Real time quantitative PCR (qPCR) is the method of choice for miRNA expression studies. For relative quantification of miRNAs, normalization to proper reference genes is mandatory. Currently, no validated reference genes for miRNA qPCR in prostate cancer are available. In this study, the expression of four putative reference genes (hsa-miR-16, hsa-miR-130b, RNU6-2, SNORD7) was examined with regard to their use as normalizer. After SNORD7 was already shown an inappropriate reference gene in preliminary experiments using total RNA pools, we studied the expression of the putative reference genes in tissue and normal adjacent tissue sample pairs from 76 men with untreated prostate carcinoma collected after radical prostatectomy. hsa-miR-130b and RNU6-2 showed no significantly different expression between the matched malignant and non-malignant tissue samples, whereas hsa-miR-16 was significantly underexpressed in malignant tissue. Softwares geNorm and Normfinder predicted hsa-miR-130b and the geometric mean of hsa-miR-130b and RNU6-2 as the most stable reference genes. Normalization of the four miRNAs hsa-miR-96, hsa-miR-125b, hsa-miR-205, and hsa-miR-375, which were previously shown to be regulated, shows that normalization to hsa-mir-16 can lead to biased results. We recommend using hsa-miR-130b or the geometric mean of hsa-miR-130b and small RNA RNU6-2 for normalization in miRNA expression studies of prostate cancer.
Aged ; Bias (Epidemiology) ; Carcinoma/diagnosis/*genetics/pathology ; Diagnostic Errors/prevention & control ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; MicroRNAs/genetics/*metabolism ; Middle Aged ; Polymerase Chain Reaction ; Prostatic Neoplasms/diagnosis/*genetics/pathology ; *Reference Standards

BACKGROUNDIn view of the high cure rates in patients with testicular germ cell tumors (TGCT), increasing clinical importance is attached to protection of fertility. Long-term infertility due to cytostatic therapy may be expected in more than 50% of the patients at a cumulative dose of cisplatin > 0.6 g/m2. The standard procedure for fertility protection in cancer patients includes cryopreservation of ejaculated spermatozoa. Considering that some patients have tumor-induced azoospermia, we examined the usefulness of testicular sperm extraction before therapy.

METHODA survey of the literature served as a basis for investigating biological and clinical aspects of the impact of chemotherapy on male fertility. A study of our patient population also enabled us to explore the option of extracting sperm from the contralateral healthy testis prior to treatment in 14 azoospermic patients with testicular germ cell tumors.

RESULTSWe were able to successfully recover haploid germ cells in 6/14 testicular biopsies from azoospermic patients with testicular germ cell cancer prior to treatment. Maturation arrest was found in 3/14 cases and Sertoli-cell-only syndrome in the rest. None of the patients had secondary healing or a treatment delay because of the testicular biopsy.

CONCLUSIONSince the post-therapeutic fertility status is difficult to predict in cancer patients, we think that TESE should be regarded as a general option prior to cancer treatment and offered to azoospermic cancer patients. New guidelines should be established in this connection.

Antineoplastic Agents ; adverse effects ; Germinoma ; complications ; drug therapy ; pathology ; Humans ; Infertility, Male ; chemically induced ; etiology ; therapy ; Male ; Oligospermia ; pathology ; Spermatozoa ; cytology ; drug effects ; Testicular Neoplasms ; complications ; drug therapy ; pathology

AIMTo characterize the coexpression of survivin, an inhibitor of apoptosis (IAF), and human telomerase reverse transcriptase (hTERT) in human testes with varying spermatogenic function.

METHODSTranscript levels of survivin mRNA and hTERT mRNA were determined in normal testes (n=11) and testes with defective spermatogenesis (n=28) using real-time reverse-transcription polymerase chain reaction (RT-PCR). The histological work-up was performed according to a modified Johnsen score.

RESULTSExpressions of both survivin and hTERT were highest at median levels of 96.8 and 709 in normal spermatogenesis and dropped to 53.3 and 534 in testes with postmeiotic spermatogenic arrest (n=10). In severe spermatogenic failure (n=18), survivin expression was lacking in most specimens (n=16), whereas at least low levels of testicular hTERT expression were largely detectable with a normalized expression of 73 in premeiotic spermatogenic arrest (n=7) and 45 in patients with Sertoli cell-only syndrome (SCOS) (n=3). Both survivin and hTERT expressions increased with a progressing Johnsen score (P for trend=0.001).

CONCLUSIONAlthough both survivin and hTERT are correlated with spermatogenic function, they show different expression patterns in testes of infertile patients. These findings substantiate results from studies in the rodent testis suggesting a predominant expression of survivin in meiotically dividing germ cells.

Biopsy ; DNA-Binding Proteins ; biosynthesis ; Gene Expression ; physiology ; Humans ; Infertility, Male ; metabolism ; Inhibitor of Apoptosis Proteins ; Male ; Microtubule-Associated Proteins ; biosynthesis ; Neoplasm Proteins ; biosynthesis ; Spermatogenesis ; physiology ; Telomerase ; biosynthesis ; Testis ; metabolism