34. DGA-Jahrestagung

8.–10. September 2022, Gießen

Abstracts*

*Begutachtet und zusammengestellt vom wissenschaftlichen Komitee.

Freie Vorträge zu aktuellen Themen – Grundlagenforschung und klinische Anwendung in der Andrologie

A novel diagnostic test to identify patients suffering from loss of CatSper function

S. Young1, C. Schiffer1, T. Pock1, A. Wagner1, B. Stallmeyer2, C. Krallmann1, J. Patz1, A. Potapenko1, L. Herrmann1, A. Röpke2, T. Sperlbaum3, H. M. Behre3, V. Nordhoff1,
S. Kliesch1, F. Tüttelmann2, C. Brenker1, T. Strünker1

1University of Münster, Centre of Reproductive Medicine and Andrologie, Münster; 2University of Münster, Institute of Reproductive Genetics, Münster; 3University of Münster, University Hospital Münster Fertility Clinic, Münster, Germany

Introduction There is a growing body of evidence that human sperm require the activity of the sperm-specific Ca2+ channel CatSper (cation channel of sperm) for fertilization. Despite being essential for fertilization, no test is available to routinely identify­patients with defective CatSper function.

Methods We developed the prototype of a novel in vitro diagnostic test to assess the function of CatSper in sperm from patients undergoing semen analysis.

Results Using this “CatSper-Activity-Test”, we identified in a cohort of 576 men seeking medical advice for suspected male infertility seven patients suffering from a loss of CatSper function. Standard semen analysis revealed that the CatSper-deficient patients are (with one exception) normozoospermic and were diagnosed with unexplained infertility. Notably, their sperm not only failed to fertilize the egg naturally but also upon intrauterine insemination (IUI) and in vitro fertilization (IVF), whereas intra-cytoplasmic sperm injection (ICSI) was successful. Two additional CatSper-deficient patients were identified among patients visiting our clinics for other reasons. We show that the loss of CatSper function is predominantly caused by a homo­zygous deletion of the CATSPER2 gene; one patient featured compound heterozygous variants of the CATSPERE gene.

Conclusion We conclude that loss of ­CatSper function is a common cause of unexplained male infertility. Affected patients are prone to experience failing cycles of medically assisted reproduction (MAR) using IUI and IVF – if the infertility is identified at all. The novel CatSper-Activity-Test faithfully identifies CatSper-deficient patients with a hands-on-time of only a few minutes and no special equipment or training required. Therefore, we envisage the CatSper-Activity-Test as a novel tool for the early diagnosis of male infertility allowing evidence-based treatment decisions in MAR, thus, sparing patients the burden of unnecessary medical and financial risks.

Oxytocin as a new treatment option for patients desiring to father children in case of spinal cord injury

A. Mietens, B. Stadler, C. Nowell, M. Whittaker, A. Pilatz, F. Wagenlehner, B. Exintaris, R. Middendorff

Justus-Liebig-University, Institute of Anatomy and Cell ­Biology, Gießen, Deutschland

During the emission phase of ejaculation, sperm is driven from the cauda epididymidis, its storage site, through the vas deferens by strong contractions. These contractions are mainly induced by the sympathetic nervous system and mediated by the neurotransmitter noradrenaline. Here, we investigated the effect of oxytocin (suggested to support ejaculation) on defined segments of the rat and human epididymis using live imaging. Our results indicate that the very last part of the epididymis in rat and human responds in a uniquely strong and rapid manner to oxytocin (similar to noradrenaline). The complex nature of this contractile response led us to develop a novel imaging analysis method which allowed us to quantify multidirectional contractions and to visualize them as heat maps. The response of the distal epididymis to oxytocin was concentration-dependent and could be inhibited by pretreatment with oxytocin antagonists (atosiban and cligosiban), but not with an arginine vasopressin 1A antagonist (SR49059). In both rat and human tissue, pretreatment with the alpha-1 adrenoreceptor antagonist tamsulosin inhibit­ed the response to noradrenaline, whereas the effect of oxytocin was unimpaired. Our data (from men and rodents) strongly suggest that the hormone oxytocin is involved in the ejaculatory process and support oxytocin signaling as an alternative potent pathway for emission-related contractions that is independent of noradrenaline signaling. Thus, oxyto­cin-based medications might be a promising non-adrenergic treatment option for ejaculatory disorders. In case of spinal cord injury and associated ejaculatory disorders (due to impaired sympathetic and noradrenergic signaling) stimulating the oxytocin pathway in the epididymis holds the potential of rescuing emission-related contractions in affected patients who wish to father a child.

Single cell RNA sequencing reveals cellular and molecular alterations underlying two distinct patient subgroups with cryptozoospermia

S. Di Persio1, L. Schülke1, T. Tekath2, L. Ebbert2, L. M. Siebert-Kuss1, N. Terwort1, I. Lu3, G. Meyer zu Hörste3, J.-F. Cremers1, J. Wistuba1, S. Sandmann2, C. Friedrich4, F. Tüttelmann4, S. Kliesch1, S. Schlatt1, S. Laurentino1, N. Neuhaus1

1University Hospital Münster, Centre of Reproductive Medicine and Andrology, Münster; 2University of Münster, ­Institute of Medical Informatics, Münster; 3University Hospital Münster, Department of Neurology with Institute of Trans­lational Neurology, Münster; 4University of Münster, ­Institute of Reproductive Genetics, Münster, Germany

Introduction Cryptozoospermia is a severe form of male infertility in which few sperm can be detected in the semen following centrifugation. Therefore, cryptozoospermic men (Crypto) depend on surgery to retrieve sperm for intracytoplasmic sperm injection. To date, the molecular and cellular alterations underlying cryptozoospermia remain largely unknown.

Methods To assess if there are subgroups of Crypto patients, we performed cluster analysis considering semen, histological, and endocrine parameters of 132 Crypto and 160 Control patients (obstructive ­azoospermia). Moreover, single cell RNA sequencing (­scRNA-seq; 4 Controls and 8 Crypto) and histological analyses (13 Controls, 26 Crypto) of testicular tissues were performed.

Results The clustering revealed two Crypto groups largely defined by their distinct testicular architecture. In group one, most seminiferous tubules contained spermatocytes as the most advanced germ cell type (meiotic arrest-like [MAL]), whereas the prevalent tubular phenotypes in group two were Sertoli cell only (SCO) tubules and tubular shadows (SCO-like [SCOL]). ScRNA-seq analyses of over 65,000 cells showed altered expression of genes related to the DNA packaging machinery in spermatocytes of MAL but not SCOL tissues. In contrast, Sertoli cells of SCOL but not MAL showed signs of reversion to a more immature state, as indicated by increased expression of genes involved in oxidative phosphorylation. Histological analyses revealed common alterations in the spermatogonial compartment of the two Crypto groups and specifically, a reduction of the reserve stem cells and an increased proportion of PIWIL4+ cells comparing to controls.

Conclusion In summary, our study revealed two distinct molecular alterations associated with the two groups of Crypto patients and a common alteration of the spermatogonial compartment. Future research shall address whether these two groups re­present a progressive phenotype or there are distinct underlying etiologies.

Elucidating the relevance of MCM-domain containing 2 (MCMDC2) for male (in-)fertility

N. Rotte1, B. Stallmeyer1, S. Kliesch2, F. Tüttelmann1, C. Friedrich1

1University of Münster, Institute of Reproductive Genetics, Münster; 2University Hospital Münster, Centre of Reproductive Medicine and Andrology, Münster, Germany

Introduction 7% of men are infertile and although frequently suspected, underlying genetic causes often remain unidentified. The most severe form, non-obstructive azoospermia, is frequently caused by meiotic arrest (MeiA). Recently, MCMD2, a minichromosome maintenance (MCM) paralog, was described in the context of murine sterility and meiotic disturbances. MCM family members are important for DNA replication, repair, and meiotic recombination. A recent publication suggests that this also applies to human MCMDC2.

Methods Whole-exome data from the Male Reproductive Genetics (MERGE) cohort including > 1600 men, mostly affected by azoospermia, was screened for bi-allelic rare (< 1%, gnomAD) coding variants in MCMDC2. Evaluation of testicular phenotype including CREM, yH2AX, and Caspase-3 staining identified meiotic progression and alterations.

Results Two azoospermic, otherwise healthy men from non-consanguineous parents were identified with bi-allelic variants in MCMDC2. Hormonal parameters (FSH, LH, testosterone) and testicular volumes were within reference ranges for M1226, carrying two compound heterozygous frameshift variants. M1762 and his infertile brother were homozygous for a stop-gain variant likely disturbing the MCM family domain. Decreased testosterone and increased FSH indicate testicular disturbances. Accordingly, M1762’s histology revealed MeiA at spermatocyte level, rarely showing CREM-negative round spermatids. Most cells did not proceed beyond zygotene, maintaining dispersed yH2AX signal. Testicular sperm extraction was negative.

Conclusion Our data demonstrated a recessive mode of inheritance for MCMDC2 disease-causing variants and strengthen this genes/proteins importance for male fertility. According to clinical guidelines, MCMDC2 reaches a strong level of evidence justifying the diagnostic analysis of azoospermic men.

Grants: This work was supported by the DFG Clinical Research Unit 326 ‚Male Germ Cells‘ (CRU326).

Freie Vorträge – Klinik

Elucidating the role of neddylation in cisplatin resistance of testicular germ cell tumors

K. Funke1, U. Einsfelder2, A. Hansen1, L. Arévalo1, S. Schneider1, D. Nettersheim3, V. Stein2, H. Schorle1

1University Hospital Bonn, Institute of Pathology, Department of Developmental Pathology, Bonn; 2University of Bonn, Institute of Physiology II, Bonn; 3Medical Faculty and University Hospital Düsseldorf, Heinrich-Heine-University Düsseldorf, Department of Urology, Urological Research Laboratory, Translational UroOncology, Düsseldorf, ­Germany

Introduction Testicular germ cell tumors (TGCT) are the predominant type of cancer in men between 18 and 45 years. Cisplatin (CDDP) based chemotherapies are highly effective. However, 10–30% of the patients with metastatic non-seminomas display CDDP resistance and poor survival rates demanding for novel treatment options.

Neddylation occurs as posttranslational modification modulating many important biological processes including tumorigenesis by inducing the proteasomal degradation of tumor suppressors like p21 or p27. NEDD8-activating enzyme (NAE1) is crucial for the activity of this pathway.

Methods A CRISPR/Cas9 based genome wide activation and knock out screen of TGCT cell lines was performed to identify potential factors for CDDP resistance. Further, MLN4924 (NAE1 inhibitor) and CDDP treatment was applied to TGCT cell lines and evaluated by XTT viability assay, FACS based apoptosis and cell cycle analysis as well as mRNA sequencing.

Results Using a genome scale CRISPR/Cas9 based screen NAE1 upregulation was identified to be a potential factor for CDDP resistance in JAR cells. Treatment with MLN4924 and CDDP of different parental TGCT- (2102EP, NCCIT, NT2/D1, JAR, TCam2) and CDDP resistant sub-cell lines (2102EP-R, NCCIT-R, NT2/D1-R) resulted in a strong synergistic decrease in viability over seven days. Apoptosis analysis confirmed the cytotoxicity of the combination treatment. Investigation of cell cycle distribution revealed G2/M phase arrest after MLN4924 and CDDP application. The fibroblast control cell line MPAF was not affected. Interestingly, RNA sequencing analysis for 2102EP and JAR cells after 2 days of combination treatment revealed a strong tendency for cell differentiation into mesoderm and endoderm.

Conclusion We identified NAE1 upregulation as a factor for CDDP resistance in TGCTs. Our results demonstrate that combination of MLN4924 and CDDP seems a potential therapeutic option for patients with CDDP sensitive and CDDP resistant tumors.

Immune characteristics of testicular germ cell tumors: A comparative study of seminoma and embryonal carcinoma

M. Figura1, J. Heyer1, R. Islam2, K. Hartmann2, S. Kliesch3, A. Pilatz1, F. Dittmar1, F. Wagenlehner1, M. Hedger Hudson4, B. Loveland5, K. Loveland6, H.-C. Schuppe7, D. Fietz2

1Justus-Liebig-University, Department of Urology, Pediatric Urology and Andrology, Gießen, Germany; 2Justus-Liebig-University, Department of Veterinary Anatomy, Histology and Embryology, Gießen, Germany; 3 University of Münster, Centre of Reproductive Medicine and Andrology, Münster, Germany; 4Institute for Medical Research Centre for Reproductive Health Clayton, Victoria, Australia; 5Burnet Institute Prahran, Victoria, Australia; 6Deptartment of Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia; 7Justus-Liebig-University, Hessian Center for Reproductive Medicine (HZRM), Gießen, Germany

Testicular germ cell tumors (TGCT) can present as seminoma (SE) and non-seminoma, e.g. embryonic carcinoma (EC). Tumor development, progression, and prognosis are poorly understood, but infiltrating immune cells and associated cytokines/chemokines are involved. To identify key cell and molecular components, we analyzed TGCT samples from different areas of tumor (tu)-bearing testes and contralateral “healthy” testis along with a comprehensive clinical database collected in a prospective cohort study (since 2017). Transcript analysis used cryopreserved tissue from SE/EC, with normal spermatogenesis (NSP) as control, to quantify immune and non-immune somatic cell markers, germ cell markers and cytokines/chemokines.

IHC scoring showed significantly higher immune cell numbers in different locations of SE and EC compared to NSP specimens. In SE tumor tissue (n = 33), T cells and macro­phages were most abundant, mainly in a disseminated distribution pattern, compared to NSP. In EC tumor-samples (n = 10), mostly T cells and DCs appeared in a disseminated and/or multifocal distribution pattern. qRT-PCR showed similar differences.

The prospective clinical database will allow assessments for possible correlations between IHC patterns of SE vs. EC and clinical para­meters, e.g. localized and metastatic TGCTs. By this comprehensive approach, we aim to decipher the role of „immune editing“ during TGCT development, progression and possible metastatic behaviour. Results will help to identify novel prognostic factors and immune-therapeutic concepts in human TGCTs.

Sexuelle Funktionsstörungen und assoziierte Faktoren bei Männern mittleren Alters: Ergebnisse der BMH-Study

K. Herkommer1, K. Dworschak1, V. H. Meissner1, S. Schiele1, M. Kron2, H. Schulwitz1, A. Dinkel3, J. E. Gschwend1

1Klinikum rechts der Isar der TU München, Klinik und Poliklinik für Urologie, München, 2Universität Ulm, Institut für Epidemiologie und Medizinische Biometrie, Ulm, 3Klinikum rechts der Isar der TU München, Klinik und Poliklinik für psychosomatische Medizin und Psychotherapie, München, Deutschland

Einleitung Ziel dieser Studie war die Bestimmung der Prävalenz der häufigsten sexuellen Dysfunktionen: Erektile Dysfunktion (ED), Ejaculatio praecox (EP) und verminderter Libido sowie deren Assoziationen zu Komorbiditäten, sexueller Identität und Lebensstil bei Männern mittleren Alters.

Methoden Im Rahmen der Bavarian Men’s Health- (BMH-) Study wurde von 2500 Männern die Prävalenz der o. g. Funktionsstörungen mittels validierter Fragebögen erhoben und deren Zusammenhänge untereinander sowie die Assoziationen mit der sexuellen Identität, 5 Komorbiditäten (Depression, Diabetes mellitus, Bluthochdruck, Hyperlipidämie, Symptome des unteren Harntrakts [LUTS]) und den Lebensstilfaktoren (Rauchen, Übergewicht [Bauchumfang], körperliche Aktivität) mittels univariabler Regressionen untersucht.

Ergebnisse 20,7 % des Kollektivs (Ø Alter: 50,4 ± 0,8J) hatten eine ED, 5,2 % eine EP und 7,2 % eine verminderte Libido. Die Mehrheit der Männer war heterosexuell: 94,0 %, homosexuell: 5,0 %, homosexuell und bisexuell: 1,0 %. Die ED war mit einer EP, verminderter Libido, den erhobenen Komorbiditäten außer der Hyperlipidämie, einem höheren Bauchumfang und einer geringeren körperlichen Aktivität assoziiert. Homosexuelle hatten häufiger eine ED als Heterosexuelle. Die EP war mit einer ED, LUTS und geringerer körperlicher Aktivität assoziiert. Eine verminderte Libido war mit einer ED, LUTS, Depression und geringerer körperlicher Aktivität assoziiert. Rauchen als Risikofaktor zeigte keinen Zusammenhang mit den untersuchten Funktionsstörungen.

Schlussfolgerung Die Prävalenz der sexuellen Funktionsstörungen ist bei Männern mittleren Alters hoch. Assoziierte Faktoren beinhalten nicht nur typische Risikofaktoren wie Komorbiditäten oder einen ungesunden Lebensstil, sondern auch das Vorhandensein weiterer sexueller Funktionsstörungen und Symptome des unteren Harntrakts. Die vorliegenden Ergebnisse liefern somit wichtige Erkenntnisse über das multifaktorielle Krankheitsbild jeder einzelnen Funktionsstörung.

Delayed restoration of spermatogenesis after orchidopexy of an adult man with bilateral cryptorchidism: A case report

F. Dittmar1, D. Fietz2, A. Pilatz1, T. Diemer1, F. Wagenlehner1, H.-C. Schuppe1

1Justus-Liebig-University, Department of Urology, Pediatric Urology and Andrology, Gießen; 2Justus-Liebig-University, Department of Veterinary Anatomy, Histology and Embryo­logy, Gießen, Germany

Introduction Reports on orchidopexy in case of bilateral undescended testicles (bUDT) in adult men incl. their fertility outcomes are sparse. Here, we report on a 32-year old patient with persistent bUDT and repeated non-obstructive azoospermia who underwent bilateral orchidopexy incl. synchronous M-TESE and follow-up for 3 years.

Material and Methods Upon first presentation and during follow-up, clinical examination incl. hormone status, ultrasound and semen analyses were performed. For histological analysis, four multi-focal biopsies of each testis were fixed with Bouin’s solution, processed and evaluated according to standard protocols. In addition, tissue specimen from each retrieval site were subjected to cryopreservation.

Results The patient showed non-obstructive azoospermia without signs of infection, normal male karyotype (46, XY), bilaterally reduced testicular volume (8 ml) and hypergonadotropic normogonadism (FSH 27,1 mU/ml; LH 10,2 mU/ml; testosterone 541,9 ng/dl) before surgery. Histological analysis mainly revealed an arrest of spermatogenesis, predominantly at the level of spermatogonia or primary spermatocytes and focal complete absence of germ cells (Sertoli cell only tubules). No signs of malignancy were observed. Only one focus of qualitatively preserved, but quantitatively severely reduced spermatogenesis was detected. Intracytoplasmic sperm injection using cryopreserved M-TESE material remained unsuccessful. Oligozoospermia (total sperm number 0.3M.; progressive motility PR 56%) could be demonstrated during follow-up.

Conclusion Orchidopexy can be beneficial even in adult men with persistent cryptorchidism. Restoration of spermatogenesis may occur after several years and normal pre­operative testosterone levels could be associated with a favourable outcome.

Poster

Effekte der Testosteronbehandlung auf Surrogatparameter der nicht-­alkoholischen Lebererkrankung (NAFLD) bei Männern mit funktio­nalem Hypogonadismus und Adipo­sitas

K. Sultan Haider1, A. Haider1, G. Doros2, A. Traish3

1Praxis Dr. Haider, Bremerhaven, Deutschland; 2Boston University School of Public Health, Department of Epidemiology and Statistics, Boston, USA; 3Boston University School of Medicine Department of Biochemistry and Department of Urology, Boston, USA

Einleitung Die Prävalenz von nicht-alko­holischer Lebererkrankung (NAFLD) bei Männern mit Adipositas und Hypogonadismus ist hoch. Wir untersuchten den Einfluss der Testosteronbehandlung auf Surrogatparameter der NAFLD bei dieser ­Patientengruppe.

Methoden In einer urologischen Registerstudie hatten 491 Männer funktionalen Hypogonadismus und Adipositas. 292 Männer (59,5 %) entschieden sich für eine Testosteronbehandlung mit Testosteron-Undecanoat-­Injektionen 1000 mg /12 Wochen nach anfänglichem 6-Wochen-Intervall (T-Gruppe), 199 (40,5 %) dagegen (KTRL). Die Mittelwerte und Standardabweichungen absoluter Messwerte über 13 Jahre wurden berechnet.

Ergebnisse Anfangsalter (Jahre): T-Gruppe: 59,5 ± 6,0, KTRL: 63,0 ± 5,0 (p < 0,0001). Follow-up: T-Gruppe 10,1 ± 3,1 (Median: 11), KTRL 9,4 ± 3,3 (10) Jahre.

?-GT (U/l): T-Gruppe: von 42,9 ± 22,8 auf 20,5 ± 6,4, KTRL: von 35,5 ± 11,6 auf 61,7 ± 5,9 (jeweils p < 0,0001).

Triglyzeride (mmol/l): T-Gruppe: 3,4 ± 0,5 auf 2,2 ± 0,1, KTRL: von 3,2 ± 0,5 auf 3,9 ± 0,5 (KTRL) (jeweils p < 0,0001).

Bauchumfang (cm): T-Gruppe: von 115,0 ± 13,2 auf 98,6 ± 6,3, KTRL: von 118,6 ± 11,4 auf 120,0 ± 8,0 (jeweils p < 0,0001).

BMI (kg/m²): T-Gruppe: von 36,8 ± 3,6 auf 28,3 ± 2,1, KTRL: von 33,9 ± 3,3 auf 34,9 ± 2,26 (jeweils p < 0,0001).

AST (U/l): T-Gruppe: von 38,4 ± 13,3 auf 20,8 ± 1,9, KTRL: von 28,6 ± 9,0 auf 54,1 ± 11,9 (jeweils p < 0,0001).

ALT (U/l): T-Gruppe: von 41,4 ± 13,70 auf 24,7 ± 2,1, KTRL: von 32,7 ± 9,4 auf 60,2 ± 13,8 (jeweils p < 0,0001).

Fatty Liver Index (FLI): T-Gruppe: von 95,1 ± 4,6 auf 61,3 ± 11,2. KTRL: von 94,1 ± 4,4 auf 98,0 ± 1,3 (jeweils p < 0,0001).

Schlussfolgerung Langzeitbehandlung mit Testosteron bei hypogonadalen Männern mit Adipositas verbesserte Surrogatparameter der NAFLD im Vergleich zu unbehandelten Kontrollpatienten.

Die Rolle von Androgen-regulierten microRNAs bei der Inhibition der Adipogenese

T. Greither, J. Jansen, D. Handke, H. M. Behre

Universitätsklinikum Halle (Saale), Zentrum für Reproduktionsmedizin und Andrologie, Halle, Deutschland

Einleitung Der Prozess der Differenzierung von mesenchymalen Stammzellen zu Fettgewebszellen wird durch Androgene inhibiert. Differenzierungsprozesse wie die Adipogenese werden auch durch Änderun­gen im zellulären microRNA-Expressionsprofil reguliert. microRNAs sind kleine, nicht-kodierende RNAs, die über eine Translationsinhibierung ihrer spezifischen Ziel­gene eine posttranskriptionelle Modulation des zellulären Proteoms vermitteln. Ziel des Projekts war zu eruieren, inwieweit microRNAs bei der Androgen-vermittelten Inhibition der Adipogenese eine Rolle spielen.

Methoden Die Adipogenese wurde in zwei Zellkulturmodellen (SGBS-Zellen und humane Fettgewebsstammzellen [hADSC]) per adipogenem Differenzierungsmedium mit und ohne Zusatz von Androgenen induziert und im OilRed-O-Test kontrolliert. In RNAseq-Analysen wurden die microRNA-Expressionsprofile verglichen und differentiell regulierte microRNAs zur Kontrolle in der quantitativen PCR selektiert. Anschließend erfolgten Zielgen-Analysen per Luciferase-­Reporter-Assay und die Evaluation der Effekte einer microRNA-­Modulation auf die Adipogenese.

Ergebnisse Die adipogene ­Differenzierung der beiden Zellkulturmodelle wurde durch Androgene inhibiert. Ausgehend von den RNAseq-Daten wurden 10 differentiell regulierte microRNAs in qPCR-Analysen vermessen. Diese zeigten mit Ausnahme der miR-194-5p keine deutliche Regulation in Reaktion auf eine Androgenbehandlung in SGBS- und hADSC-Zellen. Für die verringert exprimierte miR-194-5p wurden Zielgen­analysen durchgeführt. Dabei konnte IGF1R als Zielgen der miR-194-5p in Prä-Adipozyten validiert werden. Die Hemmung der miR-194-5p bei der adipogenen Differenzierung von Prä-Adipozyten ergab keine signifikante Inhibition der Adipogenese.

Schlussfolgerungen Wir konnten zeigen, dass eine Androgen-vermittelte Regulation von microRNA-Expressionsprofilen, ­darunter auch die miR-194-5p, bei der Inhibition der Adipogenese primär eine feinmodulierende Rolle spielt.

Effekte der Testosteronbehandlung auf anthropometrische Parameter bei Männern mit funktionalem Hypogonadismus und Adipositas

A. Haider1, K. Sultan Haider1, G. Doros2, A. Traish3

1Praxis Dr. Haider, Bremerhaven, Deutschland; 2Boston University School of Public Health, Department of Epidemiology and Statistics, Boston, USA; 3Boston University School of Medicine, Department of Biochemistry and Department of Urology Boston, USA

Einleitung Testosteronmangel ohne Schädigung der hypothalamisch-hypophysär­-gonadalen Achse wird als funktionaler Hypogonadismus bezeichnet und ist meistens mit Adipositas assoziiert.

Methoden Von 491 Männern mit funktionalem Hypogonadismus und Adipositas in einer Registerstudie entschieden sich 292 (59,5 %) für Testosteronbehandlung mit Testosteron-Undecanoat-­Injektionen 1000 mg /12 Wochen nach anfänglichem 6-Wochen-Intervall (T-Gruppe), 199 (40,5 %) dagegen (KTRL). Die Mittelwerte und Standardabweichungen absoluter Messwerte über 13 Jahre wurden berechnet.

Ergebnisse Anfangsalter (Jahre): T-Gruppe: 59,5 ± 6,0, KTRL: 63,0 ± 5,0 (p < 0,0001). Follow-up: T-Gruppe 10,1 ± 3,1 (Median: 11), KTRL 9,4 ± 3,3 (10) Jahre.

Gewicht (kg): T-Gruppe: von 114,5 ± 11,8 auf 87,5 ± 6,7, KTRL: von 105,6 ± 10,5 auf 107,9 ± 6,2 (jeweils p < 0,0001).

Gewichtsveränderung (%): T-Gruppe: –22,8 ± 4,9 %, KTRL: +8,4 ± 3,7 % (jeweils p < 0,0001).

Bauchumfang (cm): T-Gruppe: von 115,0 ± 13,2 auf 98,6± 6,3, KTRL: von 118,6 ± 11,4 auf 120,0 ± 8,0 (jeweils p < 0,0001).

BMI (kg/m²): T-Gruppe: von 36,8 ± 3,6 auf 28,3 ± 2,1, KTRL: von 33,9 ± 3,3 auf 34,9 ± 2,26 (jeweils p < 0,0001).

Waist-to-Height-Ratio: T-Gruppe: von 0,65 ± 0,07 auf 0,55 ± 0,03, KTRL: von 0,67 ± 0,06 auf 0,69 ± 0,03 (jeweils p < 0,0001).

In der T-Gruppe erreichten 118 Patienten (40,4 %) einen Gewichtsverlust von ? 20 % nach einer mittleren Beobachtungszeit von 92 Monaten (Minimum: 27, Maximum: 189). Kein Patient in KTRL erreichte einen Gewichtsverlust von ? 20 %. 290 Männer (99,3 %) in der T-Gruppe vs. 8 Männer (4,0 %) in KTRL erreichten einen Gewichts-verlust von ? 5 %.

Schlussfolgerung Die Langzeitbehandlung mit Testosteron bei Männern mit funktionalem Hypogonadismus und Adipositas verbesserte anthropometrische Parameter im Vergleich zu unbehandelten Kontrollpatienten.

BRD9 inhibitor as potential treatment option for testicular germ cell tumors

A. Hansen, K. Funke, L. Arévalo, A. Lapp, H. Schorle

Uniklinikum Bonn, Entwicklungspathologie, Bonn, Deutschland

Introduction Testicular germ cell tumors (TGCT) represent the most common tumor in young men. Curation rates of up to 95% are achieved by orchiectomy and/or in addition chemo- or radiotherapy. Nevertheless, 15–20% of patients with metastatic non-seminomas are resistant to the treatment. Development of novel treatment options interfering with the epigenetic landscape was already shown to be effective by targeting BET proteins (BRDT, BRD2, BRD3 and BRD4) in different cancer types. The bromodomain protein BRD9 is an epigenetic reader similar to proteins of the BET family influencing gene expression by recruiting transcription factors. Therefore, the BRD9 inhibitor I-BRD9 could be a possible treatment option for TGCTs.

Methods For testing influence of I-BRD9 on viability of cells XTT-assay was performed. Cell cycle arrest as well as apoptosis rate was analyzed by FACS after treatment with I-BRD9.

Results TGCT cell lines showed strong decrease in viability after treatment with I-BRD9 whereas the fibroblast control cell line was not strongly influenced. FACS analysis revealed increased apoptosis in cells treated with I-BRD9 after 24 and 48 hours compared to the solvent control. Cell cycle progression in TGCT cell lines was arrested after treatment with I-BRD9.

Conclusion I-BRD9 strongly reduces viability, initiates cell cycle arrest and apoptosis of TGCT cells while control cells remain mostly unaffected. The data suggest I-BRD9 as an effective treatment alternative for TGCT’s. RNA-seq analyses will pinpoint deregulated genes and pathways caused by inhibition of BRD9.

Tumor infiltrating T lymphocytes in human testis cancer – identification and functional analysis

R. Islam1,2, J. Heyer1,5, M. Figura1,5, S. Indumathy1,2, K. Hartmann1, C. Pleuger3, M. Fijak3, S. Kliesch4, F. Dittmar5, F.  Wagenlehner5, S. Herold6,7, M. Heiner6,7, M. Hedger2, B. Loveland8, K. Loveland2,9, J. Guo10,11, H.-C. Schuppe5, D. Fietz1

1Justus-Liebig-University, Department of Veterinary Anatomy, Histology and Embryology, Gießen, Germany; 2Centre for Reproductive Health, Hudson Institute for Medical Research, Clayton, Victoria, Australia; 3Justus- Liebig-University, Institute of Anatomy and Cell Biology, Gießen, Germany; 4University of Münster, Centre of Reproductive Medicine and Andrology, Münster, Germany; 5Justus-Liebig-University, Department of Urology, Pediatric Urology and Andrology, Gießen, Germany; 6Universities of Gießen and Marburg Lung Center (UGMLC), Department of Internal Medicine II for Pulmonary and Critical Care Medicine and Infectious Diseases, Germany; 7German Center for Lung Research (DZL), Gießen, Germany; 8Burnet Institute, Melbourne, Australia; 9Monash University, School of Clinical Sciences, Clayton, ­Victoria, Australia; 10State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; 11University of Utah School of Medicine Division of Urology, Department of Surgery, Salt Lake City, USA

T cells represent the major component of tumor infiltrating lymphocytes (TIL) associated with human testicular germ cell tumors (TGCT), but many subtypes, CD4+ regulatory (Treg) and follicular helper T cells (Tfh) have not been examined in TGCT. Therefore, we aimed to analyze Treg and Tfh in TGCT development and progression. Analysis of infiltration density and distribution of immune cells including TILs were performed in testis specimens with seminoma (SE; n = 28), germ cell neoplasia in situ ± lymphocytic infiltrates (ly) (n = 15, each) and compared to specimens with normal spermatogenesis (n = 10) or hypospermatogenesis + ly (n = 12) by immunohistochemistry (IHC). In addition, immune cells were analyzed by flow cyto­metry (FC) using fresh human testis samples (n = 24) from different areas of tumor-bearing and contralateral testes. Samples were categorized into SE (n = 12) and other TGCT, embryonal carcinoma (EC; n = 6) and mixed TGCT (n = 6). Finally, scRNAseq of normal donor samples (n = 3) and TGCT (SE, EC and mixed, n = 4) was performed to decipher T cell signatures and cell interactions. T cells, including Treg and Tfh cells, are more abundant in SE compared to all other groups as shown by IHC and FC. FC analysis confirmed the highest abundance of T cells within the tumor in SE compared to other localizations. Additionally, helper and cytotoxic T cells, Treg and Tfh cells were detectable in tumor sites of SE-bearing testes, suggesting that Treg and Tfh cells might be involved in SE biology. scRNAseq analysis confirmed T cells to be the most abundant immune cell in TGCT compared to normal testis, including the presence of Treg and Tfh. This study demonstrates the complexity and interindividual variability of TIL and suggested the possible importance of rarer T cell subtypes in the immune environment of TGCT. Further experiments aim to interrogate Treg and Tfh functions to identify novel prognostic factors and/or immune-therapeutic concepts for human TGCT.

Grants: Funded by DFG GRK1871/2

Vasektomie: Prävalenz und Sexual­leben

A. Rechberger, M. Jahnen, S. Schiele, H. Schulwitz, J. E. Gschwend, K. Herkommer

Klinikum rechts der Isar der TU München, Klinik und Poli­klinik für Urologie, München, Deutschland

Einleitung Obwohl die Vasektomie ein sehr sicheres und nebenwirkungsarmes Verfahren zur Empfängnisverhütung ist, besteht bei vielen Männern die Angst, dass sich dieser Eingriff negativ auf ihr Sexualleben auswirken könnte. Ziel der Arbeit war, die Prävalenz der Vasektomie bei deutschen Männern mittleren Alters zu erheben und mögliche Assoziationen zwischen der Vasektomie und Faktoren des Sexuallebens zu untersuchen.

Methoden Im Rahmen der Bavarian Men’s Health- (BMH-) Study wurden Daten von heterosexuellen Männern zu deren Lebensstil, ihrer sexuellen Aktivität, ihrer sexuellen Zufriedenheit und ihrem Sexualleben erhoben. Mittels validierter Fragebögen wurde das Vorliegen folgender sexueller Funktionsstörungen erfasst: Erektile Dysfunktion, Ejaculatio praecox und Libidostörung.

Ergebnisse In die Analyse wurden 2330 Männer mit einem Durchschnittalter von 50,4 ± 0,8 Jahren eingeschlossen. Bei 12,6 % (n = 294) wurde eine Vasektomie durchgeführt. Diese fand durchschnittlich vor 9,2 ± 5,8 Jahren statt.

88,2 % hatten eine feste Partnerin (bei Vasektomie 95,9 %, ohne Vasektomie 87,1 %; p < 0,001) und 73,3 % hatten Kinder (bei Vasektomie 86,7 %, ohne Vasektomie 71,4 %; p < 0,001). Mit ihrem Sexualleben zufrieden waren zum Zeitpunkt der Befragung 80,8 % (bei Vasektomie 85,6 %, ohne Vasektomie 80,1 %; p = 0,027).

Bei 13,5 % der Vasektomierten wurde eine erektile Dysfunktion erhoben (ohne Vasektomie 20,5 %; p = 0,005), bei 6,7 % eine Ejaculatio praecox (ohne Vasektomie 5,1 %; p = 0,289) und bei 4,8 % eine Libidostörung (ohne Vasektomie: 7,0 %; p = 0,159).

Schlussfolgerung Ca. jeder 10. Mann gab an, vasektomiert zu sein. Sexuelle Funktionsstörungen wurden unter den Vasektomierten nicht häufiger erfasst, die ED sogar signifikant seltener. Die Zufriedenheit mit dem Sexualleben war bei Vasektomierten höher als in der Vergleichsgruppe. Die Vasektomie stellt damit ein sicheres Verfahren zur Empfängnisverhütung ohne Anhalt für negative Auswirkung auf das Sexualleben von Männern dar.

Das sexuelle Selbstbild von Männern mittleren Alters und Assoziationen mit deren Sexualleben: Ergebnisse aus der BMH-Study

C. Passler1, A. Dinkel2, V. H. Meissner1, S. Schiele1, H. Schulwitz1, J. E. Gschwend1, K. Herkommer1

1Klinikum rechts der Isar der TU München, Klinik und Poli­klinik für Urologie, München; 2Klinikum rechts der Isar der TU München, Klinik und Poliklinik für Psychosomatische Medizin und Psychotherapie, München, Deutschland

Einleitung Das sexuelle Selbstbild eines Menschen umfasst psychische und physische Facetten und stellt die subjektive Repräsentation der eigenen Person dar. Ziel der Studie war, den Einfluss des Sexuallebens auf das sexuelle Selbstbild und dessen Facetten bei Männern zu untersuchen.

Methoden Eingeschlossen wurden 2349 heterosexuelle Männer mittleren Alters aus der Bavarian Men’s Health- (BMH)- Study. Parameter des Sexuallebens waren „sexuelle Aktivität innerhalb der letzten 3 Monate“ (sexuelle Aktivität), „sexuelle Zufriedenheit“ und „sexuelle Wichtigkeit“. Für das Selbstbild wurden Items aus dem Dresdner Körperbildfragebogen, der Male-Role-Norms-Scale sowie anhand Expertenmeinung neu definierte Items verwendet. Das Selbstbild wurde in die Facetten „Verständnis von Maskulinität“, „Körperbild“, „Sexuelles Selbstwertgefühl“ und „Wahrnehmung sozialen Drucks“ gegliedert. Für jede Facette wurde eine lineare Regression mit den Sexualparametern mit Adjustierung für die soziodemographische Faktoren Partnerschaft, Kinder, Bildungsniveau sowie Angst (GAD-2) und Depressivität (PHQ-2) berechnet.

Ergebnisse Zum Befragungszeitpunkt waren 18,7 % der Männer (Ø 50,4 Jahre) mindestens 2-mal pro Woche sexuell aktiv, 45,9 % gaben an, mit dem Sexualleben zufrieden zu sein und 60,9 % war Sexualität wichtig. Höhere sexuelle Aktivität, höhere Zufriedenheit und höhere Wichtigkeit waren mit einem positiveren Körperbild (jeweils p < 0,001) sowie einem positiveren sexuellen Selbstwertgefühl (jeweils p < 0,001) assoziiert. Höhere sexuelle Aktivität und höhere sexuelle Zufriedenheit waren mit einer niedrigeren Wahrnehmung sozialen Drucks (jeweils p < 0,01) assoziiert.

Schlussfolgerung Eine höhere sexuelle Zufriedenheit, höhere sexuelle Wichtigkeit und höhere sexuelle Aktivität sind mit Facetten des sexuellen Selbstbildes assoziiert. Eine positive Wahrnehmung der eigenen Sexualität geht mit einem positiven sexuellen Selbstbild einher.

„Sexualdelikt“-Projekt: Medizini­sche Soforthilfe und vertrauliche Spurensicherung im Land Brandenburg

K. Albrecht, J. Straube, N. Stanislawski

Brandenburgisches Landesinstitut für Rechtsmedizin (BLR), Medizinische Hochschule Brandenburg (MHB), Rechtsmedizin, Potsdam, Deutschland

Einleitung Klinisch-rechtsmedizinische Un­tersuchungen von Opfern körperlicher und sexualisierter Gewalt gehören zum regelmäßigen Untersuchungsspektrum der forensischen Medizin. Dabei werden Betroffene entweder im behördlichen Auftrag (z. B. Polizei/Staatsanwaltschaft) oder im Rahmen niederschwellig verfügbarer Untersuchungsmöglichkeiten („Opferambulanz“) begutachtet und damit die Gelegenheit eröffnet, genitale und extragenitale Verletzungen professionell und gerichtsverwertbar zu dokumentieren, sowie Spuren zu sichern und zu asservieren.

Methoden Im Land Brandenburg bieten 5 Partnerkliniken und das Brandenburgische Landesinstitut für Rechtsmedizin (BLR) im Rahmen eines Modellprojektes medizinische Soforthilfe und vertrauliche Spurensicherung nach einem stattgehabten Sexualdelikt an, wenn Betroffene nicht sofort eine Anzeige bei der Polizei erstatten möchten.

Ergebnisse Über die entsprechenden Rettungsstellen der Kliniken werden die Betroffenen zumeist zu gynäkologischen Kliniken weitergeleitet, wo speziell geschulte Ärztinnen und Ärzte, denen ein standardisiertes Spurensicherungs-Kit zur Verfügung steht, die Behandlung und Spurensicherung durchführen.

Schlussfolgerung In der Präsentation wird das landesmittelgeförderte Modellprojekt vorgestellt, Aufgaben und mögliche Probleme skizziert, sowie die Frage diskutiert, ob auch Urologische Kliniken in die Untersuchung und Spurensicherung, beispielsweise bei männlichen Opfern sexualisierter Gewalt, eingebunden werden sollten.

Anti-Müller-Hormon (AMH) als Indikator für testikuläre Degeneration beim Hund – ein Fallbericht

C. Otzdorff1, H. Aupperle-Lellbach2, B. Walter1

1Ludwig-Maximilians-Universität München, Chirurgische und Gynäkologische Kleintierklinik, München; 2Laboklin GmbH & Co.KG, Bad Kissingen, Deutschland

Einleitung Testikuläre Degeneration stellt neben einer Neoplasie eine häufige Verdachtsdiagnose bei älteren Zuchtrüden dar. Erhöhte AMH-Konzentrationen wurden bei Rüden mit Sertoli-Zelltumoren sowie bei Vorliegen einer testikulären Atrophie nach chemischer Kastration beschrieben. In immunhistochemischen Untersuchungen konnte AMH vermehrt in Sertoli-Zellen atrophischer Hoden nachgewiesen werden. AMH stellt daher einen möglichen Marker zur Diagnose der testikulären Degeneration dar.

Methoden Im November 2019 wurde ein 7 Jahre alter Zuchtrüde zur Spermagewinnung und Kryokonservierung vorgestellt. Bei der Untersuchung wurde eine Hämospermie bei sonst unauffälligem Spermiogramm festgestellt. Aufgrund der Diagnose einer Prostatazyste wurde der Rüde mit Osateronazetat (Ypozane®) behandelt. Im Januar 2021 wurde der Rüde erneut vorgestellt. Das untersuchte Ejakulat wies eine Hämospermie sowie eine Azoospermie auf. Der letzte erfolgreiche Deckakt des Rüden war im Oktober 2020. Die andrologische Untersuchung zeigte einen verkleinerten, weichen rechten Hoden. In der Ultraschalluntersuchung wurde eine heterogene Struktur des linken Hodens festgestellt. Die AMH-Konzentration lag bei > 22,96 ng/ml (oberer Messbereich, Laboklin). Es erging der Rat zur Kastration des Rüden.

Ergebnisse Die histologische ­Untersuchung der Hoden zeigte das Vorliegen eines Seminoms sowie eine gemischte tubuläre Atrophie. In den Nebenhoden wurden keine Spermien nachgewiesen. In der immunhistochemischen Untersuchung konnte AMH nur in den degenerierten Tubuli seminiferi und nicht im Bereich des Seminoms oder der intakten Tubuli detektiert werden.

Schlussfolgerung Diese Untersuchung zeigt, dass hohe AMH-Konzentrationen auch durch eine testikuläre Degeneration verursacht werden können. Diese sollte daher als Differentialdiagnose zu neoplastischen Veränderungen bei Rüden mit erhöhten AMH-Werten in Betracht gezogen werden.

Realisierung eines zuvor unerfüllten Kinderwunsches: Eine Analyse aus der Bavarian Men’s Health-(BMH-) Study

H. Leukers1, F.-M. Köhn2, M. Jahnen1, S. Schiele1, H. Schulwitz1, J. E. Gschwend1, K. Herkommer1

1Klinikum rechts der Isar der TU München, Klinik und Poli­klinik für Urologie, München; 2Andrologicum München, München, Deutschland

Einleitung In Deutschland sind 10–15 % der Paare ungewollt kinderlos. Ziel der Analyse war die Darstellung der Prävalenz des unerfüllten Kinderwunsches (KiWu) bzw. der Erfüllung eines unerfüllten KiWus in einem bevölkerungsbasierten Kollektiv von Männern mittleren Alters und damit assoziierte Faktoren.

Methoden Die ersten 2500 Männer (Ø Alter: 50,4 J [SD: 0,8]) der BMH-Study wurden u. a. zur Dauer und zur Erfüllung eines bestehenden bzw. eines in der Vergangenheit vorliegenden unerfüllten KiWus befragt. Die Untergruppen wurden hinsichtlich soziodemographischer Faktoren, Lebensstil, Komorbiditäten und urologischen Vorerkrankungen verglichen.

Ergebnisse Im Gesamtkollektiv hatten 69,4 % der Männer Ø 1,3 Kinder. Bei 19,5 % bestand/besteht ein unerfüllter KiWu über Ø 3,0 Jahre (SD: 2,5). 86,9 % von diesen hatten (mindestens) ein Spermiogramm, das in 53,4 % auffällig war. 63,9 % konnten ihren KiWu im Verlauf erfüllen. Diese hatten Ø 1,8 Kinder (SD: 0,7), 9,4 % hatten Mehrlinge. Rauchen war mit einem unerfüllten KiWu assoziiert. In der Gruppe unerfüllter KiWu waren weniger Raucher und vermehrt Ex-Raucher im Vergleich zur Gruppe ohne unerfüllten KiWu (p < 0,05). Eine Varikozele und ein Maldescensus testis waren ebenso assoziierte Risikofaktoren (p < 0,05). Die Männer, die ihren zuvor unerfüllten KiWu realisieren konnten, waren zum Beginn des KiWus jünger (Ø Alter: 34,5 J [SD: 5,8]) als die Männer, die ihren Kinderwunsch nicht erfüllen konnten (Ø Alter: 38,0J [SD: 6,6]). In der Gruppe erfüllter KiWu hatten deutlich mehr Männer eine Methode der assistierten Reproduktion in Anspruch genommen, verglichen zur Gruppe, die ihren Kinderwunsch nicht erfüllen konnte (p < 0,001).

Schlussfolgerung Ein unerfüllter KiWu be­steht/bestand bei 1/5 der Männer, der bei > 60 % erfüllt werden konnte mit durchschnittlich 1,8 Kindern. Mit einem unerfüllten KiWu assoziiert waren ein Maldescensus testis und eine Varikozele. Männer, die zum Beginn des unerfüllten KiWus jünger waren, konnten ihren KiWu eher erfüllen.

Vergleich dreier kommerzieller Kryo­medien bezüglich des Einflusses auf die diagnostischen Spermienparameter

E. L. Kruschel, K. Laubis, G. Hahn, S. Steinert, A. Salzbrunn, S. W. Schneider, K. von Kopylow

Universitätsklinikum Hamburg-Eppendorf, Andrologie, Hamburg, Deutschland

Einleitung Die Kryokonservierung von Spermien ist für die ART von grundlegender Bedeutung. Infertilität kann durch medizinische Behandlungen, wie Chemo- und Radiotherapie, Erkrankungen, wie Sichelzellanämie und Thalassämie, sowie genomische Anomalien, z. B. Klinefelter-Syndrom, induziert werden. Im Vorfeld der Behandlung oder unmittelbar nach Diagnosestellung wird dem erwachsenen Mann in solchen Fällen zur Fertilitätsprotektion eine Kryokonservierung von Spermien aus dem Ejakulat angeboten. Mechanische und chemische Einflüsse während der Kryokonservierung können sich jedoch schädigend auf die Spermienqualität auswirken. Bisher gibt es bei der Spermien-Kryokonservierung in den Laboren keinen einheitlichen Standard.

Methoden Zunächst wurden 4 verschiedene Kryokonservierungstechniken (Slow Freezing, Rapid Freezing sowie Vitrifizierung mit und ohne Kryomedium [CPA]) anhand von Ejakulaten gesunder Väter (< 39 Jahre, n = 10) und der Bestimmung des DNA-Fragmentationsindexes mithilfe der TUNEL-Färbung sowie der Untersuchung des Spermien-Chromatins mittels Acridin-Orange-Färbung miteinander verglichen. In einem zweiten Schritt wurden die o. g. Kryokonservierungstechniken auf ein Patientenkollektiv der Andrologie (n = 16) und die Analyse auf weitere diagnostische Spermienparameter (Motilität und Vitalität) ausgeweitet. Im dritten Schritt wurden 3 unterschiedliche kommerzielle CPAs (Sperm Freeze, Sperm Freezing Medium, TEST-Yolk-Buffer) an Ejakulaten weiterer Patienten unter Verwendung des Slow-Freezing-Protokolls getestet.

Die DNA-Färbung wurde mit der automatischen, mikroskopbasierten Bildgebungsplattform ScanR untersucht.

Ergebnisse Es lässt sich zeigen, dass CPAs einen relevanten Schutz für die kryokonservierten Spermien bieten. Beim Vergleich der 3 CPAs schnitt der TEST-Yolk-Buffer bei den Parametern Vitalität und Motilität signifikant besser ab.

Schlussfolgerung Die gewonnenen Daten geben wertvolle Hinweise zur Optimierung der Kryokonservierung humaner Spermien.

Cylicins are required for male ­fertility

A. Kovacevic1, S. Schneider2, M. Mayer1, A. Jäger1, J. N. Hansen3, D. Wachten4, H. Schorle1

1University Hospital Bonn, Developmental Pathology, Bonn; 2University Hospital Bonn, Core Facility ‚‘Gene Editing‘‘, Bonn; 3University Hospital Bonn, Department of Biophysical Imaging, Institute of Innate Immunity, Bonn; 4Medical ­Faculty, University Hospital Bonn, Department of Bio­physical Imaging, Institute of Innate Immunity, Bonn, ­Germany

Introduction The postacrosomal calyx of the sperm contains the cytoskeletal proteins such as Cylicin1 and Cylicin2. Cylicins are characterized by repetitive, lysine-rich tripeptides. CYLC2 is an actin binding protein that might serve as a cytoskeletal regulator in the acrosomal region of round spermatids and the postacrosomal region of mature sperm. However, the precise role of Cylicins remains to be elucidated.

Methods We established CYLC1 and CYLC2 deficient mouse lines using CRISPR/Cas9 gene editing. Mating experiments were performed to examine the fertility of the animals. The sperm and testis samples were analyzed by IHC and IF. The flagellar beat of the sperm was examined using SpermQ software.

Results CYLC1Y/- males are subfertile, with sperm number and vitality unaltered. CYLC2+/- males present normal fertility while CYLC2-/- male mice are sterile, with the severely reduced sperm number. Close inspection of DAPI and MitoRed staining revealed that CYLC2-/- sperm heads are bent back to the midpiece region with the tail wrapped around the sperm head. Consequently, CYLC2-/- sperm displayed stiffened mid-piece and irregular flagellar beat. Further, the acrosome of CYLC2-/- sperm is misplaced. Interestingly, CYLC1Y/- CYLC2+/- males show a similar phenotype. This suggests that Cylicins compensate for each other and 2 alleles of Cylicins are required for proper sperm function. ?-tubulin and HOOK1 staining showed that the manchette of the CYLC2-/- sperm is initiated properly but elongates excessively and fails to be dissolved during the maturation of the cells. Male CYLC1Y/- CYLC2- /- display even more severe morphological defects of the sperm.

Conclusion Due to the expression pattern and the phenotype of the Cylicin deficient mice, we speculate that Cylicins are involved in manchette organization, which is required for structural stability of the head-midpiece junction and localization of the acrosome.

Loss of Prm1 leads to defective ­chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice

G. E. Merges1, J. Meier1, S. Schneider1, A. Kruse2, A. C. Fröbius2, G. Kirfel3, K. Steger2, L. Arévalo1, H. Schorle1

1University Hospital Bonn, Department of Developmental Pathology, Institute of Pathology, Bonn; 2Justus-Liebig-University, Department of Urology, Pediatric Urology and Andrology, Section Molecular Andrology, Biomedical Research, Gießen; 3University of Bonn, Department of Molecular Cell Biology, Institute for Cell Biology, Bonn, Germany

Introduction During spermiogenesis sper­ma­tids undergo intense differentiation including a complete reorganization of the chromatin packaging. The majority of histones is substituted by protamines leading to DNA hypercondensation and protection of the paternal genome. In men and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/ Prm2), are expressed in a species-specific ratio. Contrary to PRM1, PRM2 is synthesized as a precursor protein which, upon binding to DNA is successively processed. As we showed previously, Prm2-/- male mice are infertile, while Prm2+/- males are fertile. In this study we analyzed the effect of Prm1-deficiency.

Methods Prm1-deficient mice were generated using CRISPR-Cas-mediated gene editing. Male fertility, sperm function and chromatin packaging were analyzed.

Results Prm1+/- male mice are subfertile while Prm1-/- males are infertile. Prm1-/- display high levels of reactive oxygen species- (ROS-) mediated DNA damage, while Prm1+/- sperm display only moderate DNA damage as shown by immunohistochemical staining (IHC) and agarose gel electrophoresis. Prm1+/- sperm motility is severely reduced. Mass spectrometric analysis and IHC revealed increased histone retention in Prm1-/-, but not Prm1+/- sperm. Further, IHC revealed increased transition protein (TNP) retention in Prm1-/- sperm. Of note, CMA3 staining, indicating protamine-free DNA, stained most Prm1+/- sperm. Interestingly, AU-gel analysis revealed that sperm from Prm1+/- and Prm1-/- mice contain high levels of incompletely processed PRM2 which is not detected in WT. Additionally, the PRM1:PRM2 ratio is skewed from 1:2 in WT to 1:5 in Prm1+/- animals.

Conclusion Our results reveal that loss of PRM1 interferes with fertility, leading to increased histone and TNP retention and defective chromatin condensation. PRM1 seems to be required for PRM2 processing. Of note, accumulation of ROS and sperm DNA damage is detected in testes already, which is earlier compared to Prm2-deficiency.

Activin A and CCR2 are critical regulators of testicular fibrosis

W. Peng1, A. Kepsch1, T. O. Kracht1, H. Hasan1, R. Wijayarathna2,3, E. Wahle1, C. Pleuger1, S.Bhushan1, S. Günther4, A. C. Kauerhof1,2, A. Planini?5,6, D. Fietz7, H.-C. Schuppe8, M. Wygrecka9, K. L. Loveland2,3, D. Ježek5,6, A. Meinhardt1,3, M. P. Hedger2,3, M. Fijak1

1Justus-Liebig-University, Department of Anatomy and Cell Biology, Gießen, Germany; 2Hudson Institute of Medical Research, Centre for Reproductive Health, Clayton, Victoria, Australia; 3Monash University, Department of Molecular & Translational Sciences, Clayton, Victoria, Australia; 4Max Planck Institute for Heart and Lung Research, ECCPS Bio­informatics and Deep Sequencing Platform, Bad Nauheim, Germany; 5University of Zagreb, School of Medicine, Department of Histology and Embryology, Zagreb, Croatia; 6University of Zagreb, School of Medicine, Centre of Excellence for Reproductive and Regenerative Medicine, Zagreb, Croatia; 7Justus-Liebig-University, Department of Veterinary Anatomy, Histology and Embryology, Gießen, Germany; 8Justus-Liebig-University, Department of Urology, Paediatric Uro­logy and Andrology, Gießen, Germany; 9University of Gießen and Marburg Lung Center, Center for Infection and Genomics of the Lung, Member of the German Center for Lung Research, Gießen, Germany

Experimental autoimmune orchitis (EAO) is a mouse model of chronic testicular inflammation, resembling the histopathology of some cases of human idiopathic spermatogenic disruption. Murine EAO features ­elevated inflammatory and fibrotic mediators, including TNF, CCL2 and activin A, immune cell infiltration, sloughing of seminiferous epithelium, leading to fibrosis and subsequent infertility. Activin A regulates inflammation and fibrosis, while CCR2 (CCL2 receptor) is involved in the trafficking of macrophages (M?), which together with fibrocytes contribute to fibrogenesis in several organs. Therefore, we aimed to investigate the role of the activin A-CCR2-M?-axis in EAO pathology. EAO was induced in C57BL/6J and Ccr2- /- mice (n = 5–8/group). Overall, deletion of Ccr2 led to reductions in organ damage, collagen deposition, and leukocyte infiltration, including fibronectin+ M?, concomitant with reduced activin A (Inhba), Cxcr4 and matrix metalloproteases (Mmp), in EAO, as established by immunofluorescence, flow cytometry and qRT-PCR. Notably, biopsies from patients with impaired spermatogenesis and fibrotic alterations also demonstrated the presence of fibronectin+ M?. Bone marrow-derived M? (BMDMs), as a surrogate for testicular M?, were cultured in the presence of activin A. Activin A increased CCR2, fibronectin, CXCR4 and Mmp2 expression in BMDMs (n = 6–8). Treatment with follistatin, a potent activin A antagonist, abolished these effects. Inhibition of activin A in vivo during EAO using a mouse model overexpressing follistatin, reduced tissue damage, while Mmps and Timp1 mRNA expression levels were positively correlated with Inhba mRNA, concomitant with decreased accumulation of testicular collagen I+ M? in EAO.

Altogether, our data indicate that CCR2 and activin A regulate the development of fibrosis during testicular inflammation and underline a crucial pro-fibrotic function for the M? in inflammation-associated fibrotic remodeling in EAO.

Molecular expression pattern of TOP2A and SF1 in normal and impaired spermatogenesis in human testis

J. Ott1, K. Hartmann1, M. Baker2, S. Kliesch3, A. Pilatz4, H.-C. Schuppe4, M. Bergmann5, D. Fietz5

1Justus-Liebig-University, Institute of Veterinary Anatomy, Histology and Embryology, Gießen, Germany; 2University of Newcastle, Australia School of Environmental and Life Science, Newcastle, Australia; 3University Clinic Münster, Department of Clinical Andrology, Centre for Reproductive Medicine and Andrology, Münster, Germany; 4University Clinic Gießen, Department of Urology, Pediatric Urology and Andrology, Gießen, Germany; 5Justus-Liebig-University, Institute of Veterinary Anatomy, Histology and Embryology, Gießen, Germany

Introduction In the testis, correct expres­sion and function of transcription and splicing factors are essential as aberrant splicing may lead to spermatogenic defects. We hypo­thesize that changed expression of ­topoisomerase 2? (TOP2A) and splicing factor 1 (SF1) is involved in defective spermatogenesis and assessed their expression in human testicular biopsies.

Material and Methods Histological evaluation of n = 48 biopsies showed normal spermatogenesis (NSP, n = 16), hypospermatogenesis (HYP, n = 13), maturation arrest (MA, n = 6), or Sertoli cell-only syndrome (SCO, n = 13). We performed RT-PCR from homogenates and laser-assisted microdissection material, qRT-PCR and immunohistochemistry, immune electron microscopy (iEM, only SF1), and Western Blot (only TOP2A).

Results TOP2A protein was detected ­within germ cell nuclei from spermatogonia up to step 4 elongating spermatids in NSP. No somatic cells were stained, which was verified by WB. RT-PCR showed transcripts in germ cells, but also in Sertoli cells. Quantitative analysis (qRT-PCR) showed significantly lower TOP2A expression in SCO specimens. SF1 protein was detected in nuclei of somatic cells, endothelial cells, and germ cells with distinct presence in early round spermatids. This staining pattern was confirmed using iEM and consistent for all histopathological groups. Congruent mRNA expression of SF1 was shown using RT-PCR. qRT-PCR showed no significant differences in mRNA expression for SF1.

Discussion Expression of TOP2A in intact spermatogenesis confirms its importance for germ cell development and cell division. So far, no involvement in germ cell loss or MA was detected. However, significantly lower transcript levels in SCO could hint at a potential use as biomarker for germ cell loss. The ubiquitous expression of SF1 underlines the central role of alternative splicing in the testis. Our results and previous studies suggest up- and downregulation of SF1 to have an impact on testis function and faulty development.

Development of a bead-based calibration strategy for the assessment of DNA fragmentation in viable spermatozoa

L. Teschke, R. da Costa, S. Schlatt

Centre of Reproductive Medicine and Andrology, Münster, Germany

Introduction Increased sperm DNA fragmentation has been associated with male infertility and adverse effects in natural and assisted reproduction [1]. However, methods for evaluating this parameter lack specificity and predictive power [2]. A significant reason for these limitations is the absence of accurate calibration systems and the non-consideration of viability during analysis. Recently, we proposed a co-staining called “DNA and Membrane Integrity test” (DMI test) [3], en­abling unison evaluation of these parameters by flow cytometry. To evolve this method, we aim to develop a calibration strategy based on fluorescent micro-beads.

Methods Three different types of microspheres were used: Polybead® Microspheres 3 ?m, ArC™ Compensation Beads 6 ?m and CytoFLEX Daily QC Fluorospheres 3 ?m. The first 2 were stained with Invitrogen™ LIVE/DEAD™ Green and Dynomics DY-480XL. The overtime fluorescence stability of the dyes was evaluated. Further, seminal samples from donors were used to assess the accuracy of using microbeads as a calibration tool (n = 15) and of the new DMI method in comparison to SCSA (n = 10).

Results No decline in fluorescence was found in either dye but difficulties during assay conditions made Polybead® and ArC™ beads unsuitable for calibration purposes. Contrary, CytoFLEX Fluorospheres showed superior gating facility and generated identical results when comparing the DMI test and SCSA in the same samples.

Conclusions We established a bead-based calibration strategy that can be used to evaluate DNA fragmentation and membrane integrity in clinical samples simultaneously. This sample-independent calibration method can help establish increased accuracy and reproducibility in sperm DNA fragmentation analysis in andrology.

References:

1. Simon L, et al. A systematic review and meta-analysis to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic sperm injection outcome. Asian J Androl 2017; 19: 80–90.

2. Cissen M, et al. Measuring sperm DNA fragmentation and clinical outcomes of medically assisted reproduction: A systematic review and meta-analysis. PLoS One 2016; 11: e0165125.

3. Da Costa R, et al. Simultaneous detection of sperm membrane integrity and DNA fragmentation by flow ­cytometry: A novel and rapid tool for sperm analysis. Andrology 2021. https://doi.org/10.1111/andr.13017.

Dexamethasone is a regulator of ­human testicular peritubular cells

Y. K. Stepanov1, J. D. Speidel2, C. Herrmann2, N. Schmid2, R. Behr3, F.-M. Köhn4, J. B. Stöckl1, U. Pickel5, M. Trottmann5, T. Fröhlich1*, A. Mayerhofer2*, H. Welter2*

*shared senior authorship

1Ludwig-Maximimilian-University, Laboratory for ­Functional Genome Analysis (LAFUGA), Gene Center, München; 2Ludwig-Maximimilian-University, Biomedical Center, Cell Biology, Anatomy III, Faculty of Medicine, München; 3Leibniz Institute for Primate Research, Platform Degenerative Diseases, German Primate Center, Göttingen; 4Andrologicum, München; 5Urologie und Andrologie, München, Germany

Introduction Human testicular peritubular cells (HTPCs) form a compartment between the seminiferous epithelium and the interstitial areas of the testis, contract and relax and thereby transport sperm and have immunological roles. The expression of the glucocorticoid receptor (GR) indicates that they represent a target of glucocorticoids (GCs), yet precise consequences of GC actions are unknown. Therefore, we studied how treatment with the synthetic GC dexamethasone (Dex) affects cultured HTPCs, which serve as a unique window into the human testis in health and disease.

Methods To examine changes in cytokines secretion upon treatment with Dex, mainly qPCR and ELISAs were used. GR-specific Dex actions were verified by siRNA-mediated GR knockdown. In addition, a holistic mass spectrometry-based proteome analysis of cellular and secreted proteins was performed.

Results Transcript levels of proinflammatory cytokines IL6, IL8, MCP-1, MCP3 and IL1B, were significantly lower upon Dex treatment. As shown for IL6, the effect was reduced upon GR-knockdown by siGR RNA. Dex also lowered GR levels. Proteomics further revealed strong responses, which are increased with prolonged Dex treatment intervals. Bioinformatics analysis revealed that the differentially abundant proteins include numerous extracellular matrix- (ECM-) and basement membrane components of the collagen- and laminin-families. In the context of cytoskeletal organization, proteins of the actin-related machinery were more and microtubule-related proteins were less abundant.

Conclusion Our results identify distinct GR-dependent Dex actions in HTPCs. They include inflammatory responses and regulation of the GR, as well as massive changes in the cellular phenotype. If transferable to the human testis, such changes may also result in vivo, in men upon treatment with Dex and could affect the cells, the peritubular compartment, and overall testicular functions.

Grants: Supported by DFG, project number 427588170)

In-depth analysis of protamine 1 & 2 double deficient mice

C. Wiesejahn, G. E. Merges, L. Arévalo, S. Schneider, H. Schorle

Uniklinikum Bonn, Entwicklungspathologie, Bonn, Deutschland

Introduction During spermiogenesis DNA bound histones are exchanged for protamines (PRMs) leading to hypercondensation of DNA. This exchange requires transition proteins (TNPs) to firstly interact with histones before both are evicted from the nucleus of elongating spermatids and replaced by PRMs. Some mammals, like rodents and primates, express two different protamine genes, Prm1 and Prm2. They are found in a species-specific ratio and alteration of this ratio is associated with sub- or infertility. Our lab has established mouse models deficient for PRM1 and PRM2. Here, we report on the generation and analysis of Prm1+/- Prm2+/- (dHET) mice.

Methods Acid urea gels and subsequent western blots, as well as IHC of dHET sperm were used to look for retention of proteins like histones and TNPs. Numbers of alive and dead sperm was monitored via eosin/nigro­sine (EN) staining and the head morphology was analyzed.

Results While Prm2+/- males are fertile, Prm1+/- males are subfertile and dHET males are infertile. We found that histones, TNPs and unprocessed PRM2 are retained on sperm DNA. As a consequence, nuclei appear more rounded in shape and are slightly bigger, suggestive of impaired DNA-hypercondensation. Interestingly, while DNA of Prm1-/- and Prm2-/- sperm is completely degraded, DNA from dHET sperm showed considerably less damage. Further, EN staining revealed that a moderate percentage (29%) of dHET sperm is alive.

Conclusion The data indicate, that dHET sperm should in principle be able to fertilize the oocyte, suggesting that the infertility seen might in part be due to failure of development post fertilization. The dHET males reveal, that PRMs seem to be available in limited amount only, since reduction to 50% is incompatible with fertility.

Mapping stage-specific alternative splicing patterns during human spermatogenesis

L. M. Siebert-Kuss1, H. Krenz2, T. Tekath2, M. Wöste2, S. Di Persio1, N. Terwort1, M. J. Wyrwoll3, J.-F. Cremers1, J. Wistuba1, M. Dugas4, S. Kliesch1, S. Schlatt1, F. Tüttelmann3, J. Gromoll1, N. Neuhaus1, S. Laurentino1

1Universitätsklinikum Münster, Centrum für Reproduktionsmedizin und Andrologie, Münster; 2Universitätsklinikum Münster, Institut für Medizinische Informatik, Münster; 3Universitätsklinikum Münster, Institut für Reproduktionsgenetik, Münster; 4Universitätsklinikum Heidelberg, Institut für Medizinische Informatik, Heidelberg, Deutschland

Introduction In the testis, extensive alternative splicing (AS) is one of the regulatory mechanisms determining the expression of a gene. AS leads to the production of different transcript isoforms (e.g. coding or non-coding) and its dysregulation is associated with the etiology of several diseases such as cancer. Due to lacking knowledge of germ cell specific AS events, its role in the pathology of male infertility remains unclear. To this end, we aimed at mapping stage-specific isoforms to examine the role of AS throughout human spermatogenesis.

Methods We performed total RNA-sequencing on testicular biopsies of non-obstructive azoospermia patients with a Sertoli cell-only phenotype (SCO, n = 3) and spermatogenic arrests at spermatogonial (SPG, n = 4), spermatocyte (SPC, n = 3) and spermatid (SPD, n = 3) level, respectively. Also obstructive azoospermia patients with complete spermatogenesis were included as controls (CTR, n = 3). In order to obtain germ cell-specific expression profiles we compared samples varying only in one consecutive germ cell-stage (i. e. SCO vs SPG, SPG vs SPC, SPC vs SPD, SPD vs CTR).

Results We identified 839 to 4138 differentially expressed genes (DEGs). Moreover, we evaluated the differential transcript usage (DTU) by comparing proportional contribution of the transcript isoforms to the overall expression of their reference gene. Intriguingly, genes with DTU showed limited overlap with DEGs, indicating that AS is mainly uncoupled from the level of gene expression and constitutes an independent layer of gene regulation. This was observed especially in spermiogenesis, displaying an increased number of genes with DTU (1702) compared to DEGs (839).

Conclusion Our study provides insights into the changes of transcriptomic programming during human spermatogenesis and helps increasing our knowledge about the molecular mechanisms controlling germ cell differentiation.

Grants: German Research Foundation Clinical Research Unit “Male Germ Cells” 326.

The marmoset spermatogonial compartment at single-cell RNA and protein level during development

J. M. Paturlanne1, S. Di Persio1, C. Herden1, M. Wöste2, X. Li3, G. Meyer zu Hörste3, J. Wistuba4, S. Schlatt5, S. Laurentino6, N. Neuhaus1

1Centrum für Reproduktionsmedizin und Andrologie, Keimbahnstammzellen, Münster; 2Institut für Medizinische ­Informatik, Münster; 3Klinik für Neurologie mit Institut für Translationale Neurologie, Münster; 4Centrum für Reproduk­tions­medizin und Andrologie, Entwicklung und Physiologie des Hodens, Münster; 5Centrum für Reproduktionsmedizin und Andrologie, Spermatogenese und Hodenfunktion, ­Münster; 6Centrum für Reproduktionsmedizin und Andrologie, Endokrinologie und Epigenetik, Münster, Deutschland

Introduction The adult human spermatogonial compartment is well explored at the protein and single-cell RNA (scRNA-seq) level, with PIWIL4+ spermatogonia considered the most undifferentiated germ cells. The total number of spermatogonia changes dynamically during human postnatal development before it increases upon puberty. However, due to limited access to human samples, the dynamics of spermatogonial subpopulations during development remain unsolved. In order to address those systematically, a suitable animal model is required. Using the marmoset could fill the gap, as these monkeys’ early differentiation of the male germ line is rather similar to the human situation.

Methods Testicular tissue from neonatal (NM, n = 3; 1–2 days old), prepubertal (PM, n = 3; 6 months old), and adult (AM, n = 2; 4 and 10 years old) marmosets were subjected to scRNA-seq. Fixed testicular tissues from NM, PM, pubertal (P; 8 m. o.), AM, and adult human (AH; 25–40 y. o.) (n = 3 each) were analysed by immunohistochemistry. MAGEA4, PIWIL4, and NANOS3 protein expressing spermatogonia were quantified.

Results The number of MAGEA4+ cells during marmoset development changed similarly to the human germ line. During marmoset development, PIWIL4+ and ­NANOS3+ cells are present at all stages analysed and their numbers remained stable (3,29 ± 1,72 and 1,59 ± 0,59 in NM; 3,25 ± 0,61 and 1,05 ± 0,18 in AM, respectively). According to ­scRNA-seq, PIWIL4+ cells were present throughout postnatal development but only formed a transcriptionally distinct subgroup at the PM stage. The numbers of undifferentiated spermatogonia, PIWIL4+ and NANOS3+, in AM and AH were similar (see above for AM; 2,71 ± 0,33 and 1,27 ± 0,59 in AH).

Conclusion The numbers of MAGEA4+ spermatogonia during development and of PIWIL4+ and NANOS3+ in the adult are comparable between marmoset and human testis at protein level, rendering the marmoset a valu­able model for studying human spermatogonial subpopulations during postnatal development

The role of unprocessed PRM2

L. Arévalo, G. Merges, S. Schneider, H. Schorle

Universitätsklinikum Bonn, Entwicklungspathologie, Bonn, Deutschland

Introduction Protamines are small sperm-specific proteins that play an important role in packaging and protecting the paternal genome on its way to the fertilization site. Primates and rodents express two protamines, PRM1 and PRM2. PRM2 differs from PRM1 primarily in its N-terminal domain (cP2), which is cleaved off sequentially during paternal chromatin packaging. DNA damage and infertility have been linked to alterations in this processing. However, the precise function of the cP2 domain remains unknown.

Methods Using CRISPR/Cas9, we created mice bearing a targeted deletion of cP2. Fertility, sperm count, function and testis histo­logy of the resulting cP2 deficient males was analyzed. Detailed analyses were done using immunohistochemistry, western blot, mass spectrometry, RNAseq and in vitro assays.

Results We show that cP2 deficiency causes incomplete histone-to-protamine transition. While increased histone retention cannot be observed, transition proteins are not evicted from the nucleus, resulting in incomplete protamine incorporation, sperm DNA de­gradation and infertility. Using a cP2 specific antibody, we show that not all of PRM2 is processed in WT sperm with unprocessed PRM2 accumulating in the cytoplasm of condensed spermatids. In vitro the cP2 sequence is able to interact with transition protein 1. Additionally, we find that sperm-specific histone variant H2A.L.2 retention fails in cP2 deficient sperm chromatin. Polycomb recessive complex component Pcgf5 seems to be expressed at higher levels in cP2-deficient testes and is involved in histone H2A monoubiquitination.

Conclusions Altogether, this indicates that unprocessed PRM2 with its cP2 domain is directly involved in transition protein eviction and the retention of H2A.L.2. Our ongoing investigation involves in vitro and in vivo co-IP analyses to define cP2-specific interaction partners and analyses of histone retention and modification patterns.

Bi-allelic variants in INSL3 and RXFP2 cause bilateral cryptor­chidism and male infertility

A.-K. Dicke1, M. J. Wyrwoll1, J. C. Albrethsen2, A. S. Busch3, D. Fietz4, A. Pilatz5, C. Bühlmann1, A. Juul2, S. Kliesch6, J. Gromoll6, F. Tüttelmann1, B. Stallmeyer1

1University of Münster, Institute of Reproductive Genetics, Münster, Germany; 2University of Copenhagen, Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark; 3University Children‘s Hospital Münster, Department of General Pediatrics, Münster, Germany; 4Justus-Liebig-University, Institute of Veterinary Anatomy, Histology and Embryology, Gießen, Germany; 5Justus-Liebig-University, Clinic for Urology, Paediatric Urology and Andrology, Gießen, Germany; 6University Hospital Münster, Centre of Reproductive Medicine and Andrology, Münster, Germany

Introduction Impaired testicular descent is a common birth defect, leading to cryptorchidism and predisposing to infertility and testicular cancer. In mice, the hormone insulin-like factor 3 (INSL3) and its receptor relaxin family peptide receptor 2 (RXFP2) are essential for inguinal canal opening and gubernaculum dilatation pulling the testes into the scrotum. For decades, the significance of human variants in the genes encoding these proteins has been ambiguous. Heterozygous variants in INSL3 and RXFP2 were proposed as associated with cryptorchidism but also frequently found in unaffected controls.

Methods We screened exome sequencing data of > 1600 infertile men for high impact variants in INSL3 and RXFP2. Clinical and testicular phenotypes were evaluated, as well as INSL3 serum levels in patients with identified rare (MAF < 0.01) LoF variants. For those, an additional immunohistochemical detection of INSL3 in testicular tissue was carried out.

Results Two patients with homozygous LoF variants in either INSL3 or RXFP2 were identified. The INSL3 variant c.143dup p. (­Arg50Profs*33) is located in the first exon and the RXFP2 variant c.1406del p.(Phe469Serfs*8) affects the transmembrane domain, thus encoding a non-functional protein if not degraded. Both patients showed bilateral cryptorchidism, azoospermia, elevated FSH and variably impaired spermatogenesis. An association of heterozygous LoF variants with cryptorchidism was excluded for both genes.

Conclusion This is the first report of homozygous LoF variants in INSL3 and RXFP2 in adults. Our findings support an autosomal recessive inheritance mode for both genes and indicate INSL3 to be a potential germ cell maintenance factor. Screening for pathogenic variants in INSL3/RXFP2 should be considered in cases of familial cryptorchidism.

Grants: This work was supported by the DFG Clinical Research Unit 326 “Male Germ Cells”.

C14orf39 – from a novel candidate to a strong infertility gene

S. A. Koser1, B. Stallmeyer1, S. Kliesch2, F. Tüttelmann1, C. Friedrich1

1University of Münster, Institute of Reproductive Genetics, Münster; 2University of Münster, Centre of Reproductive Medicine and Andrology, Münster, Germany

Introduction Genetics contribute to the aetiology of male infertility. Whole-exome-sequencing (WES) lately revealed candidate genes for severe quantitative spermatogenic failure, i.e. crypto-/azoospermia. Sufficient evidence for a gene-disease-relationship is needed for implementation into clinical diagnostics. C14orf39 is involved in chromosome synapsis and its disruption has recently been reported to cause infertility due to meiotic arrest (MeiA) in mice and humans.

Methods Within the Male Reproductive Genetics (MERGE) cohort, patients with unexplained infertility after andrological exam were analysed by WES. Results were filtered for rare (< 1%, gnomAD) homozygous loss-of-function or missense variants with likely high impact (CADD > 20), focussing on meiosis related genes with published infertile mouse models. Clinical validity assessment was done according to ClinGen.

Results WES identified 2 unrelated azoospermic men with the same homozygous predicted splice acceptor variant (c.1059-2A > G) in C14orf39. The testicular biopsy available from one affected man showed MeiA. Two additional patients, one being azoo- (MeiA), another cryptozoospermic (no biopsy), were compound heterozygous for two frameshift variants (c. [325_326del]; [1154_1157del]) and a frameshift along with a predicted splice region variant (c. [453_454del]; [511+3A > G]), respectively. Testis volume and hormonal levels were within the normal range except for slightly elevated FSH in one MeiA-patient.

Conclusions WES enabled to solve four cases with variants in C14orf39. Based on the available evidence, C14orf39 reaches a strong level of clinical validity justifying its use in genetic diagnostics for male infertility. Follow-up on the cryptozoospermic patient and further functional analysis of the variants will be of high relevance for predicting the chances of assisted reproduction in subsequent patients.

Grants: This work was supported by the DFG Clinical Research Unit 326 “Male Germ Cells”.