Effect of Iysoplatelet-activating factor on human sperm fertilizing ability*

Transcription

1 FERTILITY AND STERILITY Vol. 59, No.4, April 1993 Copyright!l'J 1993 The American Fertility Society Printed on acid-free paper in U.S.A. Effect of Iysoplatelet-activating factor on human sperm fertilizing ability* Marie-Helene Lachapelle, M.Sc. t:\: Renda Bouzayen, M.D.t Jean Langlais, Ph.D.t:\: Keith Jarvi, M.D.t Jacques Bourque, M.D.* Pierre Miron, M.D.*tll Institut de Medecine de la Reproduction de Montreal, Hopital Maisonneuve-Rosemont, Universite de Montreal, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada Objective: To evaluate the penetration rates in the hamster zona-free oocyte sperm penetration assay (SPA) after exposure of spermatozoa to lysoplatelet-activating factor (LPAF) and lysophosphatidyl choline (LPC). Design: Washed human spermatozoa were exposed to 1 ILM of LP AF or LPC, followed by the assessment of their fertilizing ability using the SPA. The percentage of penetration, the sperm binding in the SPA, the percentage of motile spermatozoa, and the acrosome reaction rates were quantified. Setting: Private research and university laboratories. Patients, Participants: Fresh and frozen semen samples from fertile donors with proven fertility were used as well as fresh semen from infertile patients attending a fertility clinic. All the infertile patients had abnormal semen analysis. Interventions: Human spermatozoa were incubated for 9 minutes in the presence or absence of LPAF or LPC at 1 ILM with.3% albumin in Ham's F-1 (GIBCO, Dorval, Quebec, Canada), and their fertilizing ability was evaluated using the SPA. The effect of these lysophospholipids on the percentage of acrosome reaction was evaluated with a fluorescent microscopy technique. Results: The penetration rates of the SPA in male factor increased significantly from 3% ± 6% with controls to 19% ± 9% and 34% ± 22% after incubation with LPC and LPAF, respectively. Sperm-oocyte binding was not significantly increased in this group. Sperm penetration assay penetration rates were also increased in fertile cryopreserved spermatozoa with LPC and LP AF. In this group, the acrosome reaction was significantly increased from 2% ± 1 % in controls to 1% ± 6% and 8% ± 3% after incubation with LPC and LPAF, respectively. Conclusion: Lysoplatelet-activating factor and LPC independently increased the penetration rate of spermatozoa and the percentage of acrosome reaction. Lysophosphatidylcholine and LP AF may be beneficial in the treatment of spermatozoa with male factor infertility and may increase fertilization rates in IVF. Fertil Steril 1993;59:863-8 Key Words: Lysoplatelet-activating factor, lysophosphatidyl choline, fertilizing ability, IVF, asthenozoospermia Received August 17, 1992; revised and accepted December 14, * Supported by a grant from the Institut de Medecine de la Reproduction de Montreal, Montreal, Quebec, Canada. t Institute de Medcine de la Reproduction de Montreal. :j: Department d'obstetrique-gynecologie, Hopital Maisonneuve-Rosemont, Universite de Montreal. Urology Research Laboratory, Royal Victoria Hospital, McGill University. II Reprint requests: Pierre Miron, M.D., Institut de Medecine de la Reproduction de Montreal, 11 Beaumont, Suite 35, Montreal, Quebec, Canada H3P 3E5. Approximately 5% of all human IVF attempts will end with a failure to fertilize (1). If the IVF is performed for male factor infertility (for men with an identified abnormality of the routine semen analysis parameters of count and percentage of motile spermatozoa), the chances of the IVF cycle resulting in nonfertilization rises to 5% with an overall 45% decrease in the pregnancy rates (2). In both of these groups, diminished fertilizing capacity (only 1 to 2 oocytes fertilized) will also contribute to diminished conception rates. Vol. 59, No.4, April 1993 Lachapelle et al. LP AF increases sperm penetration rate 863

2 Fertilization depends on the ability of spermatozoa to contact, bind to, and penetrate the oocyte, followed by the sperm-egg fusion. These processes are dependent primarily on sperm motility, capacitation, and acrosome reaction. Men with a low percentage of motile spermatozoa or having spermatozoa with an inability to capacitate or acrosome react have lower IVF fertilization rates than men with normal semen parameters (2). Factors that stimulate sperm motility, capacitation, or the acrosome reaction might enhance fertilization. There are several isolated compounds that stimulate sperm motility. Phosphodiesterase inhibitors (caffeine and pentoxifylline) (3, 4), and dibutylcyclic adenosine monophosphate (Burkman L, unpublished observations) have all been reported to induce increases in human sperm motility. Recent research has shown that platelet-activating factor (PAF), lysoplatelet-activating factor (LPAF), and lysophosphatidyl choline (LPC) stimulate sperm motion, even for spermatozoa with very low initial parameters (5). No other phospholipid was reported to similarly stimulate sperm motion. A number of other isolated compounds have been reported to increase sperm capacitation rates. Among others, LPC, lysophosphatidylethanolamine, and lysophosphatidylinositol reduce the capacitation time required for bovine spermatozoa (6, 7). There is a large and growing group of agents that have been found to increase the percentage of acrosome reaction for capacitated spermatozoa in vitro. A partial list includes PAF, LPC, diacyl glycerol glyceryl mono-oleate, phospholipase A 2, and fatty acids (8, 9). Although we have the ability to specifically examine the spermatozoa motility and acrosome reaction, one of the most accurate measures available in the laboratory to estimate the overall ability of human spermatozoa to fertilize human oocytes in vitro is the sperm penetration assay (SPA) of the zona-free hamster oocyte, developed by Yanagimachi in 1972 (1). This assay does not measure the specific motility, capacitation, or acrosomal reaction but is an overall measure of the functional capacity of the spermatozoa. Several isolated compounds are known to increase the percentage penetration for the SPA including substance P, and the phospholipids PAF and LPC (9, 1). The recent work in our laboratory on the effect of phospholipids on sperm motion reveals that not only P AF, but two other lipids, LPC and LP AF strongly stimulate sperm motion (5). In this present study, these latter two lipids were tested for their effects on the percentage of acrosome reaction and the SPA for human spermatozoa. Although LPC is known to increase the percentage of acrosome reaction and the SPA for capacitated spermatozoa, the effect of LPAF on these two processes was unknown. In addition, the effect of either of these lipids on spermatozoa with oligozoospermia or asthenozoospermia has never been reported. Materials MATERIALS AND METHODS Lysoplatelet activating factor (1--hexadecyl-racglycerophosphorylcholine), LPC (l-palmityl-racglycerophosphorylcholine), the constituents of Biggers, Whitten, and Whittingham media (BWW) (sodium chloride, potassium chloride, calcium dihydroxychloride, magnesium sulphate, potassium phosphate, glucose, sodium bicarbonate, sodium lactate, sodium pyruvate, and phenol red), pregnant mare serum gonadotropin (PMSG), hcg, fluorescein isothiodyanate (FITC)-conjugated pisatum sativum agglutinin, hyaluronidase, trypsin 1,4-Diazabicyclo [2.2.2] octane, and fraction V human serum albumin (HSA) were purchased from Sigma Chemical Co. (St. Louis, MO). Ham's F-I media was supplied by GIBCO Laboratories (Grand Island, NY). Petri dishes and paraffin oil were obtained from Fisher Scientific Co. (Montreal, Quebec, Canada). The Percoll was purchased from Pharmacia Fine Chemicals (Dorval, Quebec, Canada). Reagent grade solvents were obtained from John's Scientific Inc. (Lachine, Quebec, Canada) Semen Samples After a period of sexual abstinence of at least 48 hours and not >5 days, semen samples were collected from infertile patients attending an infertility clinic (n = 9), all of whom had semen abnormalities according to the World Health Organization criteria (11), and from donors with proven fertility (n = 3). Samples were collected by masturbation into sterile disposable plastic containers and allowed to liquefy for 15 to 3 minutes at room temperature (RT). Also, semen samples from donors with proven fertility, cryopreserved after a 1:1 dilution with a solution containing glycerol, egg yolk, 5.5% glucose, and 3% sodium citrate (15:2:26:39) was used and allowed to thaw at RT before use (n = 9). The concentration of spermatozoa in each sample was determined with a hemocytometer. The percentage of motile spermatozoa was visually assessed (X4). 864 Lachapelle et al. LP AF increases sperm penetration rate Fertility and Sterility

3 Spermatozoa Preparation Spermatozoa were prepared for the SPA and acrosome reaction test by a modification of the technique described by Bolanos et al. (12). Semen was layered on top of a Percoll density gradient of 9% and 45% Percoll buffered with Ham's F -1 medium, then centrifuged at 3 X g for 3 minutes. The supernatant was removed with a Pasteur pipette, and the sperm pellet was resuspended at a concentration of1 X 1 6 /ml in Ham's F-lO medium supplemented with.3% HSA. This suspension was divided into three equal volumes with one part used as control. Lysophosphatidyl choline or LPAF were added to the other parts at final concentration of 1 1LM. The LPAF concentrations and the time of incubation were based on optimal conditions that we found in a previous study (5). The three samples were then incubated for 9 minutes at 37 C with 5% CO 2 or the same conditions described by Sugkraroek et al. (13) for their work on capacitation. The sperm suspension was again washed through a Percoll gradient, and the pellet was resuspended in.3 ml of Ham's F-1 medium supplemented with.3% HSA. Sperm Penetration Assay The hamster ova were obtained by the method described by Yanagimachi (14). Briefly, female Golden hamsters, 8 to 12 weeks old, were superovulated by injection of 25 IUIP of PMSG. Fifty-two hours later, 25 IUIP of hcg was given. The animals were killed by cervical dislocation 17 hours after the hcg injection, the oviducts were removed, and the cumulus oophorus was dissolved with.1% hyaluronidase in BWW medium to free the eggs. The eggs were harvested, washed three times in BWW medium, and then transferred to a.1 % trypsin in BWW medium to remove the zona pellucida. When the zona began to dissolve, the eggs were washed again three times in BWW medium. After assessment of motility, aliquots (.1 ml) containing adjusted density of preincubated spermatozoa (1.5 X 1 9 /L) were placed in 35 X 1-mm Petri dish and covered with warmed (37 C) paraffin oil. Twelve zona-free eggs were transferred into each sperm drop and incubated at 37 C for 3 hours. Then the eggs were collected and washed twice in BWW medium supplemented with.3% HSA. The eggs were then examined under a phase-contrast microscope at X4 for the quantification of bound spermatozoa and spermatozoa that had penetrated. An egg was recorded penetrated when at least one swollen sperm head (or sperm pronucleus) and its associated tail were visible within the vitellus (14). Determination of Acrosome Reaction Acrosomal status was determined by the technique of Cross et al. (15). Briefly, spermatozoa, after a 9-minute incubation at 37 C with 1 1LM LPC or LPAF or with no added lipids for the control, were fixed in alcohol at 4 C, spotted on a slide, and air dried. The slide was coated with FITC-conjugated pisatum sativum agglutinin and washed with distilled, deionized water; then a coverslip was mounted over diazabicyclo octane (DABCO) and examined under fluorescent microscopy. A minimum of 2 spermatozoa were examined per sample. Statistical Analysis Statistical analyses were performed with the twotailed paired Student's t-test. If P <.5 the results are labeled significant. Values are expressed as means ± SD. RESULTS Effect of Pretreatment of Human Spermatozoa With LP AF or LPC on the SPA Preincubation for 9 minutes with LPC or LP AF induced increases in the penetration rates for the SPA for both patients' fresh and donors' frozen spermatozoa. Spermatozoa from a group of male infertile patients had control SPA penetration rates of 3% ± 6%, which was increased significantly to 19% ± 9% and 34% ± 22% with incubations of 1 1LM LPC or LPAF, respectively (Fig. 1A). Of note, LP AF induced higher SPA penetration rates than LPC. Frozen donor sperm penetrated 14% ± 19% of the hamster oocytes with no added lipids, but these penetration rates increased significantly to 65% ± 29% and 95% ± 1% when the spermatozoa were pre incubated with LPC or LPAF, respectively (Fig. 1B). Again, LPAF induced significantly greater increases in the SPA than did LPC. Spermatozoa prepared from fresh donor semen penetrated 1% of the oocytes whether the male gametes were treated with or without lipids (Table 1). Interestingly, treatment of infertile gametes with 1 1LM LP AF increased the penetration rates above that observed for the controls of donors' frozen sperm (34% versus 14%). The other parameter measured in the SPA, the number of spermatozoa bound per oocyte, was less dramatically affected by the addition of the phospholipids than was the percentage of penetration rates. In both of the groups in which fresh semen Vol. 59, No.4, April 1993 Lachapelle et al. LP AF increases sperm penetration rate 865

4 1 8 6 c tii Qj c c A Control lpc lpaf Table 1 Effect of LP AF and LPC on Fresh Donors' Spermatozoa* Penetration on SPA (%) Sperm-egg binding (%) Sperm motility (%) Control 1 ± 11 ± 6 84 ± 3 LPC 1± 6±3 83 ± 6 LPAF 1 ± 7 ± 1 84 ± 4 * Spermatozoa were incubated at 37 C with 1/lM LPAF or LPC for 9 minutes. After washing, the spermatozoa were added to the oocyte and incubated for 37 C for 3 hours (n = 3).?i tii 1 8 c 6 tii 4 Qj c 2 c.. B '// % ' Control lpc lpaf Figure 1 Effect of LP AF or LPC on the SPA for spermatozoa from infertile men and human donors with proven fertility, After a 9-minute incubation with 1 /lm LPC, LPAF, or no added lipids, the spermatozoa were washed and added to the hamster oocytes, (A), The percentage of oocytes penetrated for spermatozoa from infertile men is illustrated (n = 9), whereas B is for spermatozoa from donors with proven fertility (n = 9). *Significantly greater than control; **significantly greater than LPC with 1 J.lM LPC or LPAF, respectively. In donor spermatozoa (n = 9), after the same incubation, 2% ± 1% had acrosome reacted, whereas this was significantly increased to 1% ± 6% and 8% ± 3% with 1 J.lM LPC or LPAF, respectively (Fig. 2). Percentage of Motile Sperm The percentage of motile spermatozoa did not change significantly when spermatozoa were incubated with LPAF, LPC, or no added lipids. Percentage of motile spermatozoa after incubation for control, LPC, and LPAF was 84% ± 3%, 83% ± 6%, and 84% ± 4%, respectively, for fresh donor semen (n = 3); 77% ± 5%, 76% ± 5%, and 74% ± 7%, respectively, for frozen donor semen (n = 9); and 77% ± 8%, 79% ± 8%, and 78% ± 5%, respectively, for fresh patient semen (n = 9). was used, there were no significant changes in the number of spermatozoa bound with or without the addition of lipids. Spermatozoa from two groups of infertile patients had control spermatozoa binding rates of 1.7% ±.5%, 2.4% ±.6% with LPC and 1.6% ±.3% with LPAF. The results for the fresh donors semen are listed in Table 1. Preincubation of cryopreserved donor semen with either LPC or LPAF (8.3% ± 1.6% and 8.9% ± 1.1%, respectively) induced small but statistically significant increases in the number of sperm bound per oocyte compared with control (5.5% ±.8%). Of interest, the infertile patients' spermatozoa bound to the hamster oocytes much less avidly than either the fresh or frozen donor spermatozoa. Acrosome Reaction After incubation for 9 minutes, 2% ± 1 % of the spermatozoa from five men with male factor infertility had acrosome reacted, whereas this was increased to 22% ± 31 % and 5% ± 3% with incubations DISCUSSION A lack of fertilization occurs in approximately 5% of IVF cycles, and a low number of fertilized oocytes (1 to 2 fertilized oocytes), possibly representing some form of fertilization defect, occurs in up to 3% of IVF cycles (1, 2). In regard to the outcome of IVF, 6 c 5 U 4., a: 3 E 2 In 1 u c:( Control lpc lpaf IZl lsi DONOR PATIENT Figure 2 Effect of LP AF or LPC on the acrosome reaction of capacitated spermatozoa. Spermatozoa were incubated with 1 /lm LPAF, LPC, or no added lipids, and the acrosome reaction was assessed. *Significantly greater than control values. 866 Lachapelle et al. LP AF increases sperm penetration rate Fertility and Sterility

5 it is thus important to find new strategies that will increase the fertilizing ability of subfertile spermatozoa. In the past, attempts have been made to increase fertilization rates by adding agents to the incubation media (e.g., pentoxifylline) or by methods of micromanipulation (16, 17). The former has resulted in only a mild improvement in fertilization rates, whereas the latter is highly invasive, requires highly skilled micromanipulation techniques, and remains labor intensive and costly. Clinically, we are still looking for a widely applicable method to overcome fertilization defects or to increase fertilization rates in IVF. To test compounds for their potential effects on human IVF fertilization rates, we have chosen the SPA as a test assay. Although there is controversy, the SPA results have been correlated to IVF fertilization and conception rates (18). This series has shown that incubating spermatozoa (from patients with male factor infertility or cryopreserved donor semen) with LPC or LPAF caused a significant increase in the percentage of penetration for the SPA. Lysoplatelet-activating factor induced a greater increase in the percentage of penetration for the SPA than LPC. All patients had control SPA penetration rates of <11 % (the reported cutoff for normal), whereas eight of nine had penetration rates in the normal range with LP AF incubation. These lipids may also facilitate fertilization for fresh semen from fertile donors, but because we found a 1% SPA penetration rate for these specimens without added lipids (unchanged with the addition of lipids), it was not possible to document any increase in the SPA for these donors under our test conditions. These compounds, LPC and LP AF, appear to be acting on the spermatozoa and not on the oocyte. After incubation with the lipids, the sperm suspensions were rewashed through a Percoll density gradient to remove the LPC or LPAF still in solution, before incubation of the spermatozoa with the - cytes. With this technique, the concentration of LPC or LP AF in the incubation media with the oocytes should be very low. Successful penetration with the SPA requires contact between the spermatozoa and the oocytes, sperm binding, and penetration. The sperm-oocyte contact as well as the penetration is aided by more vigorous sperm motion, whereas the acrosomal reaction is important for penetration. In previous studies it was shown that both LPC and LPAF induce increases in the sperm linear and curvilinear velocities, with greater increases at 37 C noted with Vol. 59, No.4, April 1993 LPAF (6). The incubation with either ofthese lipids did not alter the percentage of motile spermatozoa. The present study demonstrated that both LPC and LPAF induce an acrosome reaction, with LPC inducing a slightly greater increase in the acrosome reaction than LP AF. The exogenous LPC appears to facilitate the acrosome reaction more than LPAF, but the functional study with the SPA indicates that LP AF has a greater effect on the types of sperm parameters that are more closely associated with human fertilization than LPC. The question of whether these lipids are physiologically active remains unanswered. Previous reports have provided evidence for an increase in the LPC concentration of spermatozoa with capacitation and the acrosomal reaction (19). It was postulated that the accumulation of LPC is partly due to an up regulation of sperm phospholipase A2 with capacitation, resulting in an increased hydrolysis of phosphatidyl choline to a fatty acid and LPC on the spermatozoa membrane. Lysophosphatidyl choline is a detergent with fusogenic properties, and the terminal event in the acrosomal reaction is thought to be an accumulation of LPC in the acrosomal membrane leading to a vesiculation of the membrane and a release of acrosomal contents (19). We postulate in our experiments that the exogenous LPC is taken up nonspecifically onto the spermatozoa membrane, leading to acrosomal membrane destabilization, vesiculation, and release of acrosomal contents. Alternatively, LPC may be acting through a specific receptor to induce the acrosomal reaction. Lysoplatelet-activating factor, with its close structural similarity to LPC, is also a fusogenic lipid and may be inducing the acrosomal reaction by either method proposed for LPC. Although there is no evidence for an accumulation of LPAF during capacitation, it is known that spermatozoa produce PAF during capacitation and that P AF is very rapidly hydrolyzed to LPAF and acetate by PLA2 and a PAF-acetylhydrolase-like enzyme found in the seminal plasma and on the spermatozoa (19-21). Thus, it is possible that the normal LP AF concentrations on the sperm membrane may be within the range of LP AF used in experimental conditions. Why LPAF induces a greater increase in the SPA penetration rates than LPC remains unanswered. Interestingly, LPC may be a physiological capacitation factor in vivo, given that LPC has been shown to accumulate in human follicular fluid (22) in concentrations similar to those used in the present study. Both LPC and LP AF induce significant increases in the functional assay for human spermatozoa fer- Lachapelle et al. LP AF increases sperm penetration rate 867

6 tilizing capacity, the SPA. Although both lipids induce significant increases in the penetration rates for the SPA, LP AF had a significantly greater effect than LPC. Of note, all semen specimens from patients with male factor infertility had control SPA penetrations rates of less than the cutoff for normal, but eight of the nine specimens had normal penetration rates when incubated with LP AF. These data indicate that these phospholipids may be of value in an IVF program for the treatment of spermatozoa with a fertilization defect. Weare presently testing the effect of LP AF on human IVF for male factor infertility. Acknowledgments. The authors are grateful for Ms. Linda Ward's assistance in the laboratory and for Ms. Nicole Dubuc's secretarial skills in the preparation of this manuscript. REFERENCES 1. Oehninger S, Acosta AA, Kruger T, Veeck LL, Flood J. Failure of fertilization in in vitro fertilization: the occult male factor. J Vitro Fert Embryo Transf 1988;5: Tournaye H, Devroey P, Camus M, Staessen C, Bollen N, Smitz J, et al. Comparison of in vitro fertilization in male and tubal infertility: a 3 year survey. Hum Reprod 1992;7: Ruzich JV, Gill H, Wein AJ, Van Arsdalen K, Hypolite J, Levin RM. Objective assessment of the effect of caffeine on sperm motility and velocity. Fertil SteriI1987;48: Aparicio NJ, de Turner EA, Schwarzstein L, Turner D. Effect of the phosphodiesterase inhibitor pentoxifylline on human sperm motility. Andrologia 198;12: Jarvi K, Langlais J, Roberts KD, Gagnon C. Effect of platelet activating factor (PAF) Lyso-P AF and lysophosphatidylcholine (LPC) on sperm motion: importance of albumin for motility stimulation. Fertil Steril. In press. 6. Ehrenwald E, Parks JE, Foote RH. Cholesterol efflux from bovine sperm. I. Induction of the acrosome reaction with lysophosphatidylcholine after reducing sperm cholesterol. Gamete Res 1988;2: Wheeler MB, Seidel GE Jr. Capacitation of bovine spermatozoa by lysophospholipids and trypsin. Gamete Res 1989;22: Fleming AD, Yanagimachi R. Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig sper- matozoa with special reference to the possible involvement of lysophospholipids in the acrosome reaction. Gamete Res 1981;4: Kyono K, Hoshi K, Saito A, Tsuiki A, Hoshiai H, Suzuki M. Effects of phospholipase A 2, lysophosphatidylcholine, and fatty acid on the acrosome reaction of human spermatozoa. J Exp Med 1984;144: Yanagimachi R. Penetration of guinea-pig spermatozoa into hamster eggs in vitro. J Reprod FertiI1972;28: World Health Organization. WHO Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge, Bolanos JR, Overstreet JW, Katz DF. Human sperm penetration of zona-free hamster eggs after storage of the semen for 48 hours at 2 C to 5 C. Fertil Steril1983;39: Sugkraroek P, Kates M, Leader A, Tanphaichitr N. Levels of cholesterol and phospholipids in freshly ejaculated sperm and Percoll-gradient-pelleted sperm from fertile and unexplained infertile men. Fertil Steril1991;55: Yanagimachi R. Mechanisms of fertilization in mammals. In: Mastroianni L, Biggers JD, editors. Fertilization and embryonic development in vitro. New York: Plenum Press, 1981: Cross NL, Morales P, Overstreet JM, Hanson FW. Two simple methods for detecting acrosome-reacted human sperm. Gamete Res 1986;15: Yovich JM, Edirisinghe WR, Cummins JM, Yovich JL. Influence of pentoxifylline in severe male factor infertility. Fertil Steril 199;53: Ng Soon-Chye, Bongso A, Sathananthan H, Ratnam SS. Micromanipulation: its relevance to human in vitro fertilization. Fertil Steril 199;53: Yanagimachi R, Yanagimachi H, Rogers JB. The use of zonafree animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Bioi Reprod 1976;15: Langlais J, Roberts KD. A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res 1985;12: Kuzan FB, Geissler FT, Henderson WR Jr. Role of spermatozoa platelec activating factor in fertilization. Prostaglandins 199;39: Letendre ED, Miron P, Roberts KD, Langlais J. Plateletactivating factor acetylhydrolase in human seminal plasma. Fertil SteriI1992;57: Perret B-P, Parinaud J, Ribbes H, Moatti JP, Pontonnier G, Chap H, et al. Lipoprotein and phospholipids distribution in human follicular fluid. Fertil Steril 1985;43: Lachapelle et al. LPAF increases sperm penetration rate Fertility and Sterility