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Treatment of Sperm With Platelet-activating Factor Does Not Improve Intrauterine Insemination Outcome in Unselected Cases of Mild Male Factor Infertility: A Prospective Double-blind Randomized Crossover Study
Infertility Treatment of Sperm With Platelet-activating Factor Does Not Improve Intrauterine Insemination Outcome in Unselected Cases of Mild Male Factor Infertility: A Prospective Double-blind Randomized Crossover Study Stavroula Baka, Odysseas Grigoriou, Dimitris Hassiakos, Socrates Konidaris, Konstantinos Papadias, and Evangelos Makrakis OBJECTIVES
METHODS
RESULTS
CONCLUSIONS
To evaluate the effect of sperm treatment with exogenous platelet-activating factor (PAF) on intrauterine insemination (IUI) clinical pregnancy rate in cases of mild male factor infertility. PAF is a phospholipid mediator, which is present in human sperm. The study was performed in the Assisted Reproduction Unit of the 2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieion Hospital, Athens, Greece, and included 92 couples who presented with mild male factor infertility—all candidates for IUI. A maximum of 4 IUI cycles per couple with or without exogenous PAF treatment were performed and the main outcome measure was the clinical pregnancy rate (pregnancies confirmed by ultrasonography per 100 cycles). The overall clinical pregnancy rate after a maximum of 4 IUI cycles was comparable in cases with and without sperm treatment with PAF (12.24% vs 11.11%). Addition or exclusion of PAF sperm treatment in the same patients did not significantly alter the outcome. The generalized use of exogenous PAF for the preparation of sperm in unselected cases of mild male infertility does not improve the clinical outcome of IUI. UROLOGY 74: 1025–1028, 2009. © 2009 Elsevier Inc.
T
he use of exogenous supplementation to enhance sperm function constitutes an attractive option. To achieve such an important goal, the role of possible adjuvants must be well defined, especially when dealing with specific patient population. Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine) is an important phospholipid mediator, which is produced by a variety of cells.1 Apart from platelet activation and various other effects,2,3 PAF seems to have an important role in reproduction by being involved in ovulation, sperm capacitation, fertilization, preimplantation embryo development, implantation, and parturition.4 PAF, being present in human sperm, is one of the endogenous factors directly correlated to sperm From the Assisted Reproduction Unit, 2nd Department of Obstetrics and Gynecology, Aretaieion Hospital, University of Athens, Athens, Greece; and “Embryo ART”, IVF Unit, 2nd Department of Obstetrics and Gynecology, Aretaieion Hospital, University of Athens, Athens, Greece Reprint requests: Stavroula Baka, M.D., 104, Pindou St, 15669 Athens, Greece. E-mail: [email protected] Submitted: March 14, 2009, accepted (with revisions): June 2, 2009
© 2009 Elsevier Inc. All Rights Reserved
motility, forward progression, and is responsible for the regulation of spermatozoa fertilization capacity.5 Its mechanism of action on spermatozoa, even though not exactly known, is probably mediated by a specific receptor.6 The expression, presence, and distribution of this receptor have been found to be distorted in abnormal spermatozoa,7 whereas there is a positive relation between the PAF content in human spermatozoa and their motility as well as pregnancy outcome.8 Treatment of human sperm with exogenous PAF stimulates sperm motility rate and fertilization potential.9,10 Intrauterine insemination (IUI) can be the first-line treatment for the management of infertile couples with unexplained infertility, mild and moderate male factor, anovulatory disorders, or cervical hostility; until now, no method of sperm preparation has been shown to be superior with regard to pregnancy rate after IUI, whereas whether supplementation of culture media with substances such as antioxidants may improve the results, remains the subject of further research.11 By elucidating 0090-4295/09/$34.00 doi:10.1016/j.urology.2009.06.023
1025
the role of PAF in cases of IUI, the scientific interest in this molecule can be extended to other areas of assisted reproductive technologies to improve the therapeutic approach. The aim of the present study was to evaluate the effect of sperm treatment with exogenous PAF on IUI clinical outcomes in cases of mild male factor infertility.
MATERIAL AND METHODS Subjects The study included 92 couples presenting with primary infertility in the Assisted Reproduction Unit of the 2nd Department of Obstetrics and Gynecology, University of Athens, Greece, between May 2006 and December 2007. All the female study subjects met the following criteria: age ⱕ38 years, duration of infertility ⱖ1 year, regular menstrual cycle of 26-32 days, unremarkable clinical findings on gynecological examination, ovulatory basal body temperature chart, midluteal serum progesterone levels ⱖ16 ng/mL, levels of follicle-stimulating hormone, luteinizing hormone, androstenedione, testosterone, dehydroepiandrosterone sulfate, and prolactin within normal range on cycle day-3, normal thyroid function tests, nonsignificant transvaginal ultrasound scan, and normal hysterosalpingogram. All the male study subjects met the following criteria: age ⱕ40 years, unremarkable medical history and findings on physical examination, impaired semen parameters on 2 occasions according to the World Health Organization criteria,12 with a sperm concentration of 10-20 ⫻ 106, and/or forward progressive motility of 30%-50%, and/or sperm morphology of ⬍14% normal forms (strict criteria). The Ethical Committee of Aretaieion Hospital approved the study and informed consent forms were obtained from all subjects.
Treatment The first-step treatment decision for all couples was that of intrauterine insemination in an ovarian stimulation cycle. In all cases, clomiphene citrate was administered on days 3-7 of the cycle in a daily dose of 100 mg. Ovarian response was monitored by serial ultrasound scans, and when the mean diameter of the leading follicle was ⱖ18 mm, 10 000 IUs of human chorionic gonadotropin were administered. Intrauterine insemination was scheduled to be performed 34-38 h after the human chorionic gonadotropin injection. A double-blind randomization was performed on the day of the first IUI attempt for each couple in predetermined blocks (8 consecutive blocks of 10 followed by 1 block of 6) using random number tables and the study couples were allocated to 2 groups: In Group A-PAF (46 couples), the sperm for IUI was treated with PAF, whereas in Group B-non PAF (46 couples), such a treatment was not offered (Fig. 1). If pregnancy was not achieved, a similar IUI cycle was organized. After completing 2 consecutive IUI cycles with no success, Group A failures had a maximum of 2 new IUI cycles with no PAF treatment (Group A-non-PAF), whereas Group B failures had a maximum of 2 new IUI cycles with PAF treatment of the sperm (Group B-PAF). A positive pregnancy outcome was recorded when a gestational sac containing a fetal pole was observed by ultrasound scans.
Sperm Preparation In Groups A-PAF and B-PAF, semen specimens were processed (400g, 10 min) through a 90% density silane-coated colloidal 1026
Figure 1. Flow diagram of the population included in the prospective double-blind randomized cross-over study.
silica suspension (1:1; Fertipro, Belgium), then were treated with an exogenous mixture of PAF (final concentration, 10⫺7 mol/L), (Calbiochem-Novabiochem, La Jolla, CA), in sperm washing medium (SWM; Sydney IVF sperm medium, Cook, Australia). After an exposure period of 15 min at 37°C, the sperm was washed free of unbound PAF (300g, 8 min) and suspended in SWM. In Groups A-non-PAF and B-non-PAF, semen specimens were processed through a 90% density suspension (400g, 10 min), washed with 4 mL of SWM, centrifuged (300g, 8 min), and resuspended.
Statistical Analysis Statistical analysis was performed with the SPSS 10.7 statistical software for Windows with the appropriate use of 2-test/Fisher exact, Student t test, and Kruskal-Wallis test; a binary logistic regression model was used to evaluate whether different categories of male factor could influence the IUI outcome (presence or absence of clinical pregnancy) in cases with or without sperm treatment with PAF. A minimal sample size of 80 IUI cycles per arm was required to achieve a study power of 80% for the lowest increase in IUI pregnancy rates after sperm treatment with PAF (100%), as reported in published studies. Statistical significance was set at ⬍.05.
RESULTS Table 1 shows data regarding subjects’ age, duration of infertility, type of semen abnormality in the study groups, and sperm parameters on recruitment. Age of females, age of males, and duration of infertility were comparable between the 2 groups (P ⫽ .11, P ⫽ .2, and P ⫽ .11, respectively). In addition, no significant differences regarding the type of semen abnormality were observed between the 2 groups. Finally, no significant differences were observed between the 2 groups regarding sperm concentration (P ⫽ .37), progressive motility (P ⫽ .33), and morphology (P ⫽ .77). UROLOGY 74 (5), 2009
Table 1. Characteristics of study groups Characteristic Female age* (y) Male age* (y) Duration of infertility* (mon) Oligospermia (%) Asthenospermia (%) Teratozoospermia (%) Oligo-asthenospermia (%) Oligo-asthenoteratozoospermia (%) Concentration* (⫻106/mL) Progressive motility* (%) Normal morphology* (%)
Group A
Group B
29.2 ⫾ 4.1 31.5 ⫾ 3.1 16.2 ⫾ 3.7 23.91 15.21 4.34 45.65 10.86
28.0 ⫾ 2.9 32.4 ⫾ 3.5 17.4 ⫾ 3.5 17.39 21.73 6.52 52.17 2.17
17.3 ⫾ 3.4 18.6 ⫾ 7.8 38.7 ⫾ 9.8 40.7 ⫾ 9.6 24.1 ⫾ 10.1 23.5 ⫾ 11.1
* Values are means ⫾ SD.
Table 2. Pregnancy rates and statistical comparisons in the study groups Group
Pregnancy Rate (%) †
A-PAF* A-non-PAF*‡ B-PAF§‡ B-non-PAF§†
11.90 12.06 12.69 10.46
* A-PAF vs A-non-PAF, P ⫽ .58. † A-PAF vs B-non-PAF, P ⫽ .47. ‡ B-PAF vs A-non-PAF, P ⫽ .56. § B-PAF vs B-non-PAF, P ⫽ .43.
The mean ⫾ SD of inseminating motile count was 7.1 ⫾ 2.9 (⫻106/mL) in IUI cycles with PAF treatment and 7.9 ⫾ 3.7 (⫻106/mL) in IUI cycles without PAF treatment; the inseminating motile count was comparable between the 2 groups (P ⫽ .07). The number of IUI cycles in the study groups, the number of pregnancies achieved as well as the discontinuation from the study can be seen in Figure 1. There were 10 pregnancies achieved in Group A-PAF, 9 pregnancies in Group B-non-PAF, 7 pregnancies in Group B-PAF and 7 pregnancies in Group A-non-PAF. The PAF arm of the study included 147 IUI cycles, whereas the non-PAF arm included a total of 144 IUI cycles; these numbers of cycles were adequate for the initial study power calculation. The clinical pregnancy rate (clinical pregnancies per 100 cycles) in the 4 groups studied and statistical comparisons between these groups are presented in Table 2. Clinical pregnancies in cases with and without sperm treatment with PAF were comparable during the first 2 IUI attempts (Group A-PAF vs Group B-non PAF, P ⫽ .47), the subsequent 2 IUI attempts (Group B-PAF vs Group A-non PAF, P ⫽ .56) as well as in the total of the 4 attempts (Groups A-PAF and B-PAF: clinical pregnancy rate 12.24% vs Groups A-non-PAF and B-nonPAF: clinical pregnancy rate 11.11%, P ⫽ .45). Within the 2 Groups A and B, sperm treatment with PAF resulted in a comparable number of clinical pregnancies (Group A-PAF vs Group A-non-PAF, P ⫽ .58 and Group B-PAF vs Group B-non-PAF, P ⫽ .43). The order of IUI attempts did not influence the outcome (clinical pregnancies, P ⫽ .94). Finally, the type of semen abnorUROLOGY 74 (5), 2009
mality could not significantly influence the outcome in cases with PAF treatment (Groups A-PAF and B-PAF, P ⫽ .38) as well as in cases without such a treatment (Groups A-non-PAF and B-non-PAF, P ⫽ .84).
COMMENT PAF is present in human sperm, and the spermatozoa demonstrate enzymatic pathways to synthesize and metabolize PAF.13 By binding to specific surface receptors,14 PAF could possibly trigger the formation of inositol triphosphate and diacylglycerol and could increase the intracellular calcium by stimulating the release from intracellular calcium stores as well as the entry of extracellular calcium.15-18 The elevated intracellular calcium, by causing depolymerization of the membrane actin network and activation of phospholipases, could result in acrosome reaction,19 enhanced sperm motility, and, theoretically, in increased fertilization potential.9 The distribution of PAF receptors is not equal among various spermatozoa regions with the highest concentrations being present at the neck and midpiece.8 The midpiece region contains the mitochondria and is important for the spermatozoa motility, whereas the neck region contains the proximal centriole and is important for the preimplantation embryo development.20 Finally, PAF antagonists inhibit the above-mentioned PAF receptor– mediated effects.21,22 Cumulative data support the positive effects of sperm treatment with exogenous PAF. Such a treatment increases the motility of fresh and frozen-thawed sperm,23 enhances the in vitro fertilization rates of mouse and rabbit oocytes, and results in increased rabbit embryo development.22,24-26 Finally, the use of exogenous PAF during sperm preparation for IUI appears to be an attractive option. This treatment in 10 sperm samples for IUI resulted in a double pregnancy rate (40%) compared with 10 controls.27 In a study of similar design with the present one, but a maximum of 3 IUI cycles with PAF treatment and 3 with direct swim-up technique per patient, including cases of unexplained infertility, we reported a significantly increased overall clinical pregnancy rate when sperm was treated with exogenous PAF compared to the direct swim-up technique (23.07% vs 7.92%).28 Unfortunately, we could not show similar positive outcomes in IUI cycles when PAF treatment was applied in sperm with impaired parameters. Clinical pregnancy rates were comparable not only between treatment and control groups, but within the same groups after application or removal of PAF treatment. Our results agree with those of other investigators, similar to those of Roudebush et al29 who reported in their prospective randomized study that PAF treatment enhances IUI pregnancy rates in cases of non-male factor infertility (53.9% vs 28.6% in controls), but not in cases of male factor infertility (11.8% vs 9.7% in controls). The absence of an advantageous effect of PAF treatment in male factor cases could be attributed to defective or low numbers of PAF 1027
receptors in abnormal sperm, which may result in nonresponsiveness to endogenous or exogenous PAF. According to several observations, the allocation of PAF receptors is affected in abnormal sperm, whereas the PAF-receptor mRNA sequences and expression differs between motile and nonmotile sperm.29,30 Nevertheless, this study carries certain limitations, such as the skepticism for cross-over designs in subfertility research, and the lack of information on concentrations of semen PAF, sperm PAF-receptors, semen PAF-acetylhydrolase, and endometrial PAF status; the knowledge of specific insufficiencies regarding the above-mentioned factors, might have individualized the use of exogenous PAF treatment, allowing for better outcomes. Furthermore, another limitation of the study is the lack of a concurrent positive control, which could demonstrate that the PAF used had full bioactivity. Finally, the present study was powered to detect a quite high increase in clinical pregnancy rate after the use of exogenous PAF; obviously, a larger study population could possibly allow for valid detection of a more moderate enhancement. Additional experimental studies are warranted to further clarify the importance of sperm treatment with exogenous PAF on IUI outcomes in cases of mild male factor infertility.
CONCLUSIONS The generalized use of exogenous PAF for the preparation of sperm in cases of mild male infertility does not improve the clinical outcome of intrauterine inseminations; this does not necessarily mean that there is no place for exogenous PAF use in a more specific and well-defined group of subfertile males.
References 1. Benveniste J, Henson PM, Cochrane CG. Leukocyte dependent histamine release from rabbit platelets: the role of Ig-E, basophils, and platelet-activating factor. J Exp Med. 1972;136:1356-1377. 2. Hanahan DJ. Platelet activating factor: a biologically active phosphoglyceride. Annu Rev Biochem. 1986;55:483-509. 3. Braquet P, Touqui L, Shen TY, et al. Perspectives in platelet activating factor research. Pharmacol Rev. 1987;39:97-145. 4. Harper MJ. Platelet activating factor: a paracrine factor in preimplantation stages of reproduction? Biol Reprod. 1989;40:907-913. 5. Minhas BS, Kumar R, Ricker DD, et al. The presence of plateletactivating factor-like activity in human spermatozoa. Fertil Steril. 1991;55:372-376. 6. Ahmed A, Sage SO, Plevin R, et al. Functional platelet-activating receptors linked to inositol lipid hydrolysis, calcium mobilization and tyrosine kinase activity in the human endometrial HEC-lB cell line. J Reprod Fertil. 1994;101:459-466. 7. Roudebush WE, Wild MD, Maguire EH. Expression of the plateletactivating factor receptor in human spermatozoa: differences in mRNA content and protein distribution between normal and abnormal spermatozoa. Fertil Steril. 2000;73:967-971. 8. Roudebush WE, Purnell ET. Platelet-activating factor content in human spermatozoa and pregnancy outcome. Fertil Steril. 2000;74: 257-260.
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9. Ricker DD, Minhas BS, Kumar R, et al. The effects of platelet activating factor on the motility of human spermatozoa. Fertil Steril. 1989;52:655-658. 10. Minhas BS. Platelet-activating factor treatment of human spermatozoa enhances fertilization potential. Am J Obstet Gynecol. 1993; 168:1314-1317. 11. Ombelet W, Deblaere K, Bosmans E, et al. Semen quality and intrauterine insemination. Reprod Biomed Online. 2003;7:485-492. 12. World Health Organization: WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction, 4th edn. Prepared by WHO Special Programme for Research, Development and Research Training in Human Reproduction: Cambridge University Press, Cambridge, UK, 1999. 13. Gujarati VR, Naukam RJ, Rama-Sastry BV. Enzymatic deacetylation and acetylation of ether phospholipids related to plateletactivating factor in human semen with short and long liquefaction times. Ann N Y Acad Sci. 1987;513:583-585. 14. Reinhardt JC, Cui X, Roudebush WE. Immunofluorescent evidence of the platelet-activating factor receptor on human spermatozoa. Fertil Steril. 1999;71:941-942. 15. Putney JW Jr, Poggioli J, Weiss SJ. Receptor regulation of calcium release and calcium permeability in parotid gland cells. Philos Trans R Soc Lond B Biol Sci. 1981;296:37-45. 16. Lapetina EG. PAF stimulates the phosphatidylinositol cycle. Appearance of phosphatidic acid is associated with the release of serotonin in horse platelets. J Biol Chem. 1982;257:7314-7317. 17. Putney JW. Formation and Actions of calcium-mobilizing messenger, inositol 1,4,5-triphosphate. Am J Physiol. 1987;252:G149G157. 18. Vargaftig BB, Braquet PG. PAF-acether today—relevance for acute experimental anaphylaxis. Br Med Bull. 1987;43:312-335. 19. Benoff S. Modelling human sperm-egg interactions in vitro: Signal transduction pathways regulating the acrosome reaction. Mol Hum Reprod. 1998;4:453-471. 20. Sathananthan AH, Ratnam SS, Ng SC, et al. The sperm centriole: its inheritance, replication and perpetuation in early human embryos. Hum Reprod. 1996;11:345-356. 21. Kuzan FB, Geissler FT, Henderson WR. Role of spermatozoal platelet activating factor in fertilization. Prostaglandins. 1990;39: 61-74. 22. Sengoku K, Ishikawa M, Tamate K, et al. Effects of platelet activating factor on mouse sperm function. J Assist Reprod Genet. 1992;9:447-453. 23. Hellstrom WJ, Wang R, Sikka SC. Platelet-activating factor stimulates motion parameters of cryopreserved human sperm. Fertil Steril. 1991;56:768-770. 24. Minhas BS, Kumar R, Ricker DD, et al. Effects of platelet activating factor on mouse oocyte fertilization in vitro. Am J Obstet Gynecol. 1989;31:1714-1717. 25. Roudebush WE, Minhas BS, Ricker DD, et al. Platelet activating factor enhances in vitro fertilization of rabbit oocytes. Am J Obstet Gynecol. 1990;163:1670-1673. 26. Roudebush WE, Fukuda AI, Minhas BS. Enhanced embryo development of rabbit oocytes fertilized in vitro with platelet-activating factor (PAF)-treated spermatozoa. J Assist Reprod Genet. 1993;10: 91-94. 27. Wild M, Roudebush W. Platelet-activating factor improves intrauterine insemination outcome. Am J Obstet Gynecol. 2001;184: 1064-1065. 28. Grigoriou O, Makrakis E, Konidaris S, et al. Effect of sperm treatment with exogenous platelet-activating factor on the outcome of intrauterine insemination. Fertil Steril. 2005;83:618-621. 29. Roudebush WE, Toledo A, Kort HI, et al. Platelet-activating factor significantly enhances intrauterine insemination pregnancy rates in non-male factor infertility. Fertil Steril. 2004;82:52-56. 30. Roudebush WE, Mayorov V, Adkison LR, et al. Abnormal sequence of the platelet-activating factor-receptor in nonmotile spermatozoa. Fertil Steril. 2002;78:S269.
UROLOGY 74 (5), 2009
METHODS
RESULTS
CONCLUSIONS
To evaluate the effect of sperm treatment with exogenous platelet-activating factor (PAF) on intrauterine insemination (IUI) clinical pregnancy rate in cases of mild male factor infertility. PAF is a phospholipid mediator, which is present in human sperm. The study was performed in the Assisted Reproduction Unit of the 2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieion Hospital, Athens, Greece, and included 92 couples who presented with mild male factor infertility—all candidates for IUI. A maximum of 4 IUI cycles per couple with or without exogenous PAF treatment were performed and the main outcome measure was the clinical pregnancy rate (pregnancies confirmed by ultrasonography per 100 cycles). The overall clinical pregnancy rate after a maximum of 4 IUI cycles was comparable in cases with and without sperm treatment with PAF (12.24% vs 11.11%). Addition or exclusion of PAF sperm treatment in the same patients did not significantly alter the outcome. The generalized use of exogenous PAF for the preparation of sperm in unselected cases of mild male infertility does not improve the clinical outcome of IUI. UROLOGY 74: 1025–1028, 2009. © 2009 Elsevier Inc.
T
he use of exogenous supplementation to enhance sperm function constitutes an attractive option. To achieve such an important goal, the role of possible adjuvants must be well defined, especially when dealing with specific patient population. Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine) is an important phospholipid mediator, which is produced by a variety of cells.1 Apart from platelet activation and various other effects,2,3 PAF seems to have an important role in reproduction by being involved in ovulation, sperm capacitation, fertilization, preimplantation embryo development, implantation, and parturition.4 PAF, being present in human sperm, is one of the endogenous factors directly correlated to sperm From the Assisted Reproduction Unit, 2nd Department of Obstetrics and Gynecology, Aretaieion Hospital, University of Athens, Athens, Greece; and “Embryo ART”, IVF Unit, 2nd Department of Obstetrics and Gynecology, Aretaieion Hospital, University of Athens, Athens, Greece Reprint requests: Stavroula Baka, M.D., 104, Pindou St, 15669 Athens, Greece. E-mail: [email protected] Submitted: March 14, 2009, accepted (with revisions): June 2, 2009
© 2009 Elsevier Inc. All Rights Reserved
motility, forward progression, and is responsible for the regulation of spermatozoa fertilization capacity.5 Its mechanism of action on spermatozoa, even though not exactly known, is probably mediated by a specific receptor.6 The expression, presence, and distribution of this receptor have been found to be distorted in abnormal spermatozoa,7 whereas there is a positive relation between the PAF content in human spermatozoa and their motility as well as pregnancy outcome.8 Treatment of human sperm with exogenous PAF stimulates sperm motility rate and fertilization potential.9,10 Intrauterine insemination (IUI) can be the first-line treatment for the management of infertile couples with unexplained infertility, mild and moderate male factor, anovulatory disorders, or cervical hostility; until now, no method of sperm preparation has been shown to be superior with regard to pregnancy rate after IUI, whereas whether supplementation of culture media with substances such as antioxidants may improve the results, remains the subject of further research.11 By elucidating 0090-4295/09/$34.00 doi:10.1016/j.urology.2009.06.023
1025
the role of PAF in cases of IUI, the scientific interest in this molecule can be extended to other areas of assisted reproductive technologies to improve the therapeutic approach. The aim of the present study was to evaluate the effect of sperm treatment with exogenous PAF on IUI clinical outcomes in cases of mild male factor infertility.
MATERIAL AND METHODS Subjects The study included 92 couples presenting with primary infertility in the Assisted Reproduction Unit of the 2nd Department of Obstetrics and Gynecology, University of Athens, Greece, between May 2006 and December 2007. All the female study subjects met the following criteria: age ⱕ38 years, duration of infertility ⱖ1 year, regular menstrual cycle of 26-32 days, unremarkable clinical findings on gynecological examination, ovulatory basal body temperature chart, midluteal serum progesterone levels ⱖ16 ng/mL, levels of follicle-stimulating hormone, luteinizing hormone, androstenedione, testosterone, dehydroepiandrosterone sulfate, and prolactin within normal range on cycle day-3, normal thyroid function tests, nonsignificant transvaginal ultrasound scan, and normal hysterosalpingogram. All the male study subjects met the following criteria: age ⱕ40 years, unremarkable medical history and findings on physical examination, impaired semen parameters on 2 occasions according to the World Health Organization criteria,12 with a sperm concentration of 10-20 ⫻ 106, and/or forward progressive motility of 30%-50%, and/or sperm morphology of ⬍14% normal forms (strict criteria). The Ethical Committee of Aretaieion Hospital approved the study and informed consent forms were obtained from all subjects.
Treatment The first-step treatment decision for all couples was that of intrauterine insemination in an ovarian stimulation cycle. In all cases, clomiphene citrate was administered on days 3-7 of the cycle in a daily dose of 100 mg. Ovarian response was monitored by serial ultrasound scans, and when the mean diameter of the leading follicle was ⱖ18 mm, 10 000 IUs of human chorionic gonadotropin were administered. Intrauterine insemination was scheduled to be performed 34-38 h after the human chorionic gonadotropin injection. A double-blind randomization was performed on the day of the first IUI attempt for each couple in predetermined blocks (8 consecutive blocks of 10 followed by 1 block of 6) using random number tables and the study couples were allocated to 2 groups: In Group A-PAF (46 couples), the sperm for IUI was treated with PAF, whereas in Group B-non PAF (46 couples), such a treatment was not offered (Fig. 1). If pregnancy was not achieved, a similar IUI cycle was organized. After completing 2 consecutive IUI cycles with no success, Group A failures had a maximum of 2 new IUI cycles with no PAF treatment (Group A-non-PAF), whereas Group B failures had a maximum of 2 new IUI cycles with PAF treatment of the sperm (Group B-PAF). A positive pregnancy outcome was recorded when a gestational sac containing a fetal pole was observed by ultrasound scans.
Sperm Preparation In Groups A-PAF and B-PAF, semen specimens were processed (400g, 10 min) through a 90% density silane-coated colloidal 1026
Figure 1. Flow diagram of the population included in the prospective double-blind randomized cross-over study.
silica suspension (1:1; Fertipro, Belgium), then were treated with an exogenous mixture of PAF (final concentration, 10⫺7 mol/L), (Calbiochem-Novabiochem, La Jolla, CA), in sperm washing medium (SWM; Sydney IVF sperm medium, Cook, Australia). After an exposure period of 15 min at 37°C, the sperm was washed free of unbound PAF (300g, 8 min) and suspended in SWM. In Groups A-non-PAF and B-non-PAF, semen specimens were processed through a 90% density suspension (400g, 10 min), washed with 4 mL of SWM, centrifuged (300g, 8 min), and resuspended.
Statistical Analysis Statistical analysis was performed with the SPSS 10.7 statistical software for Windows with the appropriate use of 2-test/Fisher exact, Student t test, and Kruskal-Wallis test; a binary logistic regression model was used to evaluate whether different categories of male factor could influence the IUI outcome (presence or absence of clinical pregnancy) in cases with or without sperm treatment with PAF. A minimal sample size of 80 IUI cycles per arm was required to achieve a study power of 80% for the lowest increase in IUI pregnancy rates after sperm treatment with PAF (100%), as reported in published studies. Statistical significance was set at ⬍.05.
RESULTS Table 1 shows data regarding subjects’ age, duration of infertility, type of semen abnormality in the study groups, and sperm parameters on recruitment. Age of females, age of males, and duration of infertility were comparable between the 2 groups (P ⫽ .11, P ⫽ .2, and P ⫽ .11, respectively). In addition, no significant differences regarding the type of semen abnormality were observed between the 2 groups. Finally, no significant differences were observed between the 2 groups regarding sperm concentration (P ⫽ .37), progressive motility (P ⫽ .33), and morphology (P ⫽ .77). UROLOGY 74 (5), 2009
Table 1. Characteristics of study groups Characteristic Female age* (y) Male age* (y) Duration of infertility* (mon) Oligospermia (%) Asthenospermia (%) Teratozoospermia (%) Oligo-asthenospermia (%) Oligo-asthenoteratozoospermia (%) Concentration* (⫻106/mL) Progressive motility* (%) Normal morphology* (%)
Group A
Group B
29.2 ⫾ 4.1 31.5 ⫾ 3.1 16.2 ⫾ 3.7 23.91 15.21 4.34 45.65 10.86
28.0 ⫾ 2.9 32.4 ⫾ 3.5 17.4 ⫾ 3.5 17.39 21.73 6.52 52.17 2.17
17.3 ⫾ 3.4 18.6 ⫾ 7.8 38.7 ⫾ 9.8 40.7 ⫾ 9.6 24.1 ⫾ 10.1 23.5 ⫾ 11.1
* Values are means ⫾ SD.
Table 2. Pregnancy rates and statistical comparisons in the study groups Group
Pregnancy Rate (%) †
A-PAF* A-non-PAF*‡ B-PAF§‡ B-non-PAF§†
11.90 12.06 12.69 10.46
* A-PAF vs A-non-PAF, P ⫽ .58. † A-PAF vs B-non-PAF, P ⫽ .47. ‡ B-PAF vs A-non-PAF, P ⫽ .56. § B-PAF vs B-non-PAF, P ⫽ .43.
The mean ⫾ SD of inseminating motile count was 7.1 ⫾ 2.9 (⫻106/mL) in IUI cycles with PAF treatment and 7.9 ⫾ 3.7 (⫻106/mL) in IUI cycles without PAF treatment; the inseminating motile count was comparable between the 2 groups (P ⫽ .07). The number of IUI cycles in the study groups, the number of pregnancies achieved as well as the discontinuation from the study can be seen in Figure 1. There were 10 pregnancies achieved in Group A-PAF, 9 pregnancies in Group B-non-PAF, 7 pregnancies in Group B-PAF and 7 pregnancies in Group A-non-PAF. The PAF arm of the study included 147 IUI cycles, whereas the non-PAF arm included a total of 144 IUI cycles; these numbers of cycles were adequate for the initial study power calculation. The clinical pregnancy rate (clinical pregnancies per 100 cycles) in the 4 groups studied and statistical comparisons between these groups are presented in Table 2. Clinical pregnancies in cases with and without sperm treatment with PAF were comparable during the first 2 IUI attempts (Group A-PAF vs Group B-non PAF, P ⫽ .47), the subsequent 2 IUI attempts (Group B-PAF vs Group A-non PAF, P ⫽ .56) as well as in the total of the 4 attempts (Groups A-PAF and B-PAF: clinical pregnancy rate 12.24% vs Groups A-non-PAF and B-nonPAF: clinical pregnancy rate 11.11%, P ⫽ .45). Within the 2 Groups A and B, sperm treatment with PAF resulted in a comparable number of clinical pregnancies (Group A-PAF vs Group A-non-PAF, P ⫽ .58 and Group B-PAF vs Group B-non-PAF, P ⫽ .43). The order of IUI attempts did not influence the outcome (clinical pregnancies, P ⫽ .94). Finally, the type of semen abnorUROLOGY 74 (5), 2009
mality could not significantly influence the outcome in cases with PAF treatment (Groups A-PAF and B-PAF, P ⫽ .38) as well as in cases without such a treatment (Groups A-non-PAF and B-non-PAF, P ⫽ .84).
COMMENT PAF is present in human sperm, and the spermatozoa demonstrate enzymatic pathways to synthesize and metabolize PAF.13 By binding to specific surface receptors,14 PAF could possibly trigger the formation of inositol triphosphate and diacylglycerol and could increase the intracellular calcium by stimulating the release from intracellular calcium stores as well as the entry of extracellular calcium.15-18 The elevated intracellular calcium, by causing depolymerization of the membrane actin network and activation of phospholipases, could result in acrosome reaction,19 enhanced sperm motility, and, theoretically, in increased fertilization potential.9 The distribution of PAF receptors is not equal among various spermatozoa regions with the highest concentrations being present at the neck and midpiece.8 The midpiece region contains the mitochondria and is important for the spermatozoa motility, whereas the neck region contains the proximal centriole and is important for the preimplantation embryo development.20 Finally, PAF antagonists inhibit the above-mentioned PAF receptor– mediated effects.21,22 Cumulative data support the positive effects of sperm treatment with exogenous PAF. Such a treatment increases the motility of fresh and frozen-thawed sperm,23 enhances the in vitro fertilization rates of mouse and rabbit oocytes, and results in increased rabbit embryo development.22,24-26 Finally, the use of exogenous PAF during sperm preparation for IUI appears to be an attractive option. This treatment in 10 sperm samples for IUI resulted in a double pregnancy rate (40%) compared with 10 controls.27 In a study of similar design with the present one, but a maximum of 3 IUI cycles with PAF treatment and 3 with direct swim-up technique per patient, including cases of unexplained infertility, we reported a significantly increased overall clinical pregnancy rate when sperm was treated with exogenous PAF compared to the direct swim-up technique (23.07% vs 7.92%).28 Unfortunately, we could not show similar positive outcomes in IUI cycles when PAF treatment was applied in sperm with impaired parameters. Clinical pregnancy rates were comparable not only between treatment and control groups, but within the same groups after application or removal of PAF treatment. Our results agree with those of other investigators, similar to those of Roudebush et al29 who reported in their prospective randomized study that PAF treatment enhances IUI pregnancy rates in cases of non-male factor infertility (53.9% vs 28.6% in controls), but not in cases of male factor infertility (11.8% vs 9.7% in controls). The absence of an advantageous effect of PAF treatment in male factor cases could be attributed to defective or low numbers of PAF 1027
receptors in abnormal sperm, which may result in nonresponsiveness to endogenous or exogenous PAF. According to several observations, the allocation of PAF receptors is affected in abnormal sperm, whereas the PAF-receptor mRNA sequences and expression differs between motile and nonmotile sperm.29,30 Nevertheless, this study carries certain limitations, such as the skepticism for cross-over designs in subfertility research, and the lack of information on concentrations of semen PAF, sperm PAF-receptors, semen PAF-acetylhydrolase, and endometrial PAF status; the knowledge of specific insufficiencies regarding the above-mentioned factors, might have individualized the use of exogenous PAF treatment, allowing for better outcomes. Furthermore, another limitation of the study is the lack of a concurrent positive control, which could demonstrate that the PAF used had full bioactivity. Finally, the present study was powered to detect a quite high increase in clinical pregnancy rate after the use of exogenous PAF; obviously, a larger study population could possibly allow for valid detection of a more moderate enhancement. Additional experimental studies are warranted to further clarify the importance of sperm treatment with exogenous PAF on IUI outcomes in cases of mild male factor infertility.
CONCLUSIONS The generalized use of exogenous PAF for the preparation of sperm in cases of mild male infertility does not improve the clinical outcome of intrauterine inseminations; this does not necessarily mean that there is no place for exogenous PAF use in a more specific and well-defined group of subfertile males.
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