Existing literature suggests evidence that protamine deficiency is related to DNA damage and male fertility. In this meta‐analysis, we analyzed the relationship between the ratio of protamine‐1 and protamine‐2 with male fertility and the association of protamine deficiency with sperm DNA damage. Quality of available cohort studies was evaluated using the Newcastle–Ottawa Scale checklist. Summary effect estimates with 95% confidence intervals (CI) were derived using a random effects model. The effect of the protamine ratio on male fertility was analyzed in nine studies demonstrating a significantly higher value of the protamine ratio in subfertile men (n = 633) when compared with controls (n = 453, SMD = 0.46, 95% CI 0.25–0.66, Z = 4.42, p < 0.00001). Both protamine mRNA (SMD = 0.45, 95% CI 0.11–0.79, Z = 2.63, p = 0.009) and protein ratio (SMD = 0.46, 95% CI 0.25–0.68, Z = 4.22, p < 0.0001) showed significantly increased values in subfertile patients. The association between protamine deficiency and DNA damage was analyzed in 12 studies (n = 845) exhibiting a combined overall correlation coefficient (COR) of 0.53 (95% CI 0.28–0.71, Z = 3.87, p < 0.001). Protamine deficiency measured by CMA3 staining was significantly associated with sperm DNA damage (COR = 0.71, 95% CI 0.48–0.85, Z = 4.87, p < 0.001), whereas the P1/P2 ratio was not (COR = 0.17, 95% CI −0.16 to 0.46, Z = 0.99, p = 0.33). It is concluded that the protamine ratio represents a suitable biomarker for the assessment of sperm quality and protamine deficiency is closely related with sperm DNA damage.

Human sperm contain similar amounts of protamine-1 (P1) and protamine-2 (P2). Although aberrant protamine ratios have been observed in infertile men, functional evidence is provided by protamine knockout mice exhibiting male infertility. As sperm DNA integrity is known to be linked with DNA fragmentation and apoptosis, we investigated whether the protamine ratio or Bcl2 content represent a reliable biomarker to discriminate fertile and infertile men. Real-time quantitative RT-PCR was used for P1, P2 and the apoptotic marker Bcl2 in testicular biopsies (TB; 74 infertile men versus 17 controls) and ejaculates (E; 95 infertile men versus 10 controls). The P1–P2 mRNA ratio differed significantly between groups, namely 1:4 versus 1:3.2 in TB (P = 0.0038) and 1:1.7 versus 1:1 in E (P = 0.0002), for infertile men and controls, respectively. Bcl2 mRNA content was correlated with protamine mRNA ratio (P = 0.0250 for TB; P = 0.0003 for E). Infertile men exhibit a more than 10-fold (P = 0.0155 for TB; P = 7.0 × 10⁻⁶ for E) higher Bcl2 mRNA content versus controls. No correlation was found between absolute sperm density and the protamine mRNA ratio or Bcl2 mRNA content. No significant correlation was demonstrated with fertilization rate after ICSI and either protamine ratio or Bcl2 content. We found significantly aberrant protamine ratios and a higher Bcl2 content in TB and E of infertile men compared to controls, suggesting that these molecules may be useful biomarkers for predicting male infertility.

Background: Most testicular germ cell tumors arise from intratubular germ cell neoplasia unclassified (IGCNU, also referred to as carcinoma in situ), which is thought to originate from a transformed primordial germ cell (PGC)/gonocyte, the fetal germ cell. Analyses of the molecular profile of IGCNU and seminoma show similarities to the expression profile of fetal germ cells/gonocytes. In murine PGCs, expression and interaction of Blimp1 and Prmt5 results in arginine 3 dimethylation of histone H2A and H4. This imposes epigenetic modifications leading to transcriptional repression in mouse PGCs enabling them to escape the somatic differentiation program during migration, while expressing markers of pluripotency. Results: In the present study, we show that BLIMP1 and PRMT5 were expressed and arginine dimethylation of histones H2A and H4 was detected in human male gonocytes at weeks 12–19 of gestation, indicating a role of this mechanism in human fetal germ cell development as well. Moreover, BLIMP1/PRMT5 and histone H2A and H4 arginine 3 dimethylation was present in IGCNU and most seminomas, while downregulated in embryonal carcinoma (EC) and other nonseminomatous tumors. Conclusion: These data reveal similarities in marker expression and histone modification between murine and human PGCs. Moreover, we speculate that the histone H2A and H4 arginine 3 dimethylation might be the mechanism by which IGCNU and seminoma maintain the undifferentiated state while loss of these histone modifications leads to somatic differentiation observed in nonseminomatous tumors.

Could the protamine-1 to protamine-2 mRNA ratio serve as a biomarker to estimate the fertilizing capacity of sperm from men taking part in an IVF/ICSI programme? The protamine mRNA ratio clearly discriminates between fertile and subfertile men and sperm with a normal protamine mRNA ratio exhibit a higher fertilizing capacity in IVF/ICSI. Aberrant sperm protamine ratios are associated with male factor infertility and mRNA ratio is comparable with protein ratio (due to transcriptional stop in elongating spermatids). The study population was drawn from subfertile men, whose female partners participated in IVF or ICSI programmes between September 2010 and February 2012. Normozoospermic healthy volunteers served as controls. Sperm cells were lysed, mRNA extracted, reverse transcribed and subjected to real-time quantitative PCR using specific primer pairs for protamine-1 and protamine-2. Relative protamine-1 and protamine-2 mRNA levels were analysed with the Mann–Whitney U-test (two-tailed). Quantitative RT–PCR for protamines 1 and 2 has been performed in ejaculates from 32 normozoospermic volunteers (control, University Clinic Giessen, Germany) and 306 patients, whose female partners took part in an IVF (n = 76; University Clinic Hamburg, Germany and Shanghai Jiaotong University, China) or an ICSI (n = 230; University Clinic Munich, Germany and Kinderwunschzentrum Wiesbaden, Germany) programme. The sperm protamine mRNA ratio in normozoospermic men (0.98 ± 0.3) differed significantly from that of ICSI patients (Munich 0.81 ± 0.1; Wiesbaden 0.78 ± 0.2; P < 0.001), while processed samples obtained from IVF patients revealed a normal protamine mRNA ratio (Hamburg 1.0 ± 0.07; Shanghai 1.0 ± 0.54). Normal protamine mRNA ratios were associated with a significantly higher total motile sperm count and a significantly higher percentage of progressively motile sperm. Sperm with a normal protamine mRNA ratio revealed a higher fertilization capacity (fc) in both IVF (53.6% of patients with fc > 80%; P = 0.017) and ICSI (65.1% of patients with fc > 70%; P = 0.028). The protamine mRNA ratio in an individual sperm cell used for ICSI may be different from the overall value obtained from a semen aliquot. Data are in line with current literature and suggest the protamine mRNA ratio as a diagnostic marker to estimate the fertilizing capacity of sperm. The German Research Foundation (DFG) to K.S., W.W. and A.P. (STE 892/9-2), as well as to A.S. and H.C.O. (SP721/1-3). None.

Disorders in genetic imprinting are discussed as potential genetic risk in assisted reproduction technology (ART), where most of the natural selection mechanisms are bypassed. As currently only limited information about genomic imprinting in disruptive spermatogenesis is available, we analysed the imprinting state of the paternally methylated gene H19 in various germ cell populations derived from seminiferous tubules exhibiting impaired spermatogenesis. Different germ cell types were isolated by laser microdissection from human testicular paraffin sections. Although the methylation state of the maternally imprinted gene SNRPN was investigated by methylation-specific PCR (M-PCR) to establish the isolation method, methylation of H19 was analysed by a single-strand conformation-based method. Contamination by somatic Sertoli cells was excluded because of Sertoli cell-specific vimentin immunohistochemistry before germ cell laser microdissection. We demonstrate correct genetic imprints for H19 even in spermatogonia selected from seminiferous tubules exhibiting spermatogenic arrest at the level of spermatogonia, providing no evidence for incorrect genomic imprinting in spermatozoa from infertile men used for ICSI.