Thomas Greither

Polycystic ovary syndrome (PCOS) is often associated with metabolic abnormalities in the affected patients such as obesity or a dysregulated glucose metabolism/insulin resistance (IR). IR affects the serum levels of several circulating microRNAs; however, studies on the association between IR-related microRNAs and PCOS are scarce. Therefore, we quantified the serum levels of the IR-associated microRNAs miR-93, miR-148a, miR-216a, miR-224 and miR-320a via qPCR in a cohort of 358 infertility patients, of whom 136 were diagnosed with PCOS. In bivariate correlation analyses, the serum levels of miR-93 and miR-216a were inversely associated with dipeptidyl peptidase 4 serum concentrations, and the miR-320a serum levels were significantly downregulated in PCOS patients (p = 0.02, Mann–Whitney U test). Interestingly, in all patients who achieved pregnancy after Assisted Reproductive Technology (ART) cycles, the serum levels of the five IR-associated microRNAs were significantly elevated compared to those of non-pregnant patients. In cell culture experiments, we detected a significant upregulation of miR-320a expression following testosterone stimulation over 24 and 48 h in KGN and COV434 granulosa carcinoma cells. In conclusion, we demonstrated a significantly reduced serum level of the IR-associated miR-320a in our patient cohort. This result once again demonstrates the close relationship between metabolic disorders and the dysregulation of microRNA expression patterns in PCOS.

Background: Although male factor accounts for 40%–50% of unintended childlessness, we are far from fully understanding the detailed causes. Usually, affected men cannot even be provided with a molecular diagnosis.Objectives: We aimed at a higher resolution of the human sperm proteome for better understanding of the molecular causes of male infertility. We were particularly interested in why reduced sperm count decreases fertility despite many normal‐looking spermatozoa and which proteins might be involved.Material and methods: Applying mass spectrometry analysis, we qualitatively and quantitatively examined the proteomic profiles of spermatozoa from 76 men differing in fertility. Infertile men had abnormal semen parameters and were involuntarily childless. Fertile subjects exhibited normozoospermia and had fathered children without medical assistance.Results: We discovered proteins from about 7000 coding genes in the human sperm proteome. These were mainly known for involvements in cellular motility, response to stimuli, adhesion, and reproduction. Numbers of sperm proteins showing at least threefold deviating abundances increased from oligozoospermia (N= 153) and oligoasthenozoospermia (N= 154) to oligoasthenoteratozoospermia (N= 368). Deregulated sperm proteins primarily engaged in flagellar assembly and sperm motility, fertilization, and male gametogenesis. Most of these participated in a larger network of male infertility genes and proteins.Discussion: We expose 31 sperm proteins displaying deviant abundances under infertility, which already were known before to have fertility relevance, including ACTL9, CCIN, CFAP47, CFAP65, CFAP251 (WDR66), DNAH1, and SPEM1. We propose 18 additional sperm proteins with at least eightfold differential abundance for further testing of their diagnostic potential, such as C2orf16, CYLC1, SPATA31E1, SPATA31D1, SPATA48, EFHB (CFAP21), and FAM161A.Conclusion: Our results shed light on the molecular background of the dysfunctionality of the fewer spermatozoa produced in oligozoospermia and syndromes including it. The male infertility network presented may prove useful in further elucidating the molecular mechanism of male infertility.

Deciphering the functional relationships of genes resulting from genome-wide screens for polymorphisms that are associated with phenotypic variations can be challenging. However, given the common association with certain phenotypes, a functional link should exist. We have tested this prediction in newly sequenced exomes of altogether 100 men representing different states of fertility. Fertile subjects presented with normal semen parameters and had naturally fathered offspring. In contrast, infertile probands were involuntarily childless and had reduced sperm quantity and quality. Genome-wide association study (GWAS) linked twelve non-synonymous single-nucleotide polymorphisms (SNPs) to fertility variation between both cohorts. The SNPs localized to nine genes for which previous evidence is in line with a role in male fertility maintenance: ANAPC1, CES1, FAM131C, HLA-DRB1, KMT2C, NOMO1, SAA1, SRGAP2, and SUSD2. Most of the SNPs residing in these genes imply amino acid exchanges that should only moderately affect protein functionality. In addition, proteins encoded by genes from present GWAS occupied peripheral positions in a protein–protein interaction network, the backbone of which consisted of genes listed in the Online Mendelian Inheritance in Man (OMIM) database for their implication in male infertility. Suggestive of an indirect impact on male fertility, the genes focused were indeed linked to each other, albeit mediated by other interactants. Thus, the chances of identifying a central player in male infertility by GWAS could be limited in general. Furthermore, the SNPs determined and the genes containing these might prove to have potential as biomarkers in the diagnosis of male fertility.

Our study aimed to assess the applicability of miR-486 in combination with soluble GP88 protein as a diagnostic and/or predictive biomarker for prostate cancer (PCa) patients. miR-486 and GP88 levels in serum samples from 136 patients undergoing MRI-guided biopsy of the prostate were assessed by qRT–PCR and ELISA, respectively. Of these, 86 patients received a histologically confirmed diagnosis of PCa. Neither marker showed an association with the diagnosis of cancer. PCa patients were separated based on (i) treatment into patients with active surveillance or patients with any type of curative treatment and (ii) age into elderly (>68 years) patients and younger patients (≤68 years). In elderly patients (N = 41) with the intention of curative treatment at optimized cut-off values, significantly higher GP88 levels (p = 0.018) and lower miR-486 levels (p = 0.014) were observed. The total PSA level and ISUP biopsy grade were used in a baseline model for predicting definitive therapy. The baseline model exhibited an area under the curve (AUC) of 0.783 (p = 0.005). The addition of the serum biomarkers miR-486 and GP88 to the baseline model yielded an improved model with an AUC of 0.808 (p = 0.002). Altogether, combined miR-486 and GP88 serum levels are associated with and are therefore suggested as supportive biomarkers for therapy decisions, particularly in elderly PCa patients.

The four human PIWI-LIKE gene family members PIWI-LIKE 1–4 play a pivotal role in stem cell maintenance and transposon repression in the human germline. Therefore, dysregulation of these genes negatively influences the genetic stability of the respective germ cell and subsequent development and maturation. Recently, we demonstrated that a lower PIWI-LIKE 2 mRNA expression in ejaculated spermatozoa is more frequent in men with oligozoospermia. In this study, we analysed how PIWI-LIKE 1–4 mRNA expression in ejaculated spermatozoa predicts ART outcome. From 160 IVF or ICSI cycles, portions of swim-up spermatozoa used for fertilization were collected, and the total RNA was isolated. PIWI-LIKE 1–4 mRNA expression was measured by qPCR using TaqMan probes with GAPDH as a reference gene. PIWI-LIKE 1 and 2 transcript levels in the spermatozoa of the swim-up fraction were positively correlated to each other (r_S = 0.78; p < 0.001). Moreover, lower PIWI-LIKE 2 mRNA levels, as well as lower PIWI-LIKE 1 mRNA levels, in these spermatozoa were positively associated with a fertilization rate ≥ 50% in the respective ART cycles (p = 0.02 and p = 0.0499, Mann–Whitney U-Test). When separately analysing IVF and ICSI cycles, PIWI-LIKE 1 and 2 transcript levels were only significantly associated to increased fertilization rates in IVF, yet not in ICSI cycles. Spermatozoal PIWI-LIKE 3 and 4 transcript levels were not significantly associated to fertilization rates in ART cycles. In conclusion, lower levels of spermatozoal PIWI-LIKE 1 and 2 mRNA levels are positively associated with a higher fertilization rate in IVF cycles.

Progranulin (PGRN)/GP88 is a growth factor that is expressed in a wide range of tumor tissues. The secreted form is involved in various biological processes including proliferation and inflammation. In several tumor types, the serum GP88 level is associated with a patient’s prognosis; however, data for oral squamous cell carcinomas (OSCCs) have not yet been reported. We measured the serum GP88 levels in 96 OSCC patients by an enzyme immunosorbent assay (EIA) and correlated these data with clinicopathological parameters and patient outcomes. The GP88 levels in the serum of OSCC patients and healthy volunteers were comparable. In OSCC patients, the levels did not correlate with age, sex, or TNM status. In a Kaplan–Meier survival analysis, a serum GP88 level < 68 ng/mL was significantly associated with worsened survival (p = 0.0005, log-rank-test) as well as in uni- and multivariate Cox regression analyses (RR = 4.6 [1.6–12.9], p = 0.004 and RR = 4.2 [1.2–12.0], p = 0.008). This effect was predominant in OSCC patients older than 60.5 years (p = 0.027), while in younger patients no significant association between serum GP88 levels and prognosis could be observed. Altogether, lower serum GP88 levels are significantly associated with a worsened outcome for an OSCC and may be an interesting candidate for risk stratification during OSCC therapy.

Impairment of male fertility is one of the major public health issues worldwide. Nevertheless, genetic causes of male sub- and infertility can often only be suspected due to the lack of reliable and easy-to-use routine tests. Yet, the development of a marker panel is complicated by the large quantity of potentially predictive markers. Actually, hundreds or even thousands of genes could have fertility relevance. Thus, a systematic method enabling a selection of the most predictive markers out of the many candidates is required. As a criterion for marker selection, we derived a gene-specific score, which we refer to as fertility relevance probability (FRP). For this purpose, we first categorized 2,753 testis-expressed genes as either candidate markers or non-candidates, according to phenotypes in male knockout mice. In a parallel approach, 2,502 genes were classified as candidate markers or non-candidates based on phenotypes in men. Subsequently, we conducted logistic regression analyses with evolutionary rates of genes (dN/dS), transcription levels in testis relative to other organs, and connectivity of the encoded proteins in a protein-protein interaction network as covariates. In confirmation of the procedure, FRP values showed the expected pattern, thus being overall higher in genes with known relevance for fertility than in their counterparts without corresponding evidence. In addition, higher FRP values corresponded with an increased dysregulation of protein abundance in spermatozoa of 37 men with normal and 38 men with impaired fertility. Present analyses resulted in a ranking of genes according to their probable predictive power as candidate markers for male fertility impairment. Thus, AKAP4, TNP1, DAZL, BRDT, DMRT1, SPO11, ZPBP, HORMAD1, and SMC1B are prime candidates toward a marker panel for male fertility impairment. Additional candidate markers are DDX4, SHCBP1L, CCDC155, ODF1, DMRTB1, ASZ1, BOLL, FKBP6, SLC25A31, PRSS21, and RNF17. FRP inference additionally provides clues for potential new markers, thereunder TEX37 and POU4F2. The results of our logistic regression analyses are freely available at the PreFer Genes website (https://prefer-genes.uni-mainz.de/).

Soft tissue sarcoma (STS) is a heterogeneous group of rare malignancies with a five-year survival rate of approximately 50%. Reliable molecular markers for risk stratification and subsequent therapy management are still needed. Therefore, we analyzed the prognostic potential of miR-155-5p and miR-203a-3p expression in a cohort of 79 STS patients. MiR-155-5p and miR-203a-3p expression was measured from tumor total RNA by qPCR and correlated with the demographic, clinicopathological, and prognostic data of the patients. Elevated miR-155-5p expression was significantly associated with increased tumor stage and hypoxia-associated mRNA/protein expression. High miR-155-5p expression and low miR-203a-3p expression, as well as a combination of high miR-155-5p and low miR-203a-3p expression, were significantly associated with poor disease-specific survival in STS patients in the Kaplan–Meier survival analyses (p = 0.027, p = 0.001 and p = 0.0003, respectively) and in the univariate Cox regression analyses (RR = 1.96; p = 0.031; RR = 2.59; p = 0.002 and RR = 4.76; p = 0.001, respectively), but not in the multivariate Cox regression analyses. In conclusion, the oncomiR miR-155-5p and the tumor suppressor-miR miR-203a-3p exhibit an association with STS patient prognosis and are suggested as candidates for risk assessment.

Objectives: Adipogenesis is the differentiation process generating mature adipocytes from undifferentiated mesenchymal stem cells. The differentiation can be inhibited by androgens, although knowledge about intracellular effectors of this inhibition is scarce. Recently, androgen-regulated microRNAs were detected as interesting candidates in this context. In this study, we analyse the role of miR-130a and miR-301 in the adipogenesis of human SGBS preadipocytes and whether they are prone to androgen regulation. Materials and Methods: microRNA expression during adipogenic differentiation with or without androgen stimulation was measured by qPCR. Putative target genes of miR-130a and miR-301 were identified by target database search and validated in luciferase reporter assays. Results: miR-130a and miR-301 are both significantly downregulated on day 3 and day 5 of adipogenic differentiation in comparison to day 0. Under androgen stimulation, a significant upregulation of miR-130a was detected after 7 days of adipogenesis lasting to day 14, while miR-301 did not change significantly until day 14. Luciferase reporter assays revealed the androgen receptor (AR), adiponectin (ADIPOQ) and tumour necrosis factor alpha (TNFα) as miR-130a target genes. Conclusions: miR-130a is an androgen-regulated microRNA that is downregulated during the early phase of adipogenesis and exerts its functions by regulating AR and ADIPOQ translation. These data may help to identify new signalling pathways associated with the androgen-mediated inhibition of adipogenesis.