Identification of Compounds That Can Potentially Enhance Sperm Motility in Vitro - Assignment Example

Identification of Compounds that Can Potentially Enhance Sperm Motility In Vitro Table of Contents 0 Introduction 3 2.0 Materials and Methods 4 2.1 Study Design 4 2.2 Ethical Approval 5 2.3 Selection of Study Subjects 5 2.4 Preparation of Semen Samples 5 3.0 Results 7 4.0 Discussion 10 References 12 APPENDICES: 15 Appendix 1: Non-Capacitated Cells (NCM) 15 Appendix 2: Capacitated Cells 17 Appendix 3: Basic Semen Analysis 18 Appendix 4: Paired t-test Output 19 1.0 Introduction One of the fastest developing fields in basic medical research is the treatment of infertility attributed to male factors. Preliminary investigations reveal that male infertility accounts for approximately half of all cases of infertility and affects about one man in every 20 in the population making it a significant medical and social problem that influences well-being. An important development in the last two decades is the discovery that sperm motility (asthenospermia) and morphology are the most critical determinants of the quality of sperms and the best factors for evaluating IVF (Karpuz et al., 2007). In particular, sperm motility is an important parameter because it indicates the integrity of the sperm tail and axoneme structures as well as the status of mitochondrial metabolic machinery (Saharkhiz et al., 2013). The World Health Organization uses a 40% cut off as the lower limit of progressive sperm motility. Although the exact cause of asthenospermia is not clear, there is growing evidence that it may arise due to hyper-viscosity of semen, varicoceles, autoimmunity of the sperm cell, and necrospermia resulting from immotile cilia (Kartagener) syndrome (Saharkhiz et al., 2013). Overall, reduced sperm motility indicates epididymal or testicular failure that can be caused by various factors. Currently, treatment of male infertility has focused mainly on the traditional in vitro fertilization (IVF) approach and newer interventions such as micro-epididymal sperm aspirations and microsurgical fertilization. However, these techniques are uncommon in andrological practice. Current research efforts in this area focus on the use of chemical stimulation of spermatozoa. A number of chemicals can stimulate sperm motility including the addition of compounds known to exhibit phosphodiesterase inhibition (PDEI) activity to enhance sperm motility in vitro without compromising sperm morphology and function. PDEIs are a class of related compounds such as pentoxifylline (PTX) that selectively catalyze the hydrolysis of 3’ phosphate bond in cyclic Adenosine Monophosphate (cAMP) phosphate or cyclic Guanosine Monophosphate (cGMP). In one of the pioneering studies on in vitro stimulation of sperm motility, Shen (1991) demonstrated that Pentoxifylline (PTX) increases motility (velocity) of ejaculated human spermertozoa both in in vitro aesthenozoospermic samples in oral therapy. Related studies have shown that PTX added in sperm suspensions increases sperm motion within 10 days with the motility characteristics persisting for two hours after removal of the drug (Tesarick et al., 1992). Recently, Dey et al., (2014) demonstrated that a glycoprotein called forward motility stimulating factor (FMSF) promotes progressive sperm cell motility by binding to the surface of mature spermatozoa and initiating cAMP-mediated FMSF activity. The present study investigates the in vitro motility of human sperms under the drug. We hypothesize that there is a difference in sperm motility between NCM and CM with treatment with the drug and without the drug. 2.0 Materials and Methods 2.1 Study Design In this study, we used Density gradient centrifugation (DGC) method to prepare sperms. We screened six donor samples to assess sperm motility under non-capacitating media (NCM): 1.8 mM CaCl2, 5.4 mM KCL, 0.8 mM MgSO4.7H20, 116.3 mM NaCl, 1.0 NaH2PO4, 5.55 mM D-glucose, 2.73 mM sodium pyruvate, 41.75 mM sodium lactate, 25 mM HEPES and 3 mg/mL BSA, pH 7.4, 300 mosmol/kg. The six donor samples progressed to the second phase of the study and sperm motility assessed with and without the drug (A+B %) in both NCM and capacitating media (NCM with 26 mM sodium bicarbonate replacing HEPES) in order to determine if the compound could be of clinical importance. 2.2 Ethical Approval We obtained written consent from each donor. All recruited sperm donors were informed the purpose of the study and informed about data handling in accordance with the University’s Ethical Research Committee regulations. 2.3 Selection of Study Subjects Six healthy subjects were asked to abstain from ejaculation for 2 days (48 h) to 72 h before samples were obtained through masturbation in the University laboratory. These semen samples were used in the first and second phases of the study. Selected samples 2.4 Preparation of Semen Samples 1.5 ml of 40% Percoll was pipetted into the bottom of a 15 ml centrifuge tube using Pasteur pipette. 1.5 mL of the 80% Percoll was carefully placed underneath the 40% layer and a clear interface observed between the two layers. Liquefied semen was overlaid on the 40% Percoll gradient without disturbing the interface. The sample was centrifuged at 0.3 RCF for 20 minutes in a swing-out rotor. The seminal plasma layer was removed using Pasteur pipette. 75 µl of the upper raft layer was removed and added to a tube of 4 ml Wash (NCM) using a pipette and the samples pooled together from all the samples into one wash tube. 40% and upper raft was pipetted and discarded to leave most of the 80% layer in place. The pellet (75%) was transferred into a single clean 15 mL centrifuge tube, and washed with 4 mL 1 X NCB. Both Wash tubes were centrifuged for 10 minutes at 0.5 RCF. The supernatant was aspirated leaving about 200 µl, which was mixed well and 50 µl pipetted into a fresh tube containing 1 mL NCM (x2) for both labeled wash tubes. The samples were incubated in a CO2 incubator at 370C for two hrs. 2.5 Compound Treatment We assessed sperm motility following incubation, then added 100 µM working solution of drug (10 mM stock solution) of Phosphodiesterase inhibitor (PDEi). The sperm cells were incubated again for 10 minutes at 370C, and motility assessed. For each donor sample, we determined sperm motility (percentage mobility) at 40% and 80% with drug and without drug. We used two media: non-capacitating media (NCM) [1.8 mM CaCl2, 5.4 mM KCl, 0.8 mM MgSO4.7H2O, 116.3 mM NaCl, 1.0 mM NaH2PO4, 5.55 mM D-glucose, 2.73 mM sodium pyruvate, 41.75 mM sodium lactate, 25 mM HEPES and 3 mg/ml BSA], and capacitating media (CM) [1.8 mM CaCl2, 5.4 mM KCl, 0.8 mM MgSO4.7H2O, 116.3 mM NaCl, 1.0 mM NaH2PO4, 5.55 mM D-glucose, 2.73 mM sodium pyruvate, 25 mM sodium lactate, 26 mM sodium bicarbonate and 3 mg/ml BSA] (see Tardif et al., 2014; Alasmari et al. (2013). 2.6 Statistical Analysis We conducted statistical analysis to test our null hypothesis that there is no difference in sperm motility between NCM and CM in both fractions with drug and without drug. We generated bar charts of each of the experiments: 40% fraction for CM and NCM with drug, 40% fraction for CM & NCM without drug, 80% fraction of CM &NCM with drug, and 80% fraction for CM & NCM without drug. We used t-test and ANOVA to compare differences between motility measurements for donor samples in CM and NCM with and without the drug for both the 40% and 80% fractions. 3.0 Results Statistical analysis did not establish any significantly reliable difference in sperm motility between 40% fractions in CM and NCM and no significant differences between 80% fractions in CM and NCM. Figure 1 below shows results for 40% fraction for CM and NCM with drug. A paired t-test analysis failed to show significant difference between the mean number of 40% fraction CM and 40% NCM (p = 0.536, ὰ = 0.05) FIGURE 1: 40% Fraction CM and NCM with Drug (A+B %) Figure 2 shows results for 40% Fraction CM and NCM without drug. Statistical analysis did not show statistically reliable differences between the means of the 40% Fractions of CM and NCM (p = 0.499, ὰ = 0.05). FIGURE TWO: 40% Fraction CM & NCM without Drug (A+B %): Figure 3 below shows results for 80% Fraction CM and NCM with drug. T-test statistical analysis did not reveal any significant difference between 80% CM and NCM with the drug (p = 0.971, ὰ = 0.05). FIGURE 3: 80% Fraction CM & NCM with drug FIGURE 4 below shows the results for 80% Fraction CM and NCM without drug. T-test analysis did not establish significant difference between 80% fraction CM and 80% NCM without the drug (p = 0.860, ὰ = 0.05) FIGURE 4: 80% Fraction CM & NCM without Drug Further analysis between mean counts of 40% CM with drug (M = 38.75) and without drug (M = 14.5) showed no significant differences (p = 0.091). Similarly, counts of 40% NCM with drug and no drug (p = 0.91), 80% CM with drug (M = 71.08) and without drug (M = 68.720) showed no difference (p = 0.0815), and 80% NCM with drug (M = 71) and no drug (M = 62.8) showed no significant difference (p = 0.93). 4.0 Discussion Currently, there is no consensus as to the best predictors of fertility in men, a problem that can contribute to inaccurate diagnosis of infertility and incorrect treatment or anxiety for couples. Nevertheless, sperm motility is considered an important predictor of male fertility (Milardi et al. 2012; Guzick et al. 2001). Rapid progressive sperm motility through the cervical mucus enables successful fertilization. Persistent poor progressive motility is a reliable predictor of fertilization failure, a vital outcome when deciding treatment options for couples. The typical procedure for calculating sperm motility involves estimating the speed with which sperm moves in a given volume (Vasan, 2011). Normal semen must contain at least 50% progressively motile spermatozoa. If more than 50% sperms are immotile, viability tests are recommended. In addition, sperm capacitation, the structural and biochemical alterations that spermatozoon go through in the female genital tract plays a vital role in preventing lytic enzymes. Sperm capacitation can be induced by incubating sperms with capacitation media (Vasan 2011). Stimulating sperm motility is a pivotal intervention in treating male infertility. Remarkably, there is paucity of drugs that can help increase effective sperm motility. Identification and discovery of compounds able to enhance sperm motility could significantly improve the treatment of male sterility. The present study investigated a novel approach of adding a stimulating compound to sperms to enhance motility in vitro rather than in vivo treatment, which has been unsuccessful. Our data confirms the ability of the compound to stimulate sperm motility in vitro with significant increase in motility. First, we used NCM screening in accordance with the conditions used for intrauterine insemination IUI (Bjorndahl et al. 2010). Experiments on individual donor samples showed similar profiles with enhanced motility obtained using capacitated cells, which is consistent with similar studies (Tardif et al., 2014). Current research shows increased use of Phosphodiesterase inhibitors (PDEIs) to increase sperm motility in in vitro experiments. Generally, capacitation does not involve any visible morphological changes rather it allows sperms to go through acrosome reactions induced by the ova in vivo (Ferrari et al., 2000). Capacitation agents/media provides a useful technique of capacitating cells in in vitro fertilization. According to Lamirande et al., (1997), the capacitation process involves extensive changes including removal of sterols from the plasma membrane of the cells followed by changes in trans-membrane flux and subsequent changes in intracellular ion concentration. In vitro systems carry many inherent advantages with many models having been developed using animal species and other using human, which elicits intense ethical concerns (Lamirande et al., 1997). Ideally, assessment of capacitation should be specific for capacitated sperms and should not affect the normal physiology. In a previous study designed to test the use of commercial capacitating agents, Martinez et al. (2012) demonstrated high progressive motility and functional state for cells under andtriladyl (TRY) treatment indicating the capacitating capacity of this compound. Further work should be undertaken in order to assess motility longevity and the functional state for spermatozoal cells after treatment with these compounds because there is a possibility of adverse effects on other cellular functions. Previous research shows that normal sperm function encompasses more than just sperm motility; it includes acrosome reactivity (AR) (Dana and Menge, 1996). Further tests should include detailed in vitro motility assessment, sperm penetration tests and acrosome reactivity tests. These tests will help augment our findings and justify the need for extensive in vivo experiments. References Alasmari, W. Barratt, C., L. Publicover, S., J. Whalley, K., M. et al. (2013). The clinical significance of calcium-signaling pathways mediating human sperm hyperactivation. Human Reproduction, 28(4), 866–876. Bjorndahl, L. Mortimer, D. Barratt, C., L., R. et al. 2010. A Practical Guide to Basic Laboratory Andrology. Cambridge, UK: Cambridge University Press Dana, O. & Menge, A. 1996. Assessment of sperm function and clinical aspect of impared sperm function. Frontiers in Bioscience, 1, 96-108 Dey, S. Roy, D. Majumder, G. & Bhattachryya, D. 2014. Extracellular regulation of sperm transmembrane adenylyl cyclase by a forward motility stimulating protein. Retrieved from http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110669 Ferrari, S. Barnabe, V. Zuge, R. & Zogno, M. 2000. Effect of two ram sperm capacitating media on acrosome reaction and zona-free hamster oocyte penetration test. Brazilian Journal of Veterinary Animal Science, 37(3), 229-233 Guzick, D., S. Overstreet, J., W. Factor-Litvak, P. et al. 2001. Sperm morphology, motility, and concentration in fertile and infertile men. New England Journal of Medicine, 345(19), 1388–1393 Karpuz, V. Gokturk, A. & Koyuturk, M. 2007. The effects of sperm morphology and motility on the outcomes of intracytoplasmic sperm injection. Marmara Medical journal, 20(2), 92-97 Lamirande, E. Leclerc, P. & Gagnon, C. 1997. Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization. Molecular Human Reproduction, 3(3), 175-194 Martinez, A. Camacho, J. & Correa, J. 2012. Motility and functional state of the membrane of caprice capacitated spermatozoa under different chemical agents. Open Journal of Veterinary Medicine, 2, 98-103 Milardi, D. Grande, G. Sacchini, D. et al. 2012. Male fertility and reduction in semen parameters: a single tertiary-care center experience. International journal of Endocrinology. Retrieved January 18, 2014 from http://www.hindawi.com/journals/ije/2012/649149/ Saharkhiz, N. Nikbakht, R. & Hemadi, M. 2013. Ketotifen, a mast cell blocker improves sperm motility in asthenospermic infertile men. Journal of Human Reproductive Science, 6(1), 19-22 Shen, M. Chiang, P. Yang, R. & Hong, C. & Chen, S. 1991. Pentoxilline stimulates sperm motility both in vitro and after oral therapy. British Pharmacological Society, 31(6), 711-714 Tardif, S. Madamidola, O. Brown, S. Frame, L. et al. 2014. Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity. Human Reproduction, Advance Access, pp. 1-13 Tesarick, J. Thebault, A. & Testart, J. 1992. Effect of Pentoxifylline on pserm movement characreistics in normozoospermic and asthenozoospermic specimens. Human Reproduction, 7(9), 1257-1263 Vasan, S. 2011. Semen analysis and sperm function tests: how much to test? Indian Journal of Urology, 27(1), 41-48. APPENDICES: Appendix 1: Non-Capacitated Cells (NCM) Value (% motility) Replicate 40/80% Treatment Media (Capacitation, C; Non capacitating, N) 34 1 40 Drug N 53.8 1 40 Drug N 13 1 40 Drug N 16 1 40 Drug N 58 1 40 Drug N 29 2 40 Drug N 41 2 40 Drug N 19 2 40 Drug N 4.4 2 40 Drug N 50 2 40 Drug N 16 1 40 No drug N 16 1 40 No drug N 5 1 40 No drug N 6 1 40 No drug N 56.5 1 40 No drug N 24 2 40 No drug N 20 2 40 No drug N 0 2 40 No drug N 14 2 40 No drug N 44 2 40 No drug N 85 1 80 Drug N 71 1 80 Drug N 67 1 80 Drug N 69.6 1 80 Drug N 49 1 80 Drug N 82 2 80 Drug N 76 2 80 Drug N 50 2 80 Drug N 71.7 2 80 Drug N 75.1 2 80 Drug N 79 1 80 No drug N 51 1 80 No drug N 82 1 80 No drug N 65 1 80 No drug N 68.2 1 80 No drug N 79 2 80 No drug N 47 2 80 No drug N 81 2 80 No drug N 59 2 80 No drug N 70.3 2 80 No drug N 45 1 40 Drug CM 71 1 40 Drug CM 19 1 40 Drug CM 20 1 40 Drug CM 65.4 1 40 Drug CM 39 2 40 Drug CM 73 2 40 Drug CM 29 2 40 Drug CM 9.6 2 40 Drug CM 57 2 40 Drug CM 12 1 40 No drug CM 23 1 40 No drug CM 11 1 40 No drug CM 12 1 40 No drug CM 44 1 40 No drug CM 15 2 40 No drug CM 36 2 40 No drug CM 7 2 40 No drug CM 3.3 2 40 No drug CM 60 2 40 No drug CM 86 1 80 Drug CM 51.4 1 80 Drug CM 78 1 80 Drug CM 74 1 80 Drug CM 66 1 80 Drug CM 89 2 80 Drug CM 55 2 80 Drug CM 74 2 80 Drug CM 77 2 80 Drug CM 60 2 80 Drug CM 70 1 80 No drug CM 85.6 1 80 No drug CM 72 1 80 No drug CM 56 1 80 No drug CM 61 1 80 No drug CM 78 2 80 No drug CM 55 2 80 No drug CM 56 2 80 No drug CM 64 2 80 No drug CM 66 2 80 No drug CM Appendix 2: Capacitated Cells       Drug (A+B %)           No drug (A+B %)                               40% Fraction 40% Fraction   80% Fraction 80% Fraction   40% Fraction 40% Fraction   80% Fraction 80% Fraction                         Donor 1 45 39   86 89 Donor 1 12 15   70 78 Donor 2 71 73   51.4 55 Donor 2 23 36   85.6 55 Donor 3 19 29   78 74 Donor 3 11 7   72 56 Donor 4 20 9.6   74 77 Donor 4 12 3.3   56 64                         Donor 6 65.4 57   66 60 Donor 6 44 60   61 66 Appendix 3: Basic Semen Analysis   %A+B Concentration (M/ml) Donor 1 67 60     80.4 81.5     Donor 2 63 62.5 58 70 76.2 69.25 60 53 Donor 3 70.7 71 38   58 52 41.5   Donor 4 65.6 54 54   96.3 108.1 96   Donor 5 33 33 40   10.1 9.8 10.5   Donor 6 60 79 68   287.5 Abigail? Kareema?   Appendix 4: Paired t-test Output 1: Paired t-test 40% CM & NCM with Drug Paired Samples Test Paired Differences t df Sig. (2-tailed) Mean Std. Deviation Std. Error Mean 95% Confidence Interval of the Difference Lower Upper Pair 1 40% CM with Drug - 40% NCM with Drug 2.5600 8.4551 3.7812 -7.9383 13.0583 .677 4 .536 2: Paired t-test for 40% CM & NCM No Drug Paired Samples Test Paired Differences t df Sig. (2-tailed) Mean Std. Deviation Std. Error Mean 95% Confidence Interval of the Difference Lower Upper Pair 1 40% CM No Drug - 40% NCM No Drug -3.2600 9.8177 4.3906 -15.4503 8.9303 -.742 4 .499 3: Paired t-test 80% CM & NCM with Drug Paired Samples Test Paired Differences t df Sig. (2-tailed) Mean Std. Deviation Std. Error Mean 95% Confidence Interval of the Difference Lower Upper Pair 1 80% CM with Drug - 80% NCM with Drug .0800 4.5532 2.0363 -5.5736 5.7336 .039 4 .971 4: Paired t-test 80% CM & NCM with No Drug Paired Samples Test Paired Differences t df Sig. (2-tailed) Mean Std. Deviation Std. Error Mean 95% Confidence Interval of the Difference Lower Upper Pair 1 80% CM No Drug - 80% NCM No Drug 2.9200 19.9983 8.9435 -21.9112 27.7512 .326 4 .760 5. Paired t-test 40% CM with Drug and Without Drug Paired Samples Test Paired Differences t df Sig. (2-tailed) Mean Std. Deviation Std. Error Mean 95% Confidence Interval of the Difference Lower Upper Pair 1 40% CM With Drug - 40% CM With No Drug 24.2500 19.7379 9.8689 -7.1573 55.6573 2.457 3 .091 6. Paired t-test 40% NCM with Drug and without drug Paired Samples Test Paired Differences t df Sig. (2-tailed) Mean Std. Deviation Std. Error Mean 95% Confidence Interval of the Difference Lower Upper Pair 1 40% CM With Drug - 40% CM With No Drug 24.2500 19.7379 9.8689 -7.1573 55.6573 2.457 3 .091 7. Paired t-test 80% CM with drug and without drug Paired Samples Test Paired Differences t df Sig. (2-tailed) Mean Std. Deviation Std. Error Mean 95% Confidence Interval of the Difference Lower Upper Pair 1 80% CM with drug - 80% CM without drug 2.3600 21.1766 9.4705 -23.9342 28.6542 .249 4 .815 8. Paired t-test 80% NCM with Drug and Without Drug Paired Samples Test Paired Differences t df Sig. (2-tailed) Mean Std. Deviation Std. Error Mean 95% Confidence Interval of the Difference Lower Upper Pair 1 80% NCM with drug - 80% NCM without drug 8.2000 8.3487 3.7336 -2.1662 18.5662 2.196 4 .093 Read More