire embryos, but instigated an aberrant program of embryonic gene expression, accelerating early embryo development. Thereafter, we documented significant changes in placental architecture and an attendant increase in embryo growth in fetuses sired by heat exposed males. Notably, increased fetal weight was linked to a significant increase in placental labyrinth zone area; the site of nutrient exchange between the maternal and fetal blood circulations.

Conclusion Our findings provide compelling evidence that the epididymis rapidly responds to paternal stressors, shaping the sperm epigenome and influencing embryo development and placental physiology, providing the impetus for continued investigation into the functional importance of epididymis.

Inherited defects of piRNA bio­genesis cause transposon de-repression, impaired spermatogenesis, and human male infertility

B. Stallmeyer1, C. Bühlmann1, R. Stakaitis2,3, A.-K. Dicke1, A. Zoch4,5, D. MacKenzie MacLeod4,5, J. Steingröver1, M. J. Wyrwoll1,4, S. Kliesch3 , D. F. Conrad2, D. O’Carroll4,5, F. Tüttelmann1

1Centre of Medical Genetics, Institute of Reproductive Genetics, University of Münster, Germany; 2Division of Genetics, Oregon National Primate Research Center, Oregon Health & Science University, Portland, USA; 3Laboratory of Molecular Neurooncology, Neuroscience Institute, Lithuanian University of Health Sciences, Kaunas, Lithuania; 4Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, UK; 5Wellcome Centre for Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh, UK

Introduction PIWI-interacting RNAs (piRNAs) represent the most abundant small non-coding RNAs in the mammalian testis. They are essential for silencing transposable elements and regulating gene expression. Biogenesis of piRNAs is a highly conserved and complex process. In mice, more than 20 different genes are involved and the knockout of any of these genes leads to male infertility. In contrast, for most of the human piRNA path­way genes, their contribution to the pathogenesis of spermatogenic failure is unknown.

Methods We screened exome/genome sequencing data of > 2,400 infertile men from the Male Reproductive Genomics (MERGE) cohort, mostly affected by azoospermia, for biallelic, rare (MAF < 0.01, gnomAD), high-impact variants (loss-of-function, missense with CADD > 15) in 24 genes of the piRNA pathway. The testicular phenotype of affected men and protein expression profile was determined. The amount of pachytene piRNAs was analysed by small RNA-seq and subsequent alignment to human pachytene piRNA loci. Immunohistochemical staining for the transposon activity marker (LINE1_ORFp1) was performed to analyse the impact on transposon silencing.

Results We identified 31 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4 as novel candidate genes for human spermatogenic failure. In some affected men, the testicular phenotypes differed from those of the respective knockout mice and ranged from complete germ cell loss to the production of a few morphologically abnormal sperm. A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers, demonstrating impaired piRNA biogenesis. Furthermore, LINE1 expression in spermatogonia linked impaired piRNA biogenesis to transposon de-silencing.

Conclusions These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insight into transposon silencing in human male germ cell.

Hypospermatogenesis in mice ­lacking iron regulatory protein 1

A. Harrer1,2, N. Ghatpande3, T. Grimaldini1,2, D. Fietz2,4,
V. Kumar1,2, C. Pleuger1,2, M. Fijak1,2, D. Föppl1,2, L. Rynio1,2, H.-C. Schuppe2,5, A. Pilatz2,5, M. Bartkuhn6, T. P. Kowalski6, N. Guttmann-Raviv3, S. Bhushan1,2, E. G. Meyron-Holtz3, A. Meinhardt1,2

1Institute of Anatomy and Cell Biology, Unit of Reproductive Biology, Justus-Liebig-University of Giessen, Giessen, ­Germany; 2Hessian Centre of Reproductive Medicine, Justus-Liebig-University of Giessen, Giessen, Germany; 3Faculty of Biotechnology and Food Engineering, Technion-Israel, Institute of Technology, Technion City, Haifa; Israel; 4Institute for Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University of Giessen, Giessen; Germany; 5­Department of Urology, Pediatric Urology and Andrology, Justus-Liebig-University of Giessen, Giessen, Germany; 6­Biomedical Informatics and Systems Medicine, Science Unit for Basic and Clinical Medicine, Justus-Liebig-University of Giessen, Giessen, Germany

Introduction Imbalances in testicular iron levels are linked to compromised sperm production and male infertility. Iron regulatory proteins (IRP) 1 and 2 play crucial roles in cellular iron regulation. IRPs are expressed in human testicular tissue, but the exact mechanisms of iron regulation and the roles of IRP1 and 2 in this process are not yet fully elucidated in these organs.

Methods We investigated the role of IRP1 on spermatogenesis using Irp1-deficient mice (Irp1-/-). To identify impaired spermato­genesis, we performed hematoxylin and eosin (H&E) staining, immunofluorescence staining using germ cell-specific (e.g. SCP3) and reactive oxygen species (ROS)-induced DNA damage-specific (8-OxoG) antibodies, and conducted deeper analysis with flow cyto­metry and bulk RNA-seq.

Results Histological analysis of the testis of Irp1-/- mice revealed significant reduction in the number of elongated spermatids and daily sperm production compared to wild-type (WT) mice. Flow cytometry of germ cells from WT and Irp1-/- mice showed reduction in spermatocytes, round and elongated spermatids, which was confirmed by histological and immunofluorescence quantification. ­Finally, stage VIII of spermatogenesis, crucial for spermatid maturation, was less frequent in Irp1-/- testicular cross-sections. This observation worsened with age despite unchanged intratesticular iron levels. Mechanistically, this was due to increased oxidative stress indicated by elevated 8-Oxoguanine (8-OxoG) levels, a DNA lesion resulting from ROS. ­Furthermore, bulk RNA-seq data indicated compromised DNA damage repair and cell cycle processes, including mitosis and ­meiosis, which may explain hypospermatogenesis.

Conclusions Our results suggest that IRP1 deletion leads to hypospermatogenesis due to impaired cell cycle progression, decreased DNA damage repair capacity, and increased oxidative damage. Overall, this study reveals a new role for IRP1 beyond its traditional function in iron regulation.

Exploring the influence of galectin-3 on testicular inflammation and fibrosis

H. Hasan1, E. Wahle1, T. Procida-Kowalski2, H.-C. Schuppe3, D. Fietz4, M. Bartkuhn2, A. Meinhardt1, M. Fijak1

1Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, Giessen; 2Biomedical Informatics and Systems Medicine Science Unit for Basic and Clinical Medicine Giessen, Justus-Liebig-University of Giessen, Giessen; 3Department of Urology, Pediatric Urology and Andrology, Justus-Liebig-University of Giessen, Giessen; 4Department of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University of Giessen, Giessen, Germany

Introduction Galectin-3 (Gal-3) is a ?-galactoside binding lectin that is widely distributed across various cells and tissues. It plays a crucial role in numerous biological activities, including cell migration, cell growth, angiogenesis, fibrosis, inflammation as well as regulation of spermatogenesis. However, the role of Gal-3 in testicular inflammation and fibrosis remains unclear.

Methods Immunofluorescence was used to identify the cells expressing Gal-3 in the mouse testis. To investigate the role of Gal- 3 in chronic testicular inflammation and fibrosis, Experimental autoimmune orchitis (EAO) was induced in C57/BL6J (WT) and Gal-3 deficient (Lgals3-/-) mice. Bulk RNA sequencing was employed to perform the transcriptomic analysis in Lgals3-/- and WT EAEO testes. Various immune cells were examined in both genotypes using immuno­fluorescence. Additionally, Gal-3 expression was investigated in testicular biopsies from azoospermic patients with impaired spermatogenesis and focal leukocytic infiltration by immunofluorescence.

Results In mouse testis, macrophages were identified as the primary cells expressing Gal-3. These Gal-3 expressing macrophages were increased in number and located in the interstitial space, but also, unexpectedly, within the seminiferous tubules of inflamed testes of both mouse and human tissue sections. Notably, the absence of Gal-3 exacerbates testicular inflammation and fibrosis as evidenced by increased numbers of macrophages and neutro­phils in the testis of Gal-3 deficient mice. Furthermore, collagen deposition was elevated in Lgals3-/- vs WT EAEO testes. These data are supported by bulk RNA sequencing data, which show an upregulation of genes associated with inflammatory responses, leukocyte activation, attraction of immune cells and fibrosis in Lgals3-/- vs WT EAEO testes.

Conclusion The absence of Gal-3 exacerbates testicular inflammation and fibrosis, indicating its potential immunoregulatory function in the testis.

Poster

Hypoxia culture of testicular tissue from 10-year-old sickle cell disease patient

H. I. Hamurcu1, R. Biemann2, U. Ceglarek2, B. Holstermann3, B. Brunne3, A. Soave4, A. Salzbrunn1, B. Braunger3, S. W. Schneider1, K. von Kopylow1

1Clinic and Polyclinic for Dermatology and Venerology, ­Andrological Section, University Medical Center Hamburg-Eppendorf, Hamburg; 2Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig, Leipzig; 3Institute of Neuroanatomy, University Medical Center Hamburg-Eppendorf, Hamburg; 4Department of Urology, University Medical Center Hamburg-­Eppendorf, Hamburg, Germany

Introduction Cryopreservation of sperm prior to gonadotoxic therapy to fulfill a later wish for a biological child is no option for prepubertal boys as no spermatids are produced before puberty. The only possibility here is cryopreservation of testis tissue containing spermatogonial stem cells (SSCs). To obtain sufficient SSC numbers, these must be multiplied in vitro. Studies indicate SSCs are more likely to grow under hypoxia than atmospheric O? concentration. SSCs transit from mitochondrial (MT) respiration to anaerobic glycolysis during testicular development, accompanied by a change in the MT size and shape. A recent paper showed 10–15% O? more favorable for culturing in general [Ogawa et al. 2024].

Methods Testis tissue of a 10-year-old sickle cell disease (SCD) patient was cultured under 10% O? (HC) vs. normoxic conditions (NC) for 2 weeks. Glycolytic activity was assessed by measuring lactate in the medium with ELISA. Leydig cell functionality was evaluated via analyzing the steroid hormone profile using LC-MS/MS. SSC and total germ cell numbers in cultured and uncultured tissue were determined by IF. TEM was used to study the MT phenotype of undifferentated SPG in uncultured tissue.

Results MT were predominantly of late prepubertal stage. In HC, lactate production doubled from day 9 compared to NC. Glycolytic activation of the SSC metabolism under HC was reflected in higher SSC numbers compared to NC. Both conditions showed comparable germ cell counts. Leydig cells were competent for testosterone (T) and precursor hormone production with slightly higher T and 17-OHP levels in NC, but twice as high progesterone in HC from day 9. Strong Spearman correlation was revealed between lactate, T, progesterone and 17-OHP in most cases.

Conclusion Data show the feasibility of organ culture using testis tissue from a prepubertal SCD patient. Results are promising for the benefit of hypoxic conditions in testicular organotypic culture.

Grants: Funded by Deutsche Krebshilfe

Wertigkeit eines erhöhten Testosteronspiegels

B. Schwindl, D. Magaryan, T. Klotz

Urologie, Kliniken Nordoberpfalz AG, Weiden, Deutschland

Einleitung Der erhöhte T-Spiegel beim erwachsenen Mann ist im Gegensatz zur Frau ein wenig beachtetes Thema.

Methode Internetrecherche (nur wenige Publikationen), eigenes Patientenklientel. Einbezogen werden Patienten mit einem morgendlichen Serum-Testosteron-Spiegel > 1000 ng/dl (bzw. 35 nmol/l).

Unterscheide:

1. Biosynthese (Hemmung der Rückkoppelung, Antiöstrogene, [minor-] AGS, T-produzierender Tumor)

2. Stoffwechsel (Aromatase-Hemmer, 5-Alpha-Reduktase-Typ2-Defekt)

3. Wechselwirkung am Androgenrezeptor (MAIS, CAG-Repeats)

4. Zufuhr (iatrogen, Doping, Androgene, Östrogene)

Für die klinische Praxis empfiehlt sich ein diagnostischer Flow-Chart. Anamnese (Medikamente, Anabolika), Standard-Labor (Zweitbestimmung, SHBG, LH, FAI). Hämatokrit, beim älteren Mann PSA. Erweitertes Labor: 17-OH-Progesteron, Cortisol, DHT, DHEA. Tumormarker (ß-HCG), Funktionstests (ACTH-Test). Sono: Tumor-Ausschluss. TART? Ggf. MRT-Hypophyse, CT-nativ Nebenniere, ggf. KM-CT bei V.a. Malignom; Genetik.

Therapie Abhängig von Ursache (Cortison? Antiandrogene? CAVE: Risiken), kardiovaskuläres Risiko? Thrombose-Risiko? Symptomatisch (z. B. Aderlass), 3-monatliche Verlaufskontrolle (idiopathisch).

Ergebnis Er ist selten, zumeist maskiert (u. a. fette Haut, Akne, Fertilitätsstörung?). Tumor-Ausschluss vorrangig.

Auswirkungen der Realisierung/Nichtrealisierung eines unerfüllten Kinderwunsches auf das sexuelle Selbstbild von Männern mittleren Alters

V. C. Schlöder1, L. J. Schmalbrock1, F.-M. Köhn2, S. Schiele1, H. Schulwitz1, A. Dinkel3, M. Kron4, J. E. Gschwend1, K. Herkommer1

1Klinik und Poliklinik für Urologie, School of Medicine and Health, Klinikum rechts der Isar der Technischen Universität München, München; 2Andrologicum München, München; 3Klinik und Poliklinik für Psychosomatische Medizin und Psychotherapie, School of Medicine and Health, Klinikum rechts der Isar der Technischen Universität München, München; 4Institut für Epidemiologie und Medizinische ­Biometrie, Universität Ulm, Ulm, Deutschland

Einleitung In Deutschland war im Jahr 2020 fast jedes zehnte Paar ungewollt kinderlos. Die mit einem unerfüllten Kinderwunsch einher­gehenden Langzeitfolgen für das sexuelle Selbstbild der männlichen Partner sind trotz hoher Betroffenenzahlen unzureichend erforscht. Ziel der Analyse war es zu untersuchen, ob sich das sexuelle Selbstbild bei Männern mittleren Alters mit und ohne Realisierung eines unerfüllten Kinderwunsches unterscheidet.

Methoden Die Daten wurden im Rahmen der Bavarian Men’s Health-Study mittels Fragebögen und in einem Arztgespräch erhoben. Das Vorliegen eines unerfüllten Kinderwunsches wurde erfragt und es wurden soziodemografische und psychologische Parameter sowie Parameter bezüglich des Lebensstils, des Gesundheitszustandes und des Sexuallebens erfasst. Vier Facetten des sexuellen Selbstbildes wurden erhoben: Körperbild, Verständnis von Maskulinität/Toughness, sexuelles Selbstwertgefühl und wahrgenommener Druck in Bezug auf die sexuelle Leistungsfähigkeit. Die Facetten wurden auf einer Likert-Skala bewertet. Zur Kontrolle von Störfaktoren wurde jeweils ein 1:1-­Matching mittels Propensity-Score durchgeführt und die Unterschiede in den Facetten des sexuellen Selbstbildes wurden mit Wilcoxon-Mann-Whitney-Test verglichen.

Ergebnisse In die Analyse wurden 1097 Männer mit unerfülltem Kinderwunsch eingeschlossen (Durchschnittsalter: 50,6 (± 0,7) Jahre). Das Alter zu Beginn des Kinderwunsches lag im Durchschnitt bei 35,9 (± 6,1) Jahren. Bei 65,9 % der Männer konnte der zuvor unerfüllte Kinderwunsch realisiert werden. Es zeigte sich kein signifikanter Unterschied im sexuellen Selbstbild zwischen Männern, deren Kinderwunsch realisiert werden konnte und jenen, bei denen dies nicht der Fall war (alle p > 0,05).

Schlussfolgerung Unsere Ergebnisse legen nahe, dass die Nichtrealisierung eines un­erfüllten Kinderwunsches bei Männern mittelfristig keine Beeinträchtigung des sexuellen Selbstbildes im Vergleich zu Männern mit realisiertem Kinderwunsch zur Folge hat.

Diagnosis and management of male hypogonadism in Germany

M. Zitzmann1, D. Ortius-Lechner2, N. Thierfelder2, C. Schindler3, D. Häckl4

1Centrum für klinische und operative Andrologie, Universitätsklinik Münster, Münster; 2Besins Healthcare GmbH, Berlin; 3WIG2 GmbH, Leipzig; 4Health Econocomis and ­Management, Fakultät Wirtschaftswissenschaften, Universität Leipzig, Leipzig, Germany

Background The extent to which male hypogonadism is diagnosed and treated remains ambiguous. This study scrutinizes the respective medical framework in Germany.

Method This observational study retrospectively analyzes data from 4 million male state insurance clients, aged 30 to 80, from 2014 to 2021. It evaluates hypogonadism prevalence, incidence, comorbidities, testosterone therapy (TTh) implementation, type 2 diabetes incidence, and work incapacity rates.

Results The prevalence of primary hypogonadism (0.62%) exceeded that of secondary (0.16%) and functional hypogonadism (0.09%), with both incidence and prevalence rising from 2015 to 2021. Common comorbidities included primary hypertension, back pain, and dyslipidemia. Within the first-year post-diagnosis, TTh (via transdermal or intramuscular administration) was initiated in 35.22%, 36.60%, and 59.55% of patients with primary, secondary, and functional hypo­gonadism, respectively. Untreated primary hypogonadism patients had higher work ­incapacity rates compared to treated patients (17.1% vs 9.71%; p < 0.001). Conversely, treated patients had a higher incidence of newly diagnosed type 2 diabetes (24.0% vs 17.7%; p < 0.001).

Conclusion Optimizing the diagnosis, treatment, and monitoring of male hypogonadism in clinical practice could enhance the quality of life for affected individuals and reduce societal costs.

Negativeres sexuelles Selbstbild bei 50-jährigen Männern mit sexuellen Funktionsstörungen

V. A. Söhne1, V. H. Meissner1, S. Schiele1, A. Dinkel1, M. Jahnen1, M. Kron2, J.E. Gschwend1, K. Herkommer1

1School of Medicine and Health Klinik und Poliklinik für Urologie, Klinikum rechts der Isar der Technischen Universität München, München; 2Institut für Epidemiologie und Medizinische Biometrie, Universität Ulm, Ulm, Deutschland

Einleitung Das sexuelle Selbstbild ist ein multidimensionales Konstrukt, welches die Selbstwahrnehmung einer Person in Bezug auf Sexualität darstellt. Ziel dieser Studie war die Untersuchung des sexuellen Selbstbildes bei Männern mittleren Alters mit erektiler Dysfunktion (ED), lebenslang/erworbener Ejaculatio praecox (l/e EP), subjektiver/variabler EP (s/v EP) und reduzierter Libido (RL).

Methoden 50-jährige Männer aus einer bevölkerungsbezogenen Zufallsstichprobe füllten validierte Fragebögen zu sexuellen Funktionsstörungen und zum sexuellen Selbstbild aus, das 4 Aspekte umfasste: Körperbild (3 Items des Dresden-Body-Image-In­ventory Questionnaires), Maskulinität/­Toughness (3 Items der Male-Role-Norm-Scale), ­sexuelles Selbstwertgefühl (3 neue Items) und wahrgenommener Druck in Bezug auf die sexuelle Leistungsfähigkeit (4 neue Items). Untergruppen wurden mittels Wilcoxon- und Kruskal-Wallis-Tests verglichen. Eine ­multi­ple lineare Regression wurde für jeden Aspekt des sexuellen Selbstbildes durchgeführt.

Ergebnisse 20,1 % der Männer (n = 5665) hatten eine ED, 6,0 % berichteten von l/e EP, 10,1 % von s/v EP und 7,1 % von RL. In der multiplen linearen Regression war ein negativeres Körperbild mit einer ED, EP und RL assoziiert. Ein geringeres sexuelles Selbstwertgefühl war mit einer ED, EP und RL assoziiert. Ein höherer wahrgenommener Druck war ebenfalls mit ED, EP und RL assoziiert. Ein eher traditionelles Verständnis von Maskulinität/Toughness war mit ED und EP assoziiert, ein Zusammenhang mit RL konnte nicht gezeigt werden (alle p < 0,05).

Schlussfolgerung Männer mit mindestens einer sexuellen Funktionsstörung zeigten ein negativeres sexuelles Selbstbild. Dies unterstreicht, dass sexuelle Funktionsstörungen möglichst frühzeitig diagnostiziert werden sollten und ggf. eine medizinische bzw. psychische Unterstützung angeboten werden sollte.

Ejaculatio praecox und damit assoziierte Faktoren bei Männern mittleren Alters: Ergebnisse der Bavarian Men’s Health- (BMH-) Study

A. S. Maier1, V. A. Söhne1, V. Henri Meissner1, S. Schiele1, L. J. Schmalbrock1, A. Dinkel1, M. Jahnen1, M. Kron2, J. E. Gschwend1, K. Herkommer1

1School of Medicine and Health, Klinik und Poliklinik für Urologie, Klinikum rechts der Isar der Technischen Universität München, München; 2Institut für Epidemiologie und Medizinische Biometrie, Universität Ulm, Ulm, Deutschland

Einleitung Ziel dieser Studie war es, die Prävalenz sowie assoziierte Faktoren der lebenslangen/erworbenen (l/e) und subjektiven/variablen (s/v) Ejaculatio praecox (EP) zu bestimmen, einschließlich des sexuellen Selbstbildes der Betroffenen.

Methoden Im Rahmen der BMH-Study wurden soziodemographische, lebensstil­bezogene, gesundheitliche und psychologische Parameter, das Sexualverhalten, vier Aspekte des sexuellen Selbstbildes (Körperbild, Verständnis von Maskulinität/Toughness, sexuelles Selbstwertgefühl und wahrgenommener Druck bezüglich sexueller Leistungsfähigkeit) sowie erektile Dysfunktion (ED), l/e und s/v EP und reduzierte Libido (RL) erhoben. Eingeschlossen wurden sexuell aktive Männer, die die Fragen zur EP beantwortet haben.

Ergebnisse 4838 Männer mit einem Durchschnittsalter von 50,6 (± 0,8) Jahren wurden eingeschlossen. 91,6 % lebten in einer Partnerschaft. Zum Zeitpunkt der Befragung gaben 6,0 % l/e EP, 10,1 % s/v EP, 17,2 % ED und 4,8 % RL an. Sowohl l/e als auch s/v EP waren mit moderaten/schweren Symptomen des unteren Harntrakts (IPSS > 7), schlechter subjektiver Gesundheit, positivem Screening auf Depression und Angst, geringer sexueller partnerschaftlicher Aktivität, seltener Solo-Masturbation, sexueller Unzufriedenheit, ED und RL assoziiert (alle p < 0,02). Sowohl Männer mit l/e als auch s/v EP hatten ein negativeres Körperbild, ein traditionelleres Verständnis von Maskulinität in Bezug auf den Aspekt „Toughness“, ein geringeres sexuelles Selbstwertgefühl und einen höheren wahrgenommenen Druck im Vergleich zu Männern ohne EP (alle p < 0,001).

Schlussfolgerung Männer mit s/v bzw. l/e EP zeigten ähnliche assoziierte Faktoren. In beiden Gruppen war die EP mit ED, RL, schlechter subjektiver Gesundheit, sexueller Unzufriedenheit und einem negativeren ­sexuellen Selbstbild assoziiert. Dies lässt eine vergleichbare psychische Belastung vermuten und unterstreicht, dass die EP frühzeitig diagnostiziert, darüber aufgeklärt und ggf. psychische bzw. medizinische Unterstützung begonnen werden sollte.

Inter- und intraindividuelle ­Schwankungen des Metaboloms des humanen Seminalplasmas

J. Blaurock1, K. Engel2, S. Grunewald1

1Leipzig Reproductive Health Research Center (LE-REP), Klinik für Dermatologie, Venerologie und Allergologie, Abteilung Andrologie, Universitätsklinikum Leipzig, Leipzig; 2Medizinische Fakultät, Institut für medizinische Physik und Biophysik, Universität Leipzig, Leipzig, Deutschland

Einleitung Metabolom-Untersuchungen er­lauben weit über das Spermiogramm hinaus Einblicke in die molekulare Zusammensetzung des Seminalplasmas. Studien versuchen mit dieser Technik ein besseres Verständnis für Fertilitätsstörungen des Mannes zu erreichen und spezifische Marker zu detektieren. Die inter- und intraindividuelle Variabilität einzelner Parameter ist bereits im konventionellen Spermiogramm nicht unerheblich. Wie ausgeprägt solche Schwankungen im Metabolom des Seminalplasmas sind, ist weitgehend unbekannt und war daher Gegenstand dieser Untersuchung.

Methoden Im Abstand von durchschnittlich 8 Wochen erfolgte in Ejakulaten 15 gesunder Spender eine vergleichende Metabolomanalytik des Seminalplasmas (Biocrates AbsoluteIDQ p180-Kit). Neben den Intraklassen-Korrelationskoeffizienten (ICC), die ein Maß für die Zuverlässigkeit darstellen, wurden für jeden Metaboliten intraindividuelle und interindividuelle Variationskoeffi­zienten berechnet.

Ergebnisse 75 % der durch gezielte LC-MS/MS gemessenen Metaboliten des Seminalplasmas waren bei zwei Spenden über acht Wochen zuverlässig nachweisbar. Signifikante intraindividuelle Schwankungen wiesen 18 Metaboliten, hauptsächlich Acylcarnitine und biogene Amine auf. Die Variabilität war höher, wenn auch die Spermienkonzentration signifikante Veränderungen aufwies. Biogene Amine waren die im Zeitverlauf am wenigsten zuverlässigen Analyten, während z. B. Sphingomyeline im Seminalplasma stabil nachweisbar waren.

Interindividuelle Unterschiede ließen sich vor allem bei den ethergebundenen Glycerophosphatidylcholinen finden, bei den Aminosäuren waren sie am niedrigsten.

Schlussfolgerung Für die überwiegende Zahl der Metaboliten ist die Messung einer einzigen Probe ausreichend. Stark variable Metaboliten sollten jedoch mit Vorsicht interpretiert werden. Wir empfehlen eine stratifizierte Analyse nach Spermienkonzentration, um einen möglichen Einfluss unterschiedlicher Spermakonzentrationen auf die Metabolitenwerte zu vermeiden.

Effect of genetic variants of the pre-DSB recombinosome on spermatogenesis and male infertility

L. Bosse1, S. Viville2, Ö. Okutman3, F.-M. Köhn4, S. Kliesch5, F. Tüttelmann6, C. Friedrich7

1Institut für Reproduktionsgenetik, Universität Münster, Münster, Germany; 2Laboratoire de Diagnostic Génétique, Unité de Génétique de l’infertilité (UF3472), Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 3Hôpital Erasme Service de Gynécologie-Obstetrique, Clinique de Fertilité, Hôpital Universitaire de Bruxelles, Université libre de ­Bruxelles, Bruxelles, Belgique; 4Andrologicum München, München, Germany; 5Centrum für Reproduktionsmedizin und Andrologie, Universitätsklinikum Münster, Münster; Germany; 6Centrum für klinische Genetik, Universitätsklinikum Münster, Münster, Germany; 7Institut für Reproduktionsgenetik, Universität Münster, Münster, Germany

Introduction Non-obstructive azoospermia has been linked to variants in genes involved in spermatogenesis. An essential aspect of spermatogenesis is meiosis. During this phase of germ cell maturation, a complex called the pre-DSB (double-strand break) recombinosome consisting of the proteins ANKRD31, IHO1, MEI1, MEI4, and REC114 is important for meiotic recombination. We hypothesise that genetic variants in all of the encoding genes lead to meiotic arrest and spermatogenic failure, thereby causing male infertility.

Methods To test our hypothesis, we screened the exome/genome data of the Male Reproductive Genomics (MERGE) cohort comprising > 2,700 datasets for rare (< 1%, gnomAD) coding variants in ANKRD31, IHO1, MEI1, MEI4, and REC114. Variants were validated using Sanger sequencing. Periodic acid–Schiff (PAS) staining, immuno­histochemistry (IHC), and splice assays were conducted to evaluate the impact of the genetic variants.

Results Within the MERGE cohort, we identified 12 patients with variants in the pre-DSB recombinosome genes, of which 3 men were heterozygous and 9 men had potentially biallelic variants including the first homozygous frameshift variant in IHO1.

PAS staining revealed different stages of ­meiotic arrest for all 5 cases investigated. IHC staining (H2AX, CREM) showed aberrant meiotic progression with no or few CREM-positive round spermatids.

The splice assay of the novel REC114 variant c.160G > A showed an effect on mRNA splicing resulting in an inframe deletion of 29 amino acids affecting the functional domain.

Conclusion Our data strengthens the clinical evidence for variants in genes of the pre-DSB recombinosome to have an impact on meiotic progression and, thus, lead to spermatogenic failure and male infertility.

Grants: This work was supported by the DFG Clinical Research Unit 326 “Male Germ Cells” (CRU326). LB was supported by the Medizinerkolleg (MedK) of the Medical Faculty, University of Münster.

G-quadruplexes (G4s): A new potential target for the treatment of testicular germ cell tumors?

R. Islam1, A. Hansen1, S. R. Roque2, A. Liesen1, K. Paeschke2, H. Schorle1

1Department of Developmental Pathology, Institute of ­Pathology, University Hospital Bonn, Bonn; 2Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany

Introduction G-quadruplexes (G4s) are four-stranded secondary structures of DNA that form in guanine-rich sequences in the genome and are critically involved in the development and progression of various cancers. Studies revealed that G4 ligand-assisted formation and/or stabilization of G4 could be an effective therapeutic target for breast-, lung- and pancreatic cancer. However, the role of G4s in testicular germ cell tumors (TGCTs) has not yet been studied. Here, we investigate the role of G4 structures in TGCTs to emphasize whether targeting G4s could be an effective target for the treatment of TGCTs.

Material and Methods The presence of G4s and the effect of G4 ligands on the formation/stabilization of G4s in different TGCT cells were analyzed by BG4-immunofluorescence and BG-flow cytometry. Subsequently, XTT cell viability assay and apoptosis assay were performed to evaluate the anti-tumor effects of different G4 ligands on different TGCT cells. Next, RNA-seq will be used to identify the molecular cascades triggered by G4 ­ligand-mediated stabilization of G4s. Furthermore, G4 mapping using CUT&Tag will be performed to explore the structure-function relationship of G4s in TGCT cells.

Results G4 structures were identified in TGCT cells, and their formation/stabilization was influenced by different G4 ligands. Subsequently, XTT cell viability assay revealed a significant dose- and time-dependent cytotoxic effect of different G4 ligands on different TGCT cells. Further experiments confirmed that G4 ligands induce apoptosis of different TGCT cells to different extents.

Conclusion Primary data confirmed that G4s are present in TGCT cells and targeting them using different ligands exhibited promising anti-tumor effects. However, further experiments are still needed to confirm the underlying mechanisms in order to understand the role of G4 structures in TCGTs which will further shed light on the efficacy of G4 ligands for TGCT treatment.

Extracellular matrix plays crucial ­roles during testicular tubulogenesis

S. Schlatt1, F. Schlag1, F. Schmalbein2, J. A. Eble2

1Institute for Reproductive and Regenerative Biology, Centre for Reproductive Medicine and Andrology at the University Münster, Münster; 2Institute of Physiological Chemistry and Pathobiochemistry of the University Münster, Münster, ­Germany

The extracellular matrix (ECM) is involved in Sertoli cell (SC) aggregation, cord formation and later germ cell homing and initiation of spermatogenesis. Here we test novel ex vivo cell culture models to generate specific topographic and biophysical features. 2D and 3D cultures are setup using primary rat testicular cells (d7pp) and mouse Sertoli cell line (SK11) as starting material. These cells are exposed to various ECM-conditions. Specific proteins are synthesized and coated on plastic, on collagen-I gels or on acrylamide/acrylic acid polymers.

We record up to ­several weeks spontaneous self-organization of Sertoli cell aggregates and establishment of cord-like structures. Addition of hyaluronic acid, testosterone or FSH as well as co-culture with myofibroblasts and germ cells occurred to create different micro­environments. In addition to histological changes, qRT-PCR revealed that expression of Sox9 was highest in primary rat Sertoli cells when cultured on laminin-211 and collagen-IV. Therefore, we coated these ECM components onto the surface of soft collagen-I gels. We produced and tested biofunctionalized cell-inert acrylic soft gels coated with a type IV-collagen fragment that harbors binding sites for integrins alpha1/2-beta1. These integrins are abundantly expressed on SK11 cells. We therefore cultured SK11 cells on collagen IV-doped acrylic soft gels. In these conditions, the cell line formed loop-like cords with a micro­porous lumen. SK11 stabilized the cords by expression of N-cadherin also enabling homo­typic cell-cell contacts, a prerequisite for the later tight junction formation to establish the blood-testis-barrier.

We conclude that synthesis of recombinant mini-laminin-211 and mini-collagens and their covalent immobilization to soft acrylic gels enables testing of ECM components during testicular cord formation. Our novel culture systems allow creation of testicular organoids in biochemically and biophysically defined ECM environments.

BRD9 inhibition as potential treatment option for testicular germ cell tumors

A. Hansen, K. Funke, L. Arévalo, F. Fronhoffs, C. Sanders, G. Kristiansen, H. Schorle

Abteilung für Entwicklungspathologie, Institut für Patho­logie, Uniklinikum Bonn, Bonn, Deutschland

Introduction Testicular germ cell tumors (TGCT) represent the most common tumor in young men. While curation rates are high, 15–20% of patients with metastatic non-semi­nomas develop resistance to cisplatin-based chemotherapy. In prostate cancer and breast cancer interfering with the epigenetic landscape by inhibiting BET proteins was already shown to be effective. The bromodomain-containing protein 9 (BRD9) is an epigenetic reader and part of a chromatin remodeling complex modulating gene expression by recruiting transcription factors.

Methods BRD9 expression in TGCT cell lines and tissues was determined by meta-analysis of microarray data and on protein level by Western Blot and by tissue micro­array. XTT-assay was performed to evaluate the impact of I-BRD9 treatment on viability of cells. Cell cycle arrest as well as apoptosis rate were analyzed by FACS. 3’mRNA-sequencing was performed after I-BRD9 treatment to determine differentially expressed genes caused by BRD9 inhibtion.

Results We tested the effect of the BRD9 inhibitor I-BRD9 on TGCTs. In TGCT cell lines as well as tissues BRD9 expression was heterogenous. Nevertheless, BRD9 inhibition led to strong decrease in viability of TGCT cell lines. FACS analysis revealed increased apoptosis and cell cycle arrest in G1-phase in TGCT cells treated with I-BRD9. Analysis of 3’mRNA-sequencing data showed prominent downregulation of the pluripotency network including NANOG, PRDM14 and KLF4 in TGCT cell lines already after 24 hours of I- BRD9 treatment. In addition, an upregulation of genes associated with epithelial differentiation was detected.

Conclusion I-BRD9 strongly reduces viability, initiates cell cycle arrest and apoptosis in TGCT cells while control cells remain mostly unaffected. Transcriptomic data indicate exit of pluripotency and differentiation towards the epithelial fate. The data suggest I-BRD9 as an effective treatment option for TGCTs.

Actin-related protein M1 (ArpM1) is required for correct acrosome biogenesis and sperm function in mice

E. Ordziniak1, A. Kovacevic1, S. Suvilla1, S. Ziaeipour1, N Umer1, G. E. Merges1, L. Arevalo2, H. Schorle1

1Entwicklungspathologie, Institut für Pathologie, Uni­klinikum Bonn, Bonn, Germany; 2Instituto Nacional de Investigación y Tecnología Agragria y Alimentaria Madrid, Madrid, Spain

Actin-related proteins (Arps) are a large family of proteins which share sequence similarities with normal actin and are involved in diverse cellular processes. Actin-related protein M1 (ArpM1) is testis-specific and localised in the perinuclear theca and acrosomal region of round to elongated spermatids. Finally, ArpM1 localizes in the posterior part of sperm head called calyx. In order to elucidate the role of ArpM1 in male fertility an ArpM1 deficient mouse model was generated by CRISPR/Cas9.

We observed that male ArpM1-/- mice were subfertile, with reduced litter size and sperm viability but unaltered sperm count. In vitro fertilisation (IVF) confirmed this result, with only 40% of fertilised oocytes. Morphologically abnormal acrosome structures were observed by PNA-FITC in approximately 50% of ArpM1 deficient mature sperm. Transmission Electron Microscopy (TEM) confirmed defects in acrosome morphology, showing short, incomplete and sometimes acrosomes detached from nuclear membrane. The acrosome biogenesis was analysed by TEM and immunofluorescence, showing disconnection of Golgi apparatus from nucleus in ArpM1-deficient round spermatids. To have a closer look in the molecular basis of disruption of acrosome bio­genesis in ArpM1-deficient mice, we further analysed the cis- and trans-Golgi trafficking. Immunofluorescence staining with the marker GM130 suggested the cis-Golgi network to be disturbed. The protein level of the trans-Golgi marker TGN46 was reduced as well in the ArpM1-deficient mice. Since autophagy is a cellular process directly connected to acrosomal formation, autophagic flux was next analysed. ArpM1-deficient sperm displayed a downregulation of LC3B and cathepsin B.

These results show that ArpM1 plays a significant role in acrosome biogenesis. At present possible interaction partners such as Actrt1, Actrt2 and Actl7a, are tested to be form functional complexes. Further studies are required to understand the molecular role in mice and human.

Investigating novel treatment ­options: Inhibition of Cullin 1, 4 and 5 reduces viability of testicular germ cell tumour cell lines

A. Liesen, A. Hansen, K. Funke, R. Islam, G. Kristiansen, H. Schorle

Department of Developmental Pathology, Institute of ­Pathology, University Hospital Bonn, Bonn, Germany

Introduction Testicular germ cell tumors (TGCT) are common tumors in young men. 10–30% of patients with metastatic non-seminoma, face low survival rates due to cisplatin resistance, so new treatment approaches are needed. Our lab applied a CRISPR-activation screen in TGCT cell lines and identified Neddylation to contribute to cisplatin resistance. We showed that the NAE1 inhibitor MLN4924 effectively reduced the viability of TGCT cells and re-sensitized them to cis­platin treatment.

Neddylation regulates the protein content by binding NAE1 activated NEDD8 via a three-step enzymatic cascade to a substrate and altering their stability. Such substrates are Cullins. Inhibition of selected Cullin ring ligases (CRLs) blocks only parts of the Neddylation cascade, opening the possibility of reduced side effects vs the inhibition of the complete Neddylation by blocking NAE1. A meta-ana­lysis of published expression datasets revealed all but Cullin 3 expression in TGCT tissue and cell lines.

Methods We tested the effect of Cullin inhibitors using an XTT viability assay on embryonal carcinoma (2102EP, NCCIT, NT2/D1), a choriocarcinoma (JAR), and a seminoma (TCam2) cell line.

Results Inhibition of Cullin4A & 4B by 33-11 and inhibition of Cullin1 & 5 by Gossypol displayed strongest cytotoxic effect on TGCT cells. Combination of Gossypol and cisplatin resensitized cisplatin-resistant (NCCIT-R, NT2/D1-R) cells. Non-resistant TGCT cells (2102EP, NCCIT, NT2/D1, JAR and TCam2) sensitivity to cisplatin increased when treated with Gossypol. The combination of 33-11 and cisplatin resulted in an increased decrease in viability to TGCT cell lines (2102EP, JAR and TCam2).

Conclusion Inhibition of Cullin4A & 4B by 33-1 and Cullin1 & 5 by Gossypol appears promising. It suggests that the effect using MLN4924 was primarily caused by inhibition of Cullin1, 4A, 4B & 5. Proteome analyses will reveal molecular cascades deregulated in germ cell tumor cell lines and further understanding of TGCT.

Oxidative stress induces changes in molecular markers associated with ferroptosis in human spermatozoa

P. Contreras-Mellado1, A. Bravo1, F. Zambrano1, R. Sánchez1, R. Boguen2, J. Risopatrón3, O. Merino3, P. Uribe1

1Center of Excellence in Translational Medicine – Ph.D. Program in Sciences mention Applied Cell and Molecular Bio­logy, Universidad de La Frontera, Temuco; 2Department of Diagnostic Processes and Evaluation, Universidad Católica de Temuco, Temuco; 3Center of Excellence of Biotechnology in Reproduction, Universidad de La Frontera, Temuco, California, USA

Introduction: Ferroptosis is a type of iron-dependent regulated cell death characterized by increased bioavailability of redox-active iron, loss of GPX4 antioxidant capacity, and oxidation of polyunsaturated fatty acid-containing phospholipids. Research on fer­roptosis in human spermatozoa is very limited, with evidence suggesting it as a potential emerging cause of male infertility. Nevertheless, the mechanisms underlying ferroptotic cell death in human sperm subjected to oxidative stress remain unexplored. The aim of this study was to evaluate the effect of oxidative stress induced by arachidonic acid (AA) on ferroptotic cell death in human spermatozoa.

Methods Spermatozoa from normozoospermic donors were exposed to AA (5, 25 and 50 ?M) for 1 h at 37 °C, including an untreated control. Oxidative stress was confirmed by evaluation of cytosolic and mitochondrial ROS production, viability, mitochondrial membrane potential (??m), ATP content and motility. Subsequently, molecular markers of ferroptosis including iron content, levels of GPX4, SLC7A11, ACSL4, ALOX15, IREB2 and lipid peroxidation were evaluated. The analyses were carried out using either flow cytometry, a microplate reader or confocal laser microscopy.

Results AA-induced oxidative stress showed increased cytosolic and mitochondrial ROS production accompanied by reduced viability, ??m, ATP content and motility in human spermatozoa. These results were associated with changes in ferroptotic cell death markers including an increase in ferrous (Fe2+) ions, lipid peroxidation, SLC7A11, ACSL4, ALOX15, IREB2, and a decrease in the level of GPX4 compared to the untreated control.

Conclusion This study revealed that AA-induced oxidative stress induces cell death with biochemical characteristics of ferroptosis in human spermatozoa, demonstrating another mechanism of alteration of sperm function induced by oxidative stress.

Novel protamine mouse models allow for new insights into sperm chromatin remodeling

G. E. Merges1, C. Wiesejahn1, I. Hamers1, S. Schneider1, L. Arévalo2, H. Schorle1

1Department of Developmental Pathology, University Hospital Bonn, Bonn; Germany; 2Department of Animal Reproduction, Instituto Nacional de Investigación y Tecnología Agraria, Madrid, Spain

Sperm chromatin is excessively remodeled and hypercondensed. First, the majority of canonical histones is replaced by testis-specific histones. Next, transition proteins (TNP1/TNP2) bind to DNA and allow for protamine (PRM1/PRM2) loading, finally replacing TNPs and most histones. The PRM1:PRM2 ratio is species-specific and deviations affect male fertility. Importantly, PRM2 is expressed as precursor, while PRM1 is expressed in its mature form. The N-terminal domain (cP2) of PRM2 is removed, leaving the mature PRM2 (mP2) bound to DNA. Unprocessed PRM2 and PRM1 are later detected in residual bodies and hypothesized to assist in removal of histones and TNPs.

We previously established and analyzed Prm1-, Prm2- and cP2-deficient mouse models. Here we present two new mouse models, which allow further insights into sperm chromatin remodeling.

First, we analyzed Prm1+/-Prm2+/- (dHET) mice, which showed a protamine ratio similar to WT. While the amount of protamines in epididymal sperm was comparable to WT, dHET males were infertile. Sperm showed incomplete DNA protamination and retained histones, TNPs and unprocessed PRM2. The levels of retained nuclear proteins is larger compared to other Prm-deficient mouse models. This suggests that protamines do not only hypercondense chromatin but are needed in excess in the final stages of spermio­genesis and assist in TNP and histone eviction.

Further, we generated mice expressing a fusion protein (Prmmut), where the N-terminal part of PRM1 is fused to the C-terminal part of PRM2 (lacking cP2). Remarkably, sperm function was partially retained and Prmmut mice were subfertile. Compared to dHET sperm, less histones and TNPs were retained in Prmmut sperm. This is in stark contrast to cP2-deficient mice, which were infertile and showed impaired chromatin remodeling. This suggests, that processing of the cP2 domain modulates PRM1-PRM2 interaction. In order to test this, we are currently mating Prmmut mice to cP2-deficient mice.

Progesterone induces the physiological opening of the mitochondrial permeability transition pore (mPTP) in human spermatozoa

M. León1, J. Parada2, A. Toro2, T. Torres2, P. Contreras1,
A. Bravo1, F. Zambrano1, R. Sánchez1, P. Uribe1

1Center of Excellence in Translational Medicine – Scientific and Technological Bioresource Nucleus (CEMT – BIOREN) – Ph.D.-Program in Sciences mention Applied Cell and Molecular Biology, Faculty of Agricultural Sciences and Enviroment, Universidad de La Frontera, Temuco; 2Medical Technology Career, Faculty of Medicine, Universidad de La Frontera, Temuco, California, USA

Background Mitochondria in eukaryotic cells participate in various cellular processes, including regulating cytosolic Ca2+ concentration. A pathway for Ca2+ release from mitochondria relies on the mitochondrial permeability transition pore (mPTP). The mPTP can open at two different configurations, one at low conductance, also called physiological mPTP flickering, which allows Ca2+ efflux from the mitochondrial matrix, participating in Ca2+-mediated signaling. In sperm, Ca2+ signaling participates in the physiological progesterone response, and the mechanisms underlying this response are not fully understood and the role of transient mPTP opening in human sperm has not been described. This study aims to characterize the mPTP opening in response to progesterone in human sperm.

Methods The mPTP opening in human sperm from normozoospermic donors was induced by different concentrations of progesterone (0.001–2000 ?M) and was assessed by the calcein-cobalt method. Controls included untreated sperm, ionomycin (inducer of mPTP opening) and vehicle. Sperm quality parameters including viability, mitochondrial membrane potential (MMP), ROS production and phosphatidylserine (PS) externalization were evaluated. All parameters were analyzed by flow cytometry.

Results Progesterone induces the mPTP opening in human sperm without affecting viability, MMP or ROS production, while PS externalization was slightly increased compared to the untreated control.

Conclusions Progesterone induces the mPTP opening in human sperm and this process is physiological since sperm quality parameters are not affected. This suggests that the mPTP opening participates in the biological response to progesterone in human sperm.

Funding: Partially funded by Dirección de Investigación, Universidad de La Frontera, Project FPP22-0022 and Agencia Nacional de Investigación y Desarrollo (ANID), ­Fondo Nacional de Investigación Científica y Tecnológica (FONDECYT), Grant number 1213410, Gobierno de Chile.

Infertile Esr1-deficient mice exhibit strong testicular Lipocalin 2 expression

S. K. Schröder, J. C. Kessel, R. Weiskirchen

Institut für Molekulare Pathobiochemie, Experimentelle Gentherapie und Klinische Chemie (IFMPEGKC), RWTH Aachen, Aachen, Deutschland

Introduction Lipocalin 2 (LCN2) was originally isolated from neutrophils and described as a transporter of small hydrophobic molecules. Today, LCN2 is discussed as a valuable biomarker and potential therapeutic target in various human reproductive pathologies which are often associated with infertility. There is evidence linking LCN2 to fertility, as global depletion of LCN2 negatively impacts reproductive capacity in mice. However, there is limited data on the expression and function of LCN2 in the testes.

Methods We studied LCN2 in the testes of mice in the context of fertility disorders using an estrogen receptor alpha (Esr1) mouse strain. Esr1-deficient (Esr1–/–) mice exhibit defects in sperm function that lead to infertility. Body and testes weights of wildtype and Esr1–/– males were documented and serum samples were collected. RT-qPCR, Western blot and immunohistochemical staining were used to analyse LCN2 expression in testicular tissues.

Results We found that depletion of Esr1 neither affected body nor testicular weight of adult male mice. Interestingly, serum LCN2 levels were significantly increased in Esr1–/– mice. In addition, our results demonstrate a significantly increased LCN2 expression in Esr1–/– testes compared with wildtype males of the same age. Immunohistochemical staining showed that LCN2 expression is restricted to the interstitial cells. Co-staining with steroido­genic acute regulatory protein revealed that the hormone-producing ­Leydig cells are the source of LCN2 in the testes. ­Isolation of primary murine Leydig cells using Percoll gradient centrifugation confirmed strong LCN2 expression in these cells.

Conclusion In summary, we found significantly increased LCN2 levels in Esr1–/– mice. Our study sheds light on the testicular expression of LCN2 which was restricted to Leydig cells. Further experiments should focus on depleting of LCN2 in the testis to uncover its precise functions.