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Platelet-activating factor improves intrauterine insemination outcome
Platelet-activating factor improves intrauterine insemination outcome Mark D. Wild, MD,a and William E. Roudebush, PhDb Charleston, South Carolina, and Atlanta, Georgia OBJECTIVE: Our purpose was to investigate intrauterine insemination pregnancy rates after human spermatozoa exposure to platelet-activating factor. STUDY DESIGN: Spermatozoa were incubated with platelet-activating factor in sperm-washing medium before intrauterine insemination. Patients whose sperm were incubated with sperm-washing medium alone served as controls. Pregnancy outcome was determined by ultrasonography (fetal heartbeat). RESULTS: Patients whose sperm were treated with exogenous platelet-activating factor had a significantly (P < .05) higher pregnancy rate (40%) than patients (20%) not receiving treatment. CONCLUSION: Inclusion of platelet-activating factor into a semen processing protocol, before intrauterine insemination, will significantly improve pregnancy rates. Platelet-activating factor may have a stimulatory effect on centriole-intact spermatozoa, enhancing their motility and fertilization success and resulting in improved pregnancy rates. Additional studies will elucidate the reproductive significance of platelet-activating factor activity in spermatozoa and its role in the establishment of pregnancy. (Am J Obstet Gynecol 2001;184:1064-5.)
Key words: Human, spermatozoa, platelet-activating factor, pregnancy
A number of endogenous factors have been attributed to regulation of the fertility potential of spermatozoa, for example, platelet-activating factor. Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) (PAF) is a unique and novel signaling phospholipid that has pleiotropic biologic properties in addition to platelet activation. Since its discovery in the early 1970s, this novel compound has been implicated in a variety of reproductive functions including fertilization, implantation, and parturition. The exact mechanism is uncertain, yet its importance in normal fertility is significant. PAF plays a significant role in mammalian reproduction. PAF is present in human spermatozoa, and its endogenous content has a significant and positive relationship with motility and pregnancy outcome.1 Exogenous PAF has been used to stimulate human sperm motility rate2 and fertilization potentials.3 Additionally, exogenous PAF will enhance the in vitro fertilization rates of mouse oocytes.4 Conversely, PAF antagonists will inhibit fertilization.5 The mechanism of PAF’s action on spermatozoa is not well known. The activity of PAF in other cell types is mediated by a specific receptor.6 The expression,
From the Medical University of South Carolinaa and Reproductive Biology Associates.b Reprint requests: William E. Roudebush, PhD, Reproductive Biology Associates, 5505 Peachtree-Dunwoody Rd, Suite 400, Atlanta, GA 30342. Copyright © 2001 by Mosby, Inc. 0002-9378/2001 $35.00 + 0 6/1/115228 doi:10.1067/mob.2001.115228
1064
presence, and distribution of the PAF receptor on human spermatozoa are significantly altered in abnormal forms.7 Although exogenous PAF has been used to enhance human sperm motility and fertilization potentials and improve in vitro fertilization rates in animal models, there are no reports on the use of exogenous PAF in enhancing human fertility outcomes. The study objective was therefore to determine the effect of exogenous PAF on intrauterine insemination pregnancy outcomes. Material and methods Human semen samples from men participating in infertility treatment by intrauterine insemination were collected by masturbation after at least 48 hours’ abstinence and were allowed to liquefy at 37°C for 30 minutes. Only patients with semen samples that had normal semen parameters according to World Health Organization criteria8 were enrolled in the study. This study was approved by the Medical University of South Carolina Institutional Review Board. Sperm were washed free of seminal fluid by centrifugation (400g, 10 minutes) and were treated with an exogenous mixture of PAF (final concentration, 10–7 mol/L) in Sperm Washing Medium (SWM; Scientific Irvine, Santa Ana, Calif). Synthetic PAF (Calbiochem-Novabiochem, La Jolla, Calif) was stored in a stock solution (1 × 10–6 mol/L) of chloroform and methanol (1:4). Just before use, 0.1 mL of stock PAF was dried under a gentle stream of nitrogen in siliconized tubes and redissolved in 1.0 mL of modified SWM. After an exposure period of 15 min-
Wild and Roudebush 1065
Volume 184, Number 6 Am J Obstet Gynecol
utes, the sperm were washed free of unbound PAF and resuspended in modified SWM. Patient sperm not receiving PAF treatment served as controls. Processed samples were delivered to the fertility clinic for routine intrauterine insemination. A positive pregnancy outcome was recorded when a fetal heartbeat was observed by ultrasonography. Data were analyzed by a 2 × 2 χ2 contingency table. Statistical calculations were performed with SigmaStat for Windows, version 2.03 (Jandel Scientific Corporation, San Rafael, Calif).
oocytes.4 PAF antagonists inhibit the motility, acrosome reaction, and hamster oocyte penetration and inhibit fertilization in exposed spermatozoa.4, 10 These data indicate the presence of a PAF-specific receptor in spermatozoa, which has been confirmed by immunofluorescence microscopy and molecular techniques.7 PAF may have a stimulatory effect on centriole-intact spermatozoa, enhancing their fertilization success and resulting in improved pregnancy rates.1 Additional studies will elucidate the reproductive significance of PAF activity in spermatozoa and PAF’s role in the establishment of pregnancy.
Results A total of 20 spermatozoa samples were processed as described, and 10 of them received PAF treatment. Patients whose sperm were treated with exogenous PAF had a significantly (P < .05) higher pregnancy rate (4/10, 40%) than patients (2/10, 20%) not receiving treatment. Comment Spermatozoa before any assisted reproductive technique must be first processed (eg, sucrose-gradient wash or swim-up). This processing of spermatozoa ensures that the most normal motile population is used to help facilitate conception. In the current study we have demonstrated that the addition of PAF to the sperm processing protocol will substantially improve intrauterine insemination pregnancy rates. PAF is present in human spermatozoa, and its endogenous level has a positive relationship with concentration and motility indices, implantation rate, and pregnancy outcome.1 PAF acetyl hydrolase is present in seminal plasma and is believed to have a role as a decapacitation factor.9 Removal of acetyl hydrolase during the capacitation process may promote PAF synthesis or utilization, which would, in turn, allow for an increase in spermatozoa motility.10 Exogenous PAF will stimulate capacitation and the acrosome reaction and is a calcium-dependent process.4 PAF plays a significant role in the fertilization process, enhancing fertilization rates of mouse and rabbit
REFERENCES
1. Roudebush WE, Purnell ET. Platelet-activating factor content in human spermatozoa: predicting pregnancy outcome. Fertil Steril 2000;74:257-60. 2. Ricker DD, Minhas BS, Kumar R, Robertson JL, Dodson MG. The effects of platelet activating factor on the motility of human spermatozoa. Fertil Steril 1989;52:655-8. 3. Minhas BS. Platelet-activating factor treatment of human spermatozoa enhances fertilization potential. Am J Obstet Gynecol 1993;168:1314-7. 4. Sengoku K, Ishikawa M, Tamate K, Shimizu T. Effects of plateletactivating factor on mouse sperm function. J Assist Reprod Genet 1992;9:447-53. 5. Angle MJ, Tom R, Jarvi K, McClure RD. Effect of plateletactivating factor on human spermatozoa-oocyte interactions. J Reprod Fertil 1989;98:541-8. 6. Ahmed A, Sage SO, Plevin R, Shoaibi MA, Sharkey AM, Smith SK. Functional platelet-activating receptors linked to inositol lipid hydrolysis, calcium mobilzation and tyrosine kinase activity in the human endometrial HEC-lB cell line. J Reprod Fertil 1994;101:459-66. 7. Roudebush WE, Wild MD, Maguire E. Platelet-activating factor receptor expression in human spermatozoa: differences in mRNA content and protein distribution between normal and abnormal spermatozoa. Fertil Steril 2000;73:967-71. 8. WHO Special Programme for Research, Development and Research Training in Human Reproduction. World Health Organization laboratory manual for the examination of human semen and semen-cervical mucus interaction. 4th ed. Cambridge (UK): Cambridge University Press; 1999. 9. Letendre ED, Miron P, Roberts KD, Langlais J. Platelet-activating factor acetylhydrolase in seminal plasma. Fertil Steril 1992; 57:193-8. 10. Kuzan FB, Geissler FT, Henderson WR. Role of spermatozoal platelet-activating factor in fertilization. Prostaglandins 1990;39: 61-74.
Key words: Human, spermatozoa, platelet-activating factor, pregnancy
A number of endogenous factors have been attributed to regulation of the fertility potential of spermatozoa, for example, platelet-activating factor. Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) (PAF) is a unique and novel signaling phospholipid that has pleiotropic biologic properties in addition to platelet activation. Since its discovery in the early 1970s, this novel compound has been implicated in a variety of reproductive functions including fertilization, implantation, and parturition. The exact mechanism is uncertain, yet its importance in normal fertility is significant. PAF plays a significant role in mammalian reproduction. PAF is present in human spermatozoa, and its endogenous content has a significant and positive relationship with motility and pregnancy outcome.1 Exogenous PAF has been used to stimulate human sperm motility rate2 and fertilization potentials.3 Additionally, exogenous PAF will enhance the in vitro fertilization rates of mouse oocytes.4 Conversely, PAF antagonists will inhibit fertilization.5 The mechanism of PAF’s action on spermatozoa is not well known. The activity of PAF in other cell types is mediated by a specific receptor.6 The expression,
From the Medical University of South Carolinaa and Reproductive Biology Associates.b Reprint requests: William E. Roudebush, PhD, Reproductive Biology Associates, 5505 Peachtree-Dunwoody Rd, Suite 400, Atlanta, GA 30342. Copyright © 2001 by Mosby, Inc. 0002-9378/2001 $35.00 + 0 6/1/115228 doi:10.1067/mob.2001.115228
1064
presence, and distribution of the PAF receptor on human spermatozoa are significantly altered in abnormal forms.7 Although exogenous PAF has been used to enhance human sperm motility and fertilization potentials and improve in vitro fertilization rates in animal models, there are no reports on the use of exogenous PAF in enhancing human fertility outcomes. The study objective was therefore to determine the effect of exogenous PAF on intrauterine insemination pregnancy outcomes. Material and methods Human semen samples from men participating in infertility treatment by intrauterine insemination were collected by masturbation after at least 48 hours’ abstinence and were allowed to liquefy at 37°C for 30 minutes. Only patients with semen samples that had normal semen parameters according to World Health Organization criteria8 were enrolled in the study. This study was approved by the Medical University of South Carolina Institutional Review Board. Sperm were washed free of seminal fluid by centrifugation (400g, 10 minutes) and were treated with an exogenous mixture of PAF (final concentration, 10–7 mol/L) in Sperm Washing Medium (SWM; Scientific Irvine, Santa Ana, Calif). Synthetic PAF (Calbiochem-Novabiochem, La Jolla, Calif) was stored in a stock solution (1 × 10–6 mol/L) of chloroform and methanol (1:4). Just before use, 0.1 mL of stock PAF was dried under a gentle stream of nitrogen in siliconized tubes and redissolved in 1.0 mL of modified SWM. After an exposure period of 15 min-
Wild and Roudebush 1065
Volume 184, Number 6 Am J Obstet Gynecol
utes, the sperm were washed free of unbound PAF and resuspended in modified SWM. Patient sperm not receiving PAF treatment served as controls. Processed samples were delivered to the fertility clinic for routine intrauterine insemination. A positive pregnancy outcome was recorded when a fetal heartbeat was observed by ultrasonography. Data were analyzed by a 2 × 2 χ2 contingency table. Statistical calculations were performed with SigmaStat for Windows, version 2.03 (Jandel Scientific Corporation, San Rafael, Calif).
oocytes.4 PAF antagonists inhibit the motility, acrosome reaction, and hamster oocyte penetration and inhibit fertilization in exposed spermatozoa.4, 10 These data indicate the presence of a PAF-specific receptor in spermatozoa, which has been confirmed by immunofluorescence microscopy and molecular techniques.7 PAF may have a stimulatory effect on centriole-intact spermatozoa, enhancing their fertilization success and resulting in improved pregnancy rates.1 Additional studies will elucidate the reproductive significance of PAF activity in spermatozoa and PAF’s role in the establishment of pregnancy.
Results A total of 20 spermatozoa samples were processed as described, and 10 of them received PAF treatment. Patients whose sperm were treated with exogenous PAF had a significantly (P < .05) higher pregnancy rate (4/10, 40%) than patients (2/10, 20%) not receiving treatment. Comment Spermatozoa before any assisted reproductive technique must be first processed (eg, sucrose-gradient wash or swim-up). This processing of spermatozoa ensures that the most normal motile population is used to help facilitate conception. In the current study we have demonstrated that the addition of PAF to the sperm processing protocol will substantially improve intrauterine insemination pregnancy rates. PAF is present in human spermatozoa, and its endogenous level has a positive relationship with concentration and motility indices, implantation rate, and pregnancy outcome.1 PAF acetyl hydrolase is present in seminal plasma and is believed to have a role as a decapacitation factor.9 Removal of acetyl hydrolase during the capacitation process may promote PAF synthesis or utilization, which would, in turn, allow for an increase in spermatozoa motility.10 Exogenous PAF will stimulate capacitation and the acrosome reaction and is a calcium-dependent process.4 PAF plays a significant role in the fertilization process, enhancing fertilization rates of mouse and rabbit
REFERENCES
1. Roudebush WE, Purnell ET. Platelet-activating factor content in human spermatozoa: predicting pregnancy outcome. Fertil Steril 2000;74:257-60. 2. Ricker DD, Minhas BS, Kumar R, Robertson JL, Dodson MG. The effects of platelet activating factor on the motility of human spermatozoa. Fertil Steril 1989;52:655-8. 3. Minhas BS. Platelet-activating factor treatment of human spermatozoa enhances fertilization potential. Am J Obstet Gynecol 1993;168:1314-7. 4. Sengoku K, Ishikawa M, Tamate K, Shimizu T. Effects of plateletactivating factor on mouse sperm function. J Assist Reprod Genet 1992;9:447-53. 5. Angle MJ, Tom R, Jarvi K, McClure RD. Effect of plateletactivating factor on human spermatozoa-oocyte interactions. J Reprod Fertil 1989;98:541-8. 6. Ahmed A, Sage SO, Plevin R, Shoaibi MA, Sharkey AM, Smith SK. Functional platelet-activating receptors linked to inositol lipid hydrolysis, calcium mobilzation and tyrosine kinase activity in the human endometrial HEC-lB cell line. J Reprod Fertil 1994;101:459-66. 7. Roudebush WE, Wild MD, Maguire E. Platelet-activating factor receptor expression in human spermatozoa: differences in mRNA content and protein distribution between normal and abnormal spermatozoa. Fertil Steril 2000;73:967-71. 8. WHO Special Programme for Research, Development and Research Training in Human Reproduction. World Health Organization laboratory manual for the examination of human semen and semen-cervical mucus interaction. 4th ed. Cambridge (UK): Cambridge University Press; 1999. 9. Letendre ED, Miron P, Roberts KD, Langlais J. Platelet-activating factor acetylhydrolase in seminal plasma. Fertil Steril 1992; 57:193-8. 10. Kuzan FB, Geissler FT, Henderson WR. Role of spermatozoal platelet-activating factor in fertilization. Prostaglandins 1990;39: 61-74.