The aim of this study was to investigate the roles of oxygen free radicals and mitochondrial signaling in semen disorders, in particular, how this induces low concentrations and reduced motility of sperm. Ejaculate samples were obtained from 120 young adult males (mean age, 28.7±5.3 years) with normal semen (n=30), oligospermia (n=30), asthenospermia (n=30) and oligoasthenozoospermia (n=30). The malondialdehyde (MDA) content, total superoxide dismutase (T‑SOD) activity and glutathione peroxidase (GSH‑Px) activity of the sperm samples were determined by enzymatic assays. Mitochondrial membrane potential (MMP) was determined by flow cytometric detection of accumulated membrane‑permeable JC‑1 fluorescent dye. The mRNA and protein expression levels of apoptosis-associated genes [Bcl‑2, Bax, cytochrome c (Cyt C) and caspase-3] were measured by quantitative polymerase chain reaction and western blotting. Intergroup differences were evaluated by Student's t‑test. The sperm samples from all semen disorder groups exhibited significantly lower T‑SOD content and GSH‑Px activity (all P oligospermia ≈ oligoasthenozoospermia). The sperm from patients with semen disorders also exhibited significantly lower MMP than normal sperm, as evidenced by lower mean ratios of JC‑1+ sperm per group. The semen disorder groups had significantly higher Bax, Cyt C and caspase-3 mRNA and protein expression levels in the sperm samples, but significantly lower levels of Bcl‑2 (all P asthenospermia). In conclusion, oxygen free radicals may be involved in reduced sperm concentration and motility, possibly through effects on the mitochondrial apoptotic signaling pathway. Perturbed mitochondrial release of Cyt C may be the distinguishing molecular feature between oligospermia and asthenospermia.

Increased inflammation of the placenta is considered as a risk factor and a promoter of preterm premature rupture of the membranes (pPROM). High-mobility group box 1 (HMGB1) is a recently identified inflammatory cytokine, and HMGB1-RAGE signaling pathway has been associated with many pathophysiological processes. This study aims to reveal the mechanisms of HMGB1-RAGE signaling pathway in pPROM. The mRNA levels of relative gene of HMGB1 pathway, HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2, were analyzed by real-time PCR in placentas collected from 60 normal term women, 60 women with PROM and 60 women with pPROM. Additionally, levels of HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 protein were detected in frozen placental specimens by western blot, and the locations of HMGB1, RAGE and NF-κBp65 were evaluated in the well-characterized tissue microarray (TMA) by immunohistochemistry. ELISA was further used to detect HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 level in maternal and cord serum. Compared with normal term and PROM women, we found that (1) The mRNA expressions of HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 in HMGB1-RAGE pathway of pPROM placentas were higher. (2) The protein levels of HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 in pPROM placentas were higher. (3) HMGB1 and RAGE immunoreactivity in pPROM placenta TMA were increased in the cytoplasm of syncytiotrophoblast (STB), extravillous trophoblast (EVT) and mesenchymal cells, while NF-κBp65 was enhanced in the nucleus of STB and EVT. (4) Maternal serum concentrations of HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 in pPROM group were greater. (5) Cord serum concentrations of HMGB1, RAGE, NF-κBp65, MMP-9 and MMP-2 among the 3 groups had no significant differences. HMGB1 nuclear-cytoplasmic translocation in pPROM placenta may lead to the binding of HMGB1 to its receptor RAGE, resulting in provoking NF-κBp65 activity, and then inducing the release of MMP-9 and MMP-2, which all above activities contributed to the process of pPROM. Consequently, HMGB1-RAGE signaling pathway may be involved in the pathogenesis of pPROM.

Preeclampsia (PE) is a pregnancy-specific syndrome that has severe implications on perinatal mortality and morbidity. Excessive apoptosis of trophoblasts induced by hypoxia may be associated with the development of PE, but the exact pathogenesis is unknown. Forkhead box O transcription factor 3a (FOXO3a) is activated under hypoxic conditions. Furthermore, hypoxia‑inducible factor‑1α (HIF‑1α) is sensitive to variations in partial oxygen pressure. Thus, the aims of the present study were to investigate the expression levels of HIF‑1α and FOXO3a in placental samples of early onset severe PE, and their effect on trophoblastic apoptosis under hypoxic conditions. Cobalt chloride was used to establish the hypoxic model. The present study examined the expression levels of HIF‑1α and FOXO3a in the placental tissues and HTR8/SVneo cells under hypoxic conditions. It was found that HIF‑1α and FOXO3a were highly expressed in placental tissues of patients with PE and in HTR8/SVneo cells under hypoxic conditions. Furthermore, knockdown of FOXO3a using a specific small interfering RNA (siRNA) decreased apoptosis in HTR8/SVneo cells. Moreover, it was found that after knockdown of HIF‑1α using siRNA, FOXO3a expression and the apoptotic rate were reduced in HTR8/SVneo cells. Therefore, the present results indicated that the elevated expression of HIF‑1α increased trophoblastic apoptosis by regulating FOXO3a, which may be involved in the pathogenesis of PE.