Catharyn Stern, M.D., a Lawrence Chamley, Ph.D., b Helen Norris, R.M., a Lyndon Hale, M.D., a and H. W. Gordon Baker, Ph.D. c

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1 FERTILITY AND STERILITY VOL. 80, NO. 2, AUGUST 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. A randomized, double-blind, placebocontrolled trial of heparin and aspirin for women with in vitro fertilization implantation failure and antiphospholipid or antinuclear antibodies Catharyn Stern, M.D., a Lawrence Chamley, Ph.D., b Helen Norris, R.M., a Lyndon Hale, M.D., a and H. W. Gordon Baker, Ph.D. c Royal Women s Hospital, Carlton, Victoria, Australia, and National Women s Hospital, Epsom, New Zealand Received August 19, 2002; revised and accepted December 23, Supported by a Royal Women s Hospital Research Foundation Grant and by the Melbourne IVF Research Fund, East Melbourne, Victoria; a private donation; and the Bonnie Babes Foundation, Croydon, Australia. Fauldings/DBL Pharmaceutical Company (Mulgrave, Australia) which generously provided aspirin and assisted with production of placebo injections and tablets. Presented at the International Federation of Fertility Societies (IFFS) Meeting, Melbourne, Australia, November Reprint requests: Catharyn Stern, M.D., Suite 3, 320 Victoria Parade, East Melbourne, Victoria 3002, Australia (FAX: ; kate.stern@rwh.org.au). a Royal Women s Hospital. b University Department of Obstetrics and Gynaecology, National Women s Hospital. c University of Melbourne Department of Obstetrics and Gynaecology, Royal Women s Hospital /03/$30.00 doi: /s (03) Objective: To investigate whether heparin and low-dose aspirin increase the pregnancy rate in antiphospholipid antibody or antinuclear antibody seropositive women with IVF implantation failure. Design: A double-blind, randomized, transfer-by-transfer of fresh or cryopreserved embryos, crossover trial. Setting: A hospital infertility clinic and associated IVF service. Patient(s): Women seropositive for at least one antiphospholipid (APA), antinuclear (ANA), or 2 glycoprotein I autoantibody and 10 embryos transferred without achieving pregnancy (n 143). Intervention(s): Subcutaneous unfractionated heparin (5,000 IU b.i.d.) and aspirin (100 mg daily) (158 transfers of 296 embryos) or placebo (142 transfers of 259 embryos) from the day of embryo transfer. Main Outcome Measure(s): Fetal heart per embryo transferred (implantation rate). Result(s): There was no significant difference in pregnancy rates or implantation rates between treated and placebo cycles; for example, fetal hearts per embryo transferred implantation rates were 6.8% (20/296) and 8.5% (22/259), respectively, and the generalized estimating equation covariate adjusted relative pregnancy rate was 0.65 (95% confidence interval, ). The implantation rate for seropositive trial participants (42/555, 7.6%) compared favorably with that for IVF implantation-failure patients continuing treatment outside the trial (147/3237, 4.5%). Conclusion(s): Heparin and aspirin did not improve pregnancy or implantation rates for APA-positive or ANA-positive patients with IVF implantation failure. (Fertil Steril 2003;80: by American Society for Reproductive Medicine.) Key Words: Antiphospholipid antibodies, antibodies to 2 glycoprotein I, IVF, implantation failure, heparin, aspirin, placebo Unfortunately, there are many infertile patients who fail to conceive with IVF treatment despite repeated transfers of good-quality embryos. In an effort to understand the causes of IVF implantation failure, much interest has centered on the role of autoimmune factors in reproduction and reproductive failure (1 4). Antiphospholipid antibodies (APAs) are a group of antibodies that react with negatively charged phospholipids. These antibodies include anticardiolipin antibody; lupus anticoagulant; antibodies to phosphatidylserine (phosphatidylserine antibodies), phosphatidylinositol, and phosphatidylethanolamine; and antibodies to the cofactor 2 glycoprotein I (a 2 glycoprotein I) (5 9). Antinuclear antibodies (ANA) may also be associated with reproductive failure, but in low titer they appear to be relatively nonspecific, because it has been recently shown that they are found in 9% of normally fertile women (10). Although it has been known for some time that this group of antibodies is associated with thrombotic disorders (7, 11), there have been many recent studies investigating their association with adverse reproductive events, including recurrent miscarriage (5, 6, 8), infertility (12 16), and IVF implantation failure (10, 17 19). 376

2 There is no consensus as to the importance of APAs in infertility and IVF implantation failure. Some groups have found a strong association between the presence of APAs and unsuccessful IVF treatment (10, 17, 19, 20), whereas others have failed to confirm this (15, 18, 21 23). A recent meta-analysis evaluated whether the presence of APAs affects the likelihood of IVF success and concluded that the measurement of APAs is not warranted in patients undergoing IVF (23). This analysis, by including a heterogeneous group of studies with differing patient populations, has fueled the debate rather than settling it (24). In particular, it has not addressed the relevance of APA testing for the specific group of patients characterized as having IVF implantation failure. Antibodies to 2 glycoprotein I have been investigated in very few studies, but the largest to date suggests a strong association between IVF implantation failure and seropositivity for antibodies to this cofactor (10). The mechanisms by which APAs and their cofactors affect reproductive performance have not yet been definitively characterized (11 13). Autoantibodies may exert actions on the trophoblast via interference with membrane surface hemostatic reactions or by reacting with antigens on cell surfaces, resulting in altered cell activity (2). Effects may include direct cellular injury or microvascular thrombosis (26, 27). Mechanisms by which antibodies to 2 glycoprotein I may inhibit phospholipid-dependent reactions have recently been described and involive inhibition of binding of other phospholipid-binding proteins and/or anticoagulants (28). Although the pathogenic mechanisms of APAs in reproductive failure are still unknown, their presumed thrombotic effects have led to the widespread use of heparin and aspirin for women with APAs and recurrent abortion or IVF implantation failure, with varying results (29 32). The rationale for heparin use relates to its inhibition of binding of phospholipids with antibodies, thus protecting the trophoblast from injury and thereby promoting both implantation and subsequent placentation (33). Aspirin appears to inhibit platelet aggregation via antithromboxane effects (34). Although it is unlikely that aspirin influences early implantation, it may counteract APA-mediated hypercoagulability in the choriodecidual space, thus protecting trophoblast from damage after placentation has been established (29, 35). Several groups have explored the role of immune suppression and anticoagulation in the treatment of APA-positive patients having IVF treatment, with varying conclusions (19, 29, 31, 36). To our knowledge, however, previously there have been no properly randomized, placebo-controlled studies evaluating the benefits of heparin and aspirin for APA-positive women with IVF implantation failure, and therapy mainly has been on an ad hoc basis to date. TABLE 1 Characteristics of patients at first trial cycle and average embryo cell number and grade during the trial. Variable Mean SD Range Age (y) Body mass index No. of embryos transferred No. of antibodies positive Gravida Average embryo grade a Average cell number a See text for description of embryo grading. We previously have reported results from a large study evaluating the seroprevalence of APA positivity in 460 women with IVF implantation failure, compared with those of women with recurrent miscarriage, women newly referred to an infertility clinic, and fertile controls (10). In that study we showed that some autoantibodies, including anticardiolipin IgM, ANA, and IgM antibodies to a 2 glycoprotein I, were significantly more prevalent in women with IVF implantation failure. We now report on the results of a randomized double-blind placebo-controlled trial of heparin and aspirin treatment for APA-positive or ANA-positive women with IVF implantation failure. MATERIALS AND METHODS The Royal Women s Hospital Research and Ethics Committees approved this study. Consenting patients were recruited mainly from the hospital clinics and Melbourne IVF; 10 patients were treated with IVF in other centers in Australia, but their trial therapy was managed from the trial center at the Royal Women s Hospital. Patient Characteristics Patients attending IVF programs who had previously had 10 embryos transferred without achieving a pregnancy were offered testing for a panel of autoantibodies including ANA, lupus anticoagulant, and IgG and IGM subtypes of anticardiolipin; antibodies to phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine; and anti 2 glycoprotein I. One hundred forty-three women with IVF implantation failure who tested positive for at least one autoantibody (average 2, range 1 9) were enrolled in this trial between January 1998 and June 2001 (see Table 1). Of these women, 65% of patients tested positive for ANA, and in 22.4% of patients, this was the only positive antibody, whereas 67.6% of patients tested positive for at least one of the APAs. Testing for antibodies to 2 glycoprotein I revealed 16.1% of women to be positive for a 2 glycoprotein I IgM, and 8.4% for a 2 glycoprotein I IgG. Infertility diagnoses were as follows: tubal disease in 33 patients FERTILITY & STERILITY 377

3 (23%), endometriosis (ASRM Stage III or IV) in 11 patients (8%), male infertility in 41 couples (29%), combined male and tubal infertility in 8 couples (6%), ovulatory disorders with other diagnoses in 6 patients (4%), and unexplained infertility in 44 couples (30%). Patients who had abnormal findings on hysteroscopic evaluation of the uterine cavity, osteoporosis, or known hematological/thrombotic disorders including thrombophilia, platelet dysfunction, or previous thrombosis were excluded from participation. Among participants, 12.6% smoked more than five cigarettes per day. Laboratory Evaluation Lupus Anticoagulant Testing was performed as described elsewhere (10) and involved a panel of coagulation tests, including activated partial thromboplastin time (normal, seconds), kaolin clotting time ratio (normal 1.2), kaolin clotting time mixing test ratio (normal 1.2), and dilute Russell viper venom ratio (normal ). Two percent of specimens were reported as unsatisfactory for assessment because of activation. Fresh samples were obtained from these patients and the results included. Antinuclear Antibodies Antinuclear antibodies were detected by immunofluorescence using Hep 2 cells as the antigen (Hep-2000ANA Test System; Immunoconcepts, California). A titer of 1/80 was classified as positive for the purpose of this study. Solid-Phase APA ELISAs The phospholipids (phosphatidyl serine IgG and IgM, phosphatidyl ethanolamine IgG and IgM, phosphatidyl inositol IgG and IgM, and cardiolipin IgG and IgM) were obtained from Sigma (Sydney, Australia). The relevant phospholipid was diluted to 50 /ml in ethanol, and 50 L was used to coat a 96-well ELISA plate (Corning High Binding, Acton, MA) by evaporation at 4 C overnight. Plates were exposed to blocking solution: 10% newborn calf serum in phosphate-buffered saline, ph 7.4, for 1 hour at room temperature. The blocking solution was discarded and the plates washed three times with phosphate-buffered saline, ph 7.4. Serum samples, diluted 1:100 in blocking solution, were then incubated on the plates for 1 hour at room temperature. The plates were then washed three times with phosphate-buffered saline, ph 7.4, and horseradish peroxidase conjugated goat anti-human -chain and -chain antiserum (Tago), diluted 1:8,500 in blocking solution and added for 1 hour at room temperature. The plates were again washed three times with phosphate-buffered saline, ph 7.4, and the assay was developed by addition of 1 mg/ml o-phenylamine diamine dihydrochloride (Sigma) in 0.1 M citrate buffer, ph 5.5, containing freshly prepared 0.005% H 2 O 2. The reaction was stopped by the addition of 10% HCl, and the optical density at 490 nm was determined. Multiples of the median were used to determine which samples were positive. Samples were considered to be positive when the optical density of a sample exceeded the multiple of the median of the 95th percentile of 284 normal serum samples (188 males and 104 fertile females). All assays included serum from normal samples. The intra-assay coefficients of variation ranged from 5% 13%, and interassay coefficients of variation ranged from 7% 17%. Solid-Phase 2 Glycoprotein I Antibody ELISAs 2 Glycoprotein I IgG and IgM was purified from normal human plasma. Enzyme-linked immunosorbent assay plates were coated overnight at 4 C with 50 L of purified 2 GPI (10 /ml in 0.1 M carbonate buffer, ph 9.0). The antigen solution was discarded and the plates blocked with 5% nonfat milk powder dissolved in phosphate-buffered saline, containing 0.05% Tween 20, ph 7.4, for 1 hour at room temperature. The plates were washed three times with phosphate-buffered saline Tween before serum samples, diluted 1:200 in the blocking solution, were added for 1 hour at room temperature. Thereafter, the method was the same as for the phospholipid antibody tests. The intra-assay and inter-assay coefficients of variation for the IgG assay were 10% and 6%, respectively, and for the IgM assay, they were 6% and 12%, respectively. Trial Design This trial was a prospective, randomized, double blind, placebo-controlled study. Computer-generated randomization was used for determination of the first embryo transfer cycle treatment. A crossover design was employed, with subsequent therapy alternating from transfer to transfer of fresh or cryopreserved embryos. Gamete intrafallopian transfer could also be performed and the results analyzed as for fresh embryo transfer. Pretreatment Assessment Pretreatment assessment included baseline platelet count (normal, per liter) and activated partial thromboplastin time (normal, seconds). Interventions After instruction in self-injection and medication, patients were randomized to receive unfractionated heparin sodium (5,000 U in 0.2 ml s.c. b.i.d.; DBL, Mulgrave, Australia) and aspirin (100 mg p.o.; Astrix 100; DBL) or placebo (NaCl 0.9% in 0.2 ml s.c., and sucrose [DBL] p.o.) from the day of transfer of embryos or of oocytes and sperm with GIFT, until results of serum hcg (Total hcg; Bayer Australia Ltd, Pymble, Australia) were available. Monitoring of Treatment Monitoring with activated partial thromboplastin time and platelet counts occurred 1 week after commencement of the medications. Each patient was given a therapy diary that included the requirement to report specific side effects of 378 Stern et al. Heparin in IVF implantation failure Vol. 80, No. 2, August 2003

4 TABLE 2 Results by embryo transfer (ET). ET Heparin and aspirin (H&A) or placebo (P) Transfers (no. embryos) Positive hcg per embryo transfer procedure (%) No. of fetal hearts (implantation rate %) No. of babies born (live birth rate per embryo %) 1 H&A 74 (144) 13 (18) 12 (8) 11 (8) P 69 (128) 13 (19) 12 (9) 10 (8) 2 H&A 45 (79) 6 (13) 4 (9) 3 (4) P 38 (67) 8 (21) 7 (10) 6 (9) 3 H&A 20 (37) 4 (20) 4 (11) 4 (11) P 18 (34) 2 (11) 2 (6) 1 (3) 4 H&A 12 (23) P 10 (19) 2 (20) 1 (5) H&A 7 (13) P 7 (11) Total H&A 158 (296) 23 (15) 20 (7) 18 (6) P 142 (259) 25 (18) 22 (8) 17 (7) bruising or bleeding, as well as any other problems. Patients were instructed to return all used and unused ampules and blister packs for scrutiny by trial coordinators. As per unit transfer policy, a maximum of two embryos were replaced in most cases. Before transfer, embryo cell numbers were counted, and embryos were graded morphologically on a scale from 1 to 5 (best to poorest). Transfer technique was recorded as easy, average, or difficult. Pregnancy was diagnosed as hcg 100 IU 17 days after transfer and was confirmed by transvaginal ultrasound 28 days after embryo transfer. In the absence of a hcg result of 100 IU, the medications were discontinued. If the hcg was 100 IU, the medications were continued either until the pregnancy reached 14 weeks of gestation or fetal demise was diagnosed. Sample Size The sample size calculation was based on results of 498 patients in our IVF unit from 1994 to 1997 who had 10 (range 10 23) embryos transferred before the first live-birth pregnancy. We estimated that recruiting 140 patients in whom we would expect to transfer 663 embryos would give 80% power of detecting a doubling of the implantation rate, from 5% to 10%, at the 5% ( 0.05, 1 tailed) level of statistical significance. End Points Primary end points included positive pregnancy tests ( hcg 100 IU) and live-birth pregnancies with one or more babies per transfer, and fetal hearts per embryo transferred (fetal heart implantation rate) and babies born per embryo transferred (live birth rate). Statistical Analysis Primary endpoints for the therapy trial were tabulated. Because some women had several cycles of treatment and thus often more than one embryo was transferred, the pregnancy and implantation rates are correlated, therefore 95% confidence intervals (95% CIs) were estimated by the bootstrap method. Binomial generalized estimating equation with exchangeable correlation structure were used for the unbalanced repeated measures design of the trial and to allow for covariates of pregnancy and implantation rates. The effect of female age on implantation rate was best fitted as constant to age 31 years and thereafter, by the square root of the age. Gamete intrafallopian transfer cycles were included as IVF fresh transfers with other patients embryo average cell number and grade. The 95% CI estimated by the bootstrap method of implantation rates and other characteristics of the trial cycles were used to compare with the averages of other IVF implantation-failure patients who were attending the hospital clinics and Melbourne IVF in the same time period who did not participate in the trial. RESULTS Comparison of Treatment and Placebo Cycles In this trial, 143 women underwent between one and seven embryo transfer cycles. There were 300 transfers of 555 embryos in total (Table 2), including five three-oocyte GIFT cycles in four women. There were 116 fresh transfers (227 embryos) and 184 thaw transfers (328 embryos). There were 10 biochemical pregnancies, 3 ectopic pregnancies, 6 fetal heart miscarriages, 23 singleton live-birth pregnancies, and 6 twin live-birth pregnancies. There was no significant difference between treatment and placebo cycles in combined positive pregnancy tests per transfer (14.6% and 17.6%, respectively), fetal heart implantation rates per embryo (6.8% and 8.5%), or live birth rates per embryo (6.1% and 6.6%; Table 3). Generalized estimat- FERTILITY & STERILITY 379

5 TABLE 3 Implantation rate summary. Treatment Total embryos transferred Fetal hearts Fetal heart implantation rate (95% CI), % No. of babies born Live-birth rate (95% CI), % Heparin and aspirin (4 10) 18 6 (3 10) Placebo (5 12) 17 7 (4 10) ing equation analyses of the primary endpoints showed that for positive hcg and live-birth pregnancy rates, significant covariates included average cell number in the embryos transferred, diagnosis of ovulatory disorder, single vs. multiple embryo transfer, status as a current smoker, diagnosis of endometriosis, and number of antibodies positive. Difficult embryo transfer procedure was also a significant negative factor for live-birth pregnancy rate (Table 4). The relative pregnancy rates per transfer rates for heparin and aspirin treatment vs. placebo in these models incorporating the statistically significant covariates were 0.65 (95% CI, ) for positive pregnancy tests and 0.60 (95% CI, ) for live birth. The unadjusted relative pregnancy rates were 0.80 (95% CI, ) and 0.82 (95% CI, ), respectively. For fetal heart implantation rates and live-birth rates per embryo transferred (Table 5) the significant covariates were average cell number, diagnosis of ovulatory disorder, current smoker, number of antibodies positive, and difficult transfer. Female age and transfer cycles 4 7 in the trial were also significant negative factors in the live birth per embryo transferred rate. There was no significant interaction between treatment and number of antibodies positive. The relative rates for heparin and aspirin treatment vs. placebo treatment in these models were 0.64 (95% CI ) and 0.77 (95% CI ), respectively. The unadjusted relative implantation rates were 0.76 (95% CI ) and 0.89 (95% CI ). The following factors had no significant effect on pregnancy, fetal heart implantation or livebirth rates: GIFT, embryo grade, fresh vs. thawed embryos, endometrial thickness, type of luteal phase support, specific antibody positivity including ANA-alone positivity, parity, gravidity, number of previous cycles or embryos transferred before the trial, BMI, and transfer clinician or clinic. Although superficial bruising at the sites of injection was commonly noted (64% with heparin and 6% with placebo), there were no significant or serious side effects reported in TABLE 4 Binomial generalized estimating equation analysis of pregnancy rates: relative effects (odds ratios, ORs) and 95% confidence limits (95% CL) of significant (P.05) factors, adjusted treatment effect, and within-subject correlation. Factor Clinical pregnancy per transfer Live birth pregnancy per transfer OR 95% CL OR 95% CL Difficult transfer 0 ( ) Embryo cell number 1.3 ( ) 1.5 ( ) Smoker ( ) 0.12 ( ) No. positive antibody 0.73 ( ) 0.62 ( ) tests Ovulatory disorder 15 ( ) 14 (1.9 98) Endometriosis 4.0 (1.4 12) 4.7 (1.2 18) Single embryo 0.23 ( ) 0.22 ( ) transferred Treatment 0.65 ( ) 0.60 ( ) Within-subject correlation TABLE 5 Binomial generalized estimating equation analysis of implantation rates: relative effects (odds ratios, ORs) and 95% confidence limits (95% CL) of significant (P.05) factors, adjusted treatment effect, and within-subject correlation. Factor Fetal heart per embryo transferred Live birth per embryo transferred OR 95% CL OR 95% CL Difficult transfer ( ) ( ) Treatment cycles ( ) Embryo cell number 1.5 ( ) 1.5 ( ) Smoker 0.11 ( ) 0.19 ( ) No. of positive antibody tests 0.72 ( ) 0.74 ( ) Ovulatory disorder 13 ( ) 5.8 (1.9 18) Female age (sqrt 31) 0.20 ( ) Treatment 0.64 ( ) 0.77 ( ) Within-subject correlation Sqrt square root. 380 Stern et al. Heparin in IVF implantation failure Vol. 80, No. 2, August 2003

6 either treatment group; in particular, there were no episodes of significant bleeding. Comparison of Trial and Nontrial Patients The fetal heart implantation rate (7.6%; 95% CI, ) for trial participants was significantly higher than the 4.5% (147 fetal hearts per 3,237 embryos transferred) for 1,447 IVF implantation failure patients not enrolled in the trial who had 1,788 embryo transfers within the same time period. The characteristics of patients in the trial were similar to those of the other IVF implantation failure patients, particularly for infertility diagnoses, embryo grade, or cell number. Also, 16 known-seropositive IVF implantation-failure patients who did not wish to participate in the trial and who did not receive heparin and aspirin from their treating physician had 25 further transfers of 47 embryos, with two clinical pregnancies resulting (fetal heart implantation rate, 4.2%). DISCUSSION Although APA, including anticardiolipin antibody and lupus anticoagulant, are found in low-risk obstetric populations (10, 37), they are more prevalent in women with reproductive dysfunction, including recurrent miscarriage (5, 6, 8) and infertility (14 16). There is much controversy, however, regarding the association between APAs and IVF success. Some authors have reported reduced IVF pregnancy rates in the presence of APAs (10, 17, 19), whereas a recent meta-analysis, which examined the association between APA seropositivity and IVF success, failed to confirm this association (23). It is likely that the lack of consensus relates, at least in part, to a lack of uniformity with respect to testing procedures and statistical methodologies (13, 18, 22, 23, 38). With the exception of anticardiolipin (39), there is no universally accepted standard for the determination of APA levels. Of importance, few studies have actually evaluated a group of patients with true, accurately defined IVF implantation failure, and those that have done so have suggested a clear association (10, 40). It appears that autoantibodies exert their pathogenetic effects either by interfering with cell membrane hemostatic reactions with consequent microthrombus formation or by reacting with antigens on cell surfaces, resulting in altered cell activity (25). Much interest has centered on the target of APA activity and in vitro and animal models have examined endothelial cells (41), trophoblast cells (25, 42) and the preembryo itself (43). As summarized by Ware Branch and Hatasaka (13), several nonrandomized studies have attempted to answer the question of whether infertile women who are APA positive should be treated with heparin and aspirin (Schenk et al., abstract) (31, 36). Kutteh et al. (31) showed no difference in pregnancy rates between treated and untreated women, whereas Sher et al. (29, 36) and Schenk et al. (abstract) have both reported improved pregnancy rates in APA-positive women having treatment. Unlike studies evaluating the use of these medications in women with recurrent miscarriage that show a clear benefit (30, 32), no consensus exists regarding IVF patients. This may well relate at least in part to the fact that patient groups have not been well characterized in the above studies. Although there is a lack of evidence regarding the use of heparin and aspirin, and despite the report of a pregnancy-related death from cerebral hemorrhage associated with aspirin and heparin use in IVF (44), treatment on an ad hoc basis has become commonplace in clinical practice. To our knowledge, this report describes the first placebocontrolled, randomized trial to evaluate heparin and aspirin usage in patients with IVF implantation failure. The aim of this trial was to determine whether heparin and aspirin treatment would increase the implantation rates in patients with previous poor IVF results with 10 embryos transferred without a pregnancy. We included patients with any phospholipid antibodies and also some who were positive for ANA alone, as some clinicians would regard a positive ANA as suggestive of an autoimmune factor operating in the implantation failure. Otherwise, no restraints were put on patients, and subsequent treatments were left to the discretion of the clinician, apart from the randomized administration of heparin and aspirin or of placebo injections in the first transfer cycle. Thus, there was considerable heterogeneity in patient characteristics, particularly for age and BMI. Also, five GIFT cycles were included. A crossover design is attractive for patients as it allows them to be confident that they can receive the active treatment. The design also allows the patient variability to be managed to some extent, as participants will generally be in both arms of the trial. However, some potential problems conducting and analyzing the trial require attention. Sample size calculations can be complex. We took a pragmatic approach by using results from previous patients with IVF implantation failure and took a simple approach to calculating sample size, using a conservative estimate of increase in fetal heart implantation rate per embryo transferred, from 5% to 10%. Although we entered the target number of patients, the number of embryos transferred was fewer than we expected. This may be related to another problem of a crossover trial in IVF: not all patients continue for the same number of transfer cycles. Those who are pregnant stop treatment, and also those who may recognize that they had the active treatment, from the appearance of side effects such as bruising, may not come back for a subsequent placebo cycle. In this trial, there were slightly more heparin and aspirin treated than placebo-treated cycles. Statistical analysis must allow for the correlated outcomes, as patients may have more than one embryo transferred at a time, and the same woman may have several transfer cycles within the FERTILITY & STERILITY 381

7 trial. We used the bootstrap technique to estimate the 95% confidence limits, and the intervals were slightly ( 0.5%) wider than exact binomial confidence intervals. For regression analysis, a repeated-measures analysis is required to allow for the correlated outcomes, and this must be able to cope with missing data. Generalized estimating equations for binomial outcomes are appropriate for this purpose. In this trial, confidence interval for the treatment vs. placebo effects, after allowing for statistically significant prognostic factors for the outcomes pregnancy and implantation, were somewhat narrower than for the unadjusted relative effects. Also after adjustment for the statistically significant covariates, there was a slight but not statistically significant negative effect of heparin and aspirin on implantation rates. Thus, we have been unable to show that the use of heparin and aspirin for APA-seropositive or ANA-seropositive IVF implantation failure confers any benefit, but the confidence limits are quite wide ( ). Although some groups had started the treatment before embryo transfer we do not believe this small time difference was likely to be significant in influencing implantation, which would occur some days later. In Australia in general, and in our clinic in particular, low numbers of embryos (one or two, rarely three) are transferred to avoid the risk of high-multiple pregnancy. Embryo cryopreservation techniques are highly developed. There was no significant difference in the results for fresh and cryopreserved embryos. There was no relationship between seroprevalence of particular antibodies and outcome, unlike the case in the study reported by Sher et al. (36). However, there was a significant association between seropositivity for a number of antibodies and a poorer outcome. As expected, smoking was a negative prognostic factor, and this finding reinforces the need to encourage IVF patients to stop smoking. When the results of the placebo treatment were better than expected, we examined the data for IVF implantation-failure patients who were not enrolled in the trial to determine whether there had been an improvement in the results for this group of patients during the time of the trial. However, these patients had the expected low implantation rates (4.5%). The demographics did not differ significantly between the patients in the trial and the other IVF implantation-failure patients, and the results for a small group of seropositive patients who had further transfers outside the trial were also low, suggesting that seropositivity per se did not confer a more favorable outcome. Although this was not the primary aim of this study and firm conclusions are inappropriate, we suspect that the higher implantation rates (7.6%) for embryo transfers for both drug and placebo trial arms may result from the extra care and attention that trial patients receive. Clinical trials should be used routinely to test potential innovations in ART. In conclusion, until there is clear evidence of a benefit associated with the use of heparin and aspirin therapy, these medications cannot be recommended for patients with IVF implantation failure and with seropositivity to APAs or ANAs. Acknowledgments: We thank the clinicians of the Royal Women s Hospital and Melbourne IVF (especially John McBain, M.D., Andrew Speirs, M.D., Geoff Clarke, M.D., Michael Gronow, M.D., Raphael Kuhn, M.D., Greg Fox, M.D., and Jim Tsaltas, M.D.), for referring their patients for assessment for this trial. We would also like to thank the clinicians of other IVF units who permitted us to include their patients in this trial (Drs. Thomas, Woolcott, Watkins, Kovacs, Driscoll, M. and H. Smith, and Chau). Invaluable assistance was provided by Swee Wong, pharmacist at Royal Women s Hospital for randomization and dispensation of medications. Ian Gordon, Ph.D., of the Statistical Consulting Centre, University of Melbourne, Melbourne, Australia, kindly reviewed the statistical analysis. References 1. Gleicher N, El-Roiey A. The reproductive autoimmune failure syndrome. Am J Obstet Gynecol 1988;159: Geva E, Amit A, Lerner-Geva L, Lessing JB. Autoimmunity and reproduction. Fertil Steril 1997;67: Gleicher N. Antiphospholipid antibodies and reproductive failure: what they do and what they don t do: how to, and how not to treat! Hum Reprod 1997;12: Geva E, Amit A, Lerner-Geva L, Azem F, Yovel I, Lessing JB. Autoimmune disorders: another possible cause for in-vitro fertilization and embryo transfer failure. Hum Reprod 1995;10: Matzner W, Chong P, Xu G, Ching W. Characterisation of antiphospholipid antibodies in women with recurrent spontaneous abortions. J Reprod Med 1994;39: Yetman DL, Kutteh WH. Antiphospholipid antibody panels and recurrent pregnancy loss: prevalence of anticardiolipin antibodies compared with other antiphospholipid antibodies. Fertil Steril 1996;66: Harris EN, Gharavi AE, Boey ML, Portel BM, Mackworth-Young CC, Loizou S, et al. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983;2: Cowchock S, Smith JB, Gocial S. Antibodies to phospholipids and nuclear antigens in patients with related abortions. Am J Obstet Gynecol 1986;155: McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. 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