Doi:10.1016/j.fertnstert.2003.11.057

FERTILITY AND STERILITY௡
Copyright 2004 American Society for Reproductive Medicine Printed on acid-free paper in U.S.A.
Platelet-activating factor significantly
enhances intrauterine insemination
pregnancy rates in non-male factor
infertility
William E. Roudebush, Ph.D., Andrew A. Toledo, M.D., Hilton I. Kort, M.D.,Dorothy Mitchell-Leef, M.D., Carlene W. Elsner, M.D., and Joe B. Massey, M.D. Reproductive Biology Associates, Atlanta, Georgia Objective: To determine the efficacy of treating semen specimens with platelet-activating factor (PAF) before
IUI.
Design: Prospective randomized double-blinded study of PAF treatment of sperm for patients with a history
of infertility undergoing IUI.
Setting: Private infertility center.
Intervention(s): Patients had ovulation induction therapy with clomiphene citrate (CC) or gonadotropin, two
IUIs per month with PAF treatment.
Main Outcome Measure(s): Clinical pregnancy rates.
Result(s): There was a significant difference in IUI pregnancy rates per cycle between control (10/56; 17.9%)
and PAF (14/47; 29.8%) treatment groups in the normal male study arm. There was a significant difference
in cumulative IUI pregnancy rates between control (10/35; 28.6%) and PAF (14/26; 53.9%) patient groups in
the normal male study arm. There was no significant difference in IUI pregnancy rates per cycle between
control (12/124; 9.7%) and PAF (14/119; 11.8%) treatment groups in the male factor study arm. There was
no significant difference in cumulative IUI pregnancy rates between control (12/46; 26.1%) and PAF (14/38;
36.8%) patient groups in the male factor study arm. There was a significant difference in overall cumulative
IUI pregnancy rates between control (21/81; 25.9%) and PAF (27/64; 42.2%) patient groups.
Conclusion(s): The inclusion of PAF into the IUI sperm wash procedure significantly improves pregnancy
rates. However, the significant improvement can only be shown to affect men presenting with normal semen
parameters. (Fertil Steril௡ 2004;82:52– 6. 2004 by American Society for Reproductive Medicine.)
Key Words: Platelet-activating factor, intrauterine insemination, sperm, pregnancy
IUI cycles followed by gonadotropin/IUI for up induction followed by intrauterine insemina- to three cycles. Failed IUI therapy subsequently tion (IUI) has become standard therapy for results in consideration of IVF therapy. Silver- Presented in part (O-222)at the 58th Annual Meeting nontubal factor-related infertility. The initial berg et al. have shown that two IUI proce- treatment for infertile couples is IUI and is dures per cycle have improved pregnancy rates, Reproductive Medicine,Seattle, WA, October 12– although this continues to be controversial dependent on the ovulation induction regimen Male fertility requires production of an ad- lative pregnancy rates for three cycles have sperm with sufficient motility and the ability to approached those of a single IVF treatment undergo capacitation and the acrosome reac- when gonadotropins are combined with IUI tion to penetrate the oocyte’s cumulus oopho- Atlanta, Georgia 30342(FAX: 404-256-8376; Commonly, because of lower patient costs, rus and zona pellucida for fertilization. Defects complexity and risks of high order multiple in any of these necessary events may lead to pregnancy (i.e., triplets or more), clomiphene subfertility or infertility. Recently, for exam- citrate (CC) is initially used. In our center the ple, chromatin integrity of sperm was found to doi:10.1016/j.fertnstert.2003.
11.057 typical treatment regimen involves three CC/ A number of endogenous biochemical factors have been completed. Cycle stimulation was controlled by CC or go- attributed to regulate the fertility potential of spermatozoa, nadotropins. In CC-controlled cycles, 50 –150 mg of CC was for example, platelet-activating factor (PAF). Platelet-acti- given for 5 cycle days. Timing of IUI was based on LH surge vating factor is a unique and novel signaling phospholipid or ultrasound-timed hCG administration. In cycles managed that has pleiotropic biologic properties in addition to platelet by gonadotropins, stimulations were started on cycle day 3 activation. Since its discovery in the early 1970s this novel with 75–225 IU daily (with dosages individually titrated compound has been implicated in a variety of reproductive based on patient age and previous response). Ultrasound functions including fertilization, implantation, and parturi- (one to four ultrasound follicular studies per IUI cycle) tion The exact mechanism is uncertain, yet its impor- monitoring with E (Access2, Beckman Coulter, Inc., Brea, tance in normal fertility is significant.
CA) observation was performed until the lead follicle was at Platelet-activating factor is present in human spermatozoa least 18 mm in all but exceptional cases. Preovulatory LH and its endogenous content has a significant and positive surge timing was monitored by urine LH levels. The IUI was relationship with motility and pregnancy rate Exogenous performed 12–18 hours and again in 36 –38 hours after hCG PAF has been used to stimulate human sperm motility injection. Patients taking CC who experienced a spontaneous and IVF and embryo development rates in rabbits A LH surge had only one IUI performed 24 hours later.
preliminary study demonstrated the improvement of IUI Semen Analysis
pregnancy rates after a short-term exposure of sperm to PAF.
Semen specimens were permitted to liquefy for 30 – 60 min- In the present study, our objective was to determine the utes at 37°C. Sperm concentration and motility, before and after effect of exogenous PAF on IUI pregnancy rates in couples treatment, were evaluated by computer-assisted semen analysis presenting with normal and abnormal semen analyses. The (IVOS v10.9i, Hamilton-Thorne Research, Beverly, MA) opera- study was a prospective randomized double-blinded study of ting at a sampling frequency of 60 Hz. All analyses were per- PAF treatment of sperm for patients with a history of infer- formed using MicroCell-20 Micron (Conception Technologies, tility undergoing IUI. Patients were randomized (blind num- San Diego, CA) counting chambers. A total of five random ber draw from computer-generated random number table) areas were selected and evaluated by the IVOS system at 37°C.
into either receiving PAF treatment or serving as controls for Semen Processing and PAF Exposure for IUI
up to three IUI cycles. Furthermore, couples were catego-rized into either normal or male factor groups.
Control Group
MATERIALS AND METHODS
Semen specimens were processed (400 g; 12 minutes) through a 90% density silane-coated silica suspension (1:1; Study Population and Management
Promotor; CERES Fertility, San Diego, CA), washed with 4 Healthy, infertile patients with nontubal factor infertility mL of sperm wash medium (InVitroCare, San Diego, CA), were made aware of the study and informed of the study centrifuged (300 g; 8 minutes) and resuspended with 0.5 mL design with the possibility of improved results based on a of PureSperm-Wash (Nidacon International AB, Goteborg, small clinical trial The PAF treatment option was not made available outside the current study. Possible risks were PAF Treatment Group
discussed and informed consent was obtained under guide-lines approved by the Western Institutional Review Board Semen specimens were processed (400 g; 12 minutes) (Seattle, WA). Inclusion criteria were as follows: basal FSH through a 90% density silane-coated silica suspension (1:1; level Ͻ15 mIU/mL, evidence of a normal uterine cavity, and Promotor), resuspended in 10Ϫ7M PAF in sperm wash medium no contraindication to pregnancy. Infertility diagnoses in- (InVitroCare) and incubated for 15 minutes at 37°C. After cluded anovulatory, endometriosis, idiopathic, tubal (single incubation, sperm were washed (300 g; 8 minutes) free of PAF or fibroids), cervical factor, and male factor. Male factor and resuspended in 0.5 mL of PureSperm-Wash (Nidacon).
infertility patients were classified as such if they failed to IUI and Pregnancy Outcomes
meet one or more reference standards Couples were Washed sperm preparations were inseminated with an IUI permitted to enter the study at any point in a series of IUI Catheter (Lifetek Medical, Inc., Portage, WI). Pregnancy treatments. Couples (normal male and male factor study outcomes were determined first by ␤-hCG serum levels groups) were randomized into one of two study groups (Access2, Beckman Coulter, Inc.) and confirmed by ultra- (group 1, control; group 2, PAF) from January 2001 to sonography (fetal heartbeat ϭ a positive outcome).
December 2002. Once patients were assigned to the respec-tive treatment group, only the RBA Andrology Laboratory Sample Size and Statistical Analysis
staff was aware of assignment. Patients were subsequently Power estimates based on published IUI pregnancy rates treated in each consecutive cycle with the same regimen (i.e., (5%–16%) and preliminary data reveal that a minimal sam- control or PAF augmented). No clinical staff (physician or ple size of 83 IUI cycles per male factor and 40 IUI cycles nurse) was informed of assignment until the study was per normal male treatment group is required for alpha to equal FERTILITY & STERILITY௡
Platelet activating factor-intrauterine insemination patient Cumulative Platelet activating factor-intrauterine Control, 36.4 (Ϯ3.01); PAF, 35.5 (Ϯ4.02) Control, 38.5 (Ϯ6.01); PAF, 36.0 (Ϯ5.94) Control, 36.2 (Ϯ4.16); PAF, 35.9 (Ϯ4.93) Control, 35.8 (Ϯ4.54); PAF, 34.1 (Ϯ4.40) a,b,cSimilar superscripts are significantly different, PϽ.05.
Roudebush. Platelet-activating factor and IUI pregnancy. Fertil Steril 2004. enrolled in the study, of these 3 (1.8%) converted to IVF and 17 (10.3%) never followed through with IUI therapy.
Patient pregnancy rates are presented in (per Roudebush. Platelet-activating factor and IUI pregnancy. Fertil Steril 2004. cycle) and (cumulative). Cumulative IUI pregnancyrate by number of attempts according to treatment group for 0.05 and to show a difference with 80% power (beta ϭ 0.80).
all patients (normal and male factor) are presented in Figure A 50% increase in pregnancy rate after treatment to the IUI 1. In couples presenting with normal semen characteristics protocol was used. Data were analyzed by ␹2. Statistical calcu- the cycle-specific pregnancy rate was 29.8% (14/47) when lations were performed with SigmaStat for Windows, ver- PAF was used. This was significantly higher (PϽ.05) when sion 2.03 (Jandel Scientific Corporation, San Rafael, CA).
compared with the control (10/56; 17.9%). Cumulative preg-nancy rates were significantly higher (PϽ.05) in the PAF treatment group (14/26; 53.90%) than the control group(10/35; 28.6%). There was no significant difference in IUI A prospective comparison of pregnancy rates was performed pregnancy rates per cycle between control (12/124; 9.7%) after sperm treatment with or without PAF, at the time of semen and PAF (14/119; 11.8%) treatment groups in the male washing just before IUI. Patient demographics are presented in factor study arm. There was no significant difference in There were no significant differences in patient cumulative IUI pregnancy rates between control (12/46; demographics between the pregnant and nonpregnant groups 26.1%) and PAF (14/38; 36.8%) patient groups in the male or control and PAF treatment groups. A total of 165 patients factor study arm. There was a significant difference (PϽ.05)in overall cumulative IUI pregnancy rates between control(22/81; 27.2%) and PAF (28/64; 43.8%) patient groups.
Platelet activating factor-intrauterine inseminationpregnancy rate per cycle.
Platelet activating factor-intrauterine inseminationmultiple pregnancy rate.
Significantly different, PϽ.05.
Roudebush. Platelet-activating factor and IUI pregnancy. Fertil Steril 2004. Roudebush. Platelet-activating factor and IUI pregnancy. Fertil Steril 2004. Roudebush et al.
Platelet-activating factor and IUI pregnancy Cumulative intrauterine insemination (IUI) pregnancy rate by Ongoing platelet activating factor-intrauterine number of attempts according to treatment group for all patients (normal and male factor). PAF ϭ platelet-activating Roudebush. Platelet-activating factor and IUI pregnancy. Fertil Steril 2004. cells’ surface, initiating intracellular calcium release, en-hancing sperm capacitation and cell motility, thus enhancingfertilization rates. Sperm with defective or low numbers ofPAF receptor may not respond to PAF (endogenous orexogenous), thus having poor motility Additional stud-ies will elucidate the reproductive significance of PAF ac- Roudebush. Platelet-activating factor and IUI pregnancy. Fertil Steril 2004. tivity and PAF’s mechanism of action in spermatozoa. Otherpossible factors may attribute to the high pregnancy ratesobserved in this study. For example, in most cases we There was no significant difference in multiple pregnancy rates performed two IUI’s per cycle, which has been demonstrated between the control and PAF treatment groups Additional clinical studies are warranted to further estab- DISCUSSION
lish the use of PAF therapy for patient’s undergoing IUI The inclusion of PAF to IUI sperm wash procedure im- therapy for infertility treatment. In particular, larger numbers proves pregnancy rates. However, this significant improve- of male factor infertility patients will determine the signifi- ment could only be demonstrated in cases where the semen cance of PAF–IUI therapy for these individuals. To summa- analysis was normal. This confirms our original results dem- rize, exposure of sperm to PAF can significantly increase IUI onstrating the effectiveness of exogenous PAF supplemen- Treatment of sperm in male factor patients with PAF APPENDUM
showed an increase in pregnancy rates, albeit not statistically On February 10, 2003, we initiated the routine washing of significant. It is possible that sperm in these individuals are semen with PAF before IUI. Our overall ongoing pregnancy incapable of responding to the exogenous PAF due to poor rate since then is 41.6% and is presented in PAF receptor levels and, or faulty PAF receptors in thesperm The PAF antagonists will inhibit the motility,acrosome reaction and hamster oocyte penetration in ex-posed sperm, thus suggesting the presence of receptors forPAF We have recently reported on the presence anddistribution of the PAF receptor in human sperm and Acknowledgments: The authors express their sincere gratitude and thanks to our preliminary data demonstrate that distribution of the the staff and physicians (Daniel Shapiro, M.D., David Keenan, M.D., and receptor is significantly altered in abnormal sperm We Scott Slayden, M.D.) at Reproductive Biology Associates for patient re- have also discovered that PAF receptor mRNA expression cruitment and laboratory work without whose assistance would have madethis study impossible.
differs significantly between motile (high content) and non-motile (low content) sperm In addition we have foundthat sperm with abnormal motility have different PAF recep- References
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FERTILITY & STERILITY௡
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Platelet-activating factor and IUI pregnancy

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