Differentiation of human male germ cells from Wharton's jelly-derived mesenchymal stem cells.

OBJECTIVE: Recapitulation of the spermatogenesis process in vitro is a tool for studying the biology of germ cells, and may lead to promising therapeutic strategies in the future. In this study, we attempted to transdifferentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into male germ cells using all-trans retinoic acid and Sertoli cell-conditioned medium. METHODS: Human WJ-MSCs were propagated by the explant culture method, and cells at the second passage were induced with differentiation medium containing all-trans retinoic acid for 2 weeks. Putative germ cells were cultured with Sertoli cell-conditioned medium at 36℃ for 3 more weeks. RESULTS: The gene expression profile was consistent with the stage-specific development of germ cells. The expression of Oct4 and Plzf (early germ cell markers) was diminished, while Stra8 (a premeiotic marker), Scp3 (a meiotic marker), and Acr and Prm1 (postmeiotic markers) were upregulated during the induction period. In morphological studies, approximately 5% of the cells were secondary spermatocytes that had completed two stages of acrosome formation (the Golgi phase and the cap phase). A few spermatid-like cells that had undergone the initial stage of tail formation were also noted. CONCLUSION: Human WJ-MSCs can be transdifferentiated into more advanced stages of germ cells by a simple two-step induction protocol using retinoic acid and Sertoli cell-conditioned medium.

Efficacy of two sperm preparation techniques in reducing non-specific bacterial species from human semen.

CONTEXT: Artificial reproductive techniques using seminal preparations with bacteria may cause pelvic inflammatory disease and its sequalae. AIMS: To assess efficacy of two sperm preparation techniques to clear bacteria and the effect of bacteriospermia on sperm recovery rates. SETTINGS AND DESIGN: A descriptive cross-sectional study was carried out among males of subfertile couples. SUBJECTS AND METHODS: Semen samples were randomly allocated into swim-up method (group S, n = 68) and density gradient method (group D, n = 50) for sperm preparation. Seminal fluid analysis and bacterial cultures were performed in each sample before and after sperm preparation. STATISTICAL ANALYSIS: McNemar's chi-squared test and independent samples t-test in SPSS version 16.0 were used. RESULTS: Organisms were found in 86 (72.88%) out of 118 samples, before sperm preparation; Streptococcus species (n = 40, 46.51% of which 14 were Group D Streptococcus species), Coagulase negative Staphylococcus species (n = 17, 19.76%), Staphylococcus aureus (n = 13, 15.11%), Coliform species (n = 11, 12.79% of which 09 were Escherichia coli) and Corynebacterium species (n = 5, 5.81%). There was a statistically significant reduction of culture positive samples in raw vs. processed samples; in group S, 49 (72.05%) vs. 16 (23.52%) and in group D, 37 (74%) vs. 18 (36%). In group S and D, mean (SD) recovery rates of culture positive vs. culture negative samples were 39.44% (SD-14.02) vs. 44.22% (SD-22.38), P = 0.39 and 52.50% (SD-37.16) vs. 49.58% (SD-40.32), P = 0.82 respectively. CONCLUSIONS: Both sperm preparation methods significantly reduced bacteria in semen, but total clearance was not achieved. Sperm recovery rate was not affected by bacteriospermia.

Relationship between seminal plasma zinc and semen quality in a subfertile population.

RATIONALE: Current knowledge on the relationship between seminal zinc levels and different parameters of human semen is inconsistent. OBJECTIVES: To assess the relationship between seminal plasma zinc and semen quality using two markers; zinc concentration (Zn-C) and total zinc per ejaculate (Zn-T). DESIGN: The study was carried out as a cross-sectional study. SUBJECTS AND METHODS: Semen parameters of 152 healthy men undergoing evaluation for subfertility were assessed. Seminal plasma zinc levels were determined using flame atomic absorption spectrometry. Zn-C, expressed as µg/mL, was multiplied by ejaculated volume to calculate Zn-T. Mann Whitney U test and Chi-square test were used to compare the zinc levels between different seminal groups when appropriate. Correlations were observed with Pearson's correlation of coefficient. Analysis was carried out using SPSS 10.0 for windows software. RESULTS: Zn-C was low in 23 (15%) samples, while in 32 (21%) of the samples Zn-T was abnormal. The number of subnormal samples was high in the low-zinc groups compared with the normal-zinc groups, 15 vs. 8 (P > 0.05) for Zn-C and 28 vs. 4 (P < 0.001) for Zn-T. Zn-C was significantly high in the asthenozoospermics compared with the normal motile group; 138.11 µg/mL (83.92) vs. 110.69 11 µg/mL (54.59) (P < 0.05). Zn-T was significantly low in samples with hyperviscosity compared with samples with normal viscosity; 220.06 µg (144.09) vs. 336.34 µg (236.33) (P < 0.05). Conversely, Zn-T was high in samples with low viability compared with those with normal viability; 437.67 µg (283.88) vs. 305.15 µg (221.19) (P < 0.05). Weak correlations were found between Zn and some semen parameters. However, the correlation was negative between pH and Zn-C (r = -0.193, P < 0.05) as well as Zn-T (r = -0.280, P < 0.01). On the other hand, correlations were positive between Zn-T and sperm count (r = 0.211, P < 0.05). CONCLUSION: Count, motility, viability, pH and viscosity are affected by variations of seminal plasma zinc. Seminal plasma Zn-T is the better marker for assessing the relationship between zinc and semen quality.

Effects of zinc supplementation on sexual behavior of male rats.

CONTEXT: Effects of zinc on male sexual competence are poorly understood. AIM: To study the effects of different doses of zinc on the sexual competence of males using a rat model. MATERIALS AND METHODS: Three subsets (eight in each subset) of sexually experienced adult male rats were supplemented with three different oral doses of zinc sulphate (a daily dose of 1 mg, 5 mg and 10 mg respectively) for two weeks. A subset of eight animals without zinc supplementation was used as the control group Sexual behavior was observed by placing them individually in cages with receptive females. Statistical Analysis : Data analysis was done using SPSS v10 for windows computer software. RESULTS: Supplementation of 5 mg of zinc/day for two weeks led to a prolongation of ejaculatory latency; 711.6 sec. (SEM 85.47) vs. 489.50 sec. (SEM 67.66), P < 0.05 and an increase in number of penile thrusting; 52.80 (SEM 11.28) vs. 26.50 (SEM 6.17), P < 0.05, compared to controls. The same group had elevated prolactin (PRL) and testosterone (T) levels compared to controls at the end of treatment period; PRL- 7.22 ng/dl (SEM 3.68) vs. 2.90 ng/dl (SEM 0.34) and T- 8.21 ng/ml (SEM 6.09) vs. 2.39 ng/ml (SEM 1.79), P < 0.05. In contrast, reduction of libido was evident in the same group, but this effect was not statistically significant ( P > 0.05). However, partner preference index was positive and 5 mg zinc supplementation did not exert a significant adverse effect on the muscle strength and co-ordination. The subset of rats supplemented with 1 mg/day did not show a difference from the control group while supplementation with 10 mg/day led to a reduction of the libido index, number of mounts and intromissions. Conclusions : Zinc therapy improves sexual competence of male rats; the effect is dose dependent. Increase in the T levels is beneficial in this regard. However, increase in PRL is responsible for the reduced libido index. Further studies on pigs and monkeys are needed to evaluate the therapeutic use of zinc in sexual dysfunction.