University of Tennessee, Division of Reproductive Endocrinology and Immunology, Department of Obstetrics and Gynecology,956 Court Avenue, Room D324, Memphis, TN 38163-2116, USA
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Abstract |
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Key words: anticardiolipin antibodies/antiphospholipid antibodies/aspirin/heparin/recurrent pregnancy loss
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Introduction |
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Low dose aspirin, heparin, prednisone and i.v. immunoglobulin have been proposed as treatments for APS with mechanisms that affect both the immune and coagulation system to counteract the effects of APA. Theoretical mechanisms for APA-induced thrombosis include decreased prostacyclin production by endothelial cells, increased thromboxane production by platelets, and decreased protein-C activation (Chamley et al., 1993; Shibata et al., 1993
). Exogenous heparin has been shown to inhibit the binding of APA in vitro in a doseresponse manner; thus, endogenous heparin produced by trophoblasts may function in a similar fashion (Ermel et al., 1995
). The antithromboxane effects of aspirin on inhibition of platelet aggregation are thought to work in concert with heparin to promote and enhance implantation (Patrono, 1994
; Hauth, 1995
). Prednisone and i.v. immunoglobulin have been proposed to alter the immune system and autoantibody action (Spinnato et al., 1995
), while low dose aspirin may improve placental blood flow by decreasing the thromboxane to prostacyclin ratio, thus enhancing implantation (Rubinstein et al., 1999
). Currently, all of these therapies are being utilized as treatments for women with RPL and APA.
In prospective studies, the use of s.c. heparin and aspirin has resulted in successful deliveries in 75% cases of women with RPL and APA with a low frequency of obstetric and maternal complications (Kutteh, 1996
; Rai et al., 1997
). However, these reports included women who were positive for the common phospholipids (CL, PS and LAC). We identified a group of women with a history of RPL and other positive APA and compared their obstetric history to women with RPL and CL, PS or LAC. We prospectively followed these women to determine if a positive test for antibodies to phosphatidyl inositol (PI), phosphatidyl glycerol (PG), and/or phosphatidyl ethanolamine (PE) would suggest a need for treatment with heparin and aspirin or if aspirin therapy alone might offer them an improved live birth rate.
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Materials and methods |
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The diagnosis of APS was based on a well-documented history of RPL (at least two spontaneous, consecutive miscarriages fathered by the same partner) and positive levels of immunoglobulin (Ig)M or IgG APA (20 phospholipid units) on two separate occasions at least 6 weeks apart. Each woman had a complete evaluation for RPL, which included a history and physical examination, karyotypes on both partners, hysterosalpingogram or hysteroscopy, mid-luteal progesterone, thyroid stimulating hormone, prolactin, lupus anticoagulant [partial thromboplastin time (aPTT), dilute Russells viper venom time (dRVVT), and platelet neutralization test], IgG and IgM APA, and cervical cultures for mycoplasma, ureaplasma and chlamydia. All women who participated were started on low dose aspirin (81 mg/day) and prenatal vitamins prior to conception. The initial dosage of heparin was 5000 IU s.c. twice daily if the womans weight was >150 lbs and 6000 IU twice daily if her weight was <150 lbs. All patients were instructed to have quantitative ß-hCG and progesterone level with any missed period and continued aspirin (81 mg/day) at the first confirmed pregnancy test. Those women with progesterone levels to <20 ng/dl were treated with vaginal progesterone 50 mg twice daily until 10 gestational weeks. For those women utilizing heparin, twice daily s.c. injections of heparin (50006000 IU) were started at the first confirmed pregnancy test. All patients were counselled, given the same information concerning treatment options, and were given literature on the risks and benefits of heparin. Those patients who did not desire heparin were advised to take low dose aspirin (81 mg/day) throughout pregnancy (group 3). The Institutional Review Boards of The University of Texas Southwestern Medical Center and University of Tennessee Memphis Health and Science Center approved the treatment protocols.
APA enzyme-linked immunosorbent assay (ELISA)
All serum samples were evaluated for IgG and IgM antibodies against CL, PS, PI, PG, and PE utilizing the ELISA method (Harris, 1990). Briefly, individual 96-well microtitre plates (Immulon-2; Dynatech Labs, Chantilly, VA, USA) were coated with 30 µl of either purified phospholipids (Sigma Chemical Co., St Louis, MO, USA) at a concentration of 45 µg/ml (CL) in ethanol or 50 µg/ml PI, PS, PG and PE in methanol. The plates were air-dried overnight at 4°C then blocked with 200 µl of 10% fetal calf serum (FCS; Gibco, Long Island, NY, USA) in 1xphosphate-buffered saline (PBS; Gibco), washed and incubated at 37°C for 2 h with 50 µl of patients sera diluted 1:50 in 10% FCS in PBS. Each unknown sample was run in duplicate. The plates were then washed to remove unbound antibody and proteins, and a secondary antibody, alkaline phosphatase-conjugated antihuman IgG (Caltag Labs, San Francisco, CA, USA) or IgM (Biosourse; Tago Immunologicals, Camarillo, CA, USA) was added to the plate. After incubation and washing, p-nitrophenyl phosphate substrate (Sigma 104; Sigma Chemical Co.) was added and used to indirectly measure the level of APA in a patients serum. The optical density of the samples, caused by the cleavage of the substrate by the enzyme, was determined at 405 nm by a Bio-Tek Microplate Reader Model EL 340 (Bio-Tek Instruments, Winooski, VT, USA) and was used to quantify the amount of APA in the sera.
Every assay plate also included a known high positive anticardiolipin sample [>100 GPL (IgG phospholipid units)] run in duplicate. Plates were incubated until the high positive wells achieved an optical density of >1.0; typically, this required an incubation of 2030 min. Referenced standard sets for cardiolipin (Louisville APL Diagnostics, Inc., Atlanta, GA, USA) and known negative sera were used on every plate. All results were defined in phospholipid units for IgG (GPL) and IgM (MPL) as follows: <10 IU, negative; 1019 IU, borderline; 2080 IU, positive; and >80 IU, high positive. PS, PI, PG and PE values were interpreted based on the multiples of the median (MoM) method as described previously (Kutteh et al., 1994; Branch et al., 1997
). Known positive samples with PI, PG, PS and PE were included in every plate.
Briefly, phospholipid units for IgG and IgM were calculated for each serum sample, and the median value was determined from the non-Gaussian distribution. The cut-off value in phospholipid units of each APA was determined by using the 99th percentile of the normal population, 3.0 times the median value (Kutteh et al., 1994
). All values reported as positive were the mean of duplicated determinations with background absorbance obtained from wells prepared without the coating phospholipid subtracted. Any values with SE >10% were discarded and reassayed. Inter-assay variation was <8%, and intra-assay variation was <6%.
Heparin and aspirin treatment
Women with RPL and APA who desired treatment initiated treatment with s.c. heparin (50006000 IU) every 12 h with the first positive pregnancy test. Platelets and PTT were obtained 6 h after heparin injection weekly for 2 weeks after the initiation of heparin therapy and 1 week following any adjustment in heparin dosage. Thereafter, platelets and PTT were checked periodically to ascertain that they were in the normal range. The heparin dosage was adjusted downward if the PTT was elevated outside the normal range or if the platelet count fell to <1x105/ml. Supplementation with calcium carbonate to achieve a total daily intake of 1.5 g/day was prescribed for all patients, an approach that has been reported to counteract the osteoporotic effects of heparin (Dahlman et al., 1994).
Each pregnancy was documented by transvaginal ultrasonography scheduled at 7 weeks gestation for the determination of fetal heart motion. Additional ultrasonography was performed as indicated, but generally baseline sonograms were obtained at 20 weeks. Antenatal testing (fetal kick counts, non-stress tests, or biophysical profiles) was initiated at 2830 weeks when indicated. Aspirin was discontinued 2 weeks before the estimated due date. Heparin was continued until full term and was discontinued when the patient initiated spontaneous labour. The evening heparin dose was omitted prior to planned amniocentesis or a scheduled operative delivery.
Statistical analyses and data collection
Statistical analyses were performed using one-way analysis of variance. A comparison of all pairs of columns was performed using TukeyKramer multiple comparison test. A post-hoc analysis was performed if P < 0.05. A power analysis was performed with the following assumptions based on previous data (Kutteh, 1996): the data were sampled from populations with identical SD; the data were sampled from populations that follow Gaussian distributions; expected delivery of live births in group 3 (aspirin alone) = 0.40; expected delivery rate in group 1 and group 2 (heparin and aspirin) = 0.75; significance
= 0.05; and ß = 0.80. For a one-tailed analysis, the number of patients required in each group to detect a 35% difference in a successful delivery was 18 women in each group.
Each patient was personally interviewed and medical records were reviewed to confirm pregnancy histories. Outcome data included maternal and obstetric complications. Preterm birth included deliveries before 37 completed menstrual weeks. Gestational diabetes included women who demonstrated glucose intolerance requiring dietary or medical control. Minor bleeding included haematuria, nosebleeds, gum bleeds, and bleeding at the injection site. Thrombocytopenia was defined as platelet counts <1x105/ml. Pre-eclampsia was diagnosed based on hypertension and proteinuria. Intrauterine growth retardation (IUGR) was diagnosed when birthweight was below the 10th percentile for gestational age. Major bleeding included abruption, blood losses at delivery requiring transfusion, or vaginal bleeds requiring hospitalization during pregnancy. Karyotype analysis was recommended on all subsequent miscarriage; however, this was not always possible.
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Results |
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Complications of treatment
Documented obstetric and maternal complications were low (Table III). There were no significant differences in preterm birth and IUGR among groups. None of the patients had thrombocytopenia, major bleeding, or fractures. In the women who had a subsequent pregnancy loss while being treated with heparin and aspirin, there were no apparent differences in the identifiable causes of pregnancy loss.
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Discussion |
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The lack of standardization among APA laboratories has made it difficult for physicians to identify patients with APS and those at risk for a miscarriage (Peaceman et al., 1993; Kutteh et al., 1994
; Carreras et al., 2000
). Thus, an international group of investigators has established both clinical and laboratory criteria for the diagnosis of APS (Wilson et al., 2001
). Yet, problems still exist when pregnancy loss patients are referred to infertility clinics that may have had testing performed at different laboratories using different control values and cut-off values to determine positive results. Also, standard testing may exclude a population of APA patients who have significant obstetric problems but test positive for other APA and negative for the most common antiphospholipids such as CL or LAC.
This ongoing debate of the clinical significance of CL, LAC and other APA has prompted some investigations to screen patients using a panel of APA for the evaluation of RPL. For example, Branch et al. analysed the 95th and 99th percentile as the positive and negative cut-off for a panel of phospholipids among 147 women with RPL, APS and fertile controls (Branch et al., 1997). By using the 99th percentile, they found that 26/147 (17.7%) had positive antibodies to CL and 13/147 women (8.8%) with RPL demonstrated binding against phospholipids other than CL or LAC. Based on comparison with controls, they concluded that this difference was not clinically significant. In a much larger study, the prevalence of APA among 866 women with RPL was investigated. In this population, 17% of women had a positive level for CL antibodies in comparison with 10% of women with antibodies other than CL (Yetman and Kutteh, 1996
). Although this study was retrospective, it suggests that 10% of women with RPL would not have been identified for a risk of APA if diagnosis was based on a test for CL and/or PS and/or LAC antibodies exclusively.
This study also evaluated heparin and aspirin therapy and aspirin therapy alone to treat a population of women who may have gone untreated, if APA other than CL, PS and/or LAC were excluded as risk factors for an adverse pregnancy outcome. We previously reported that women with RPL and CL antibodies demonstrated a successful delivery rate of 44% with aspirin therapy alone compared with 80% heparin and aspirin therapy (Kutteh, 1996). Based on these data, assumptions could be made for the expected delivery rate among treatment groups which were used to estimate the sample size.
For this study, we compared the pregnancy outcomes of women who had a normal evaluation except for positive CL, PS and/or LAC treated with heparin and aspirin (group 1); positive PI, PG and/or PE antibodies treated with heparin and aspirin (group 2); and positive PI, PG and/or PE treated with aspirin alone (group 3). The live birth rate was 76% for group 1, 64% for group 2 and 46% for group 3. These data are in agreement with prospective reports that have shown a successful obstetric outcome with heparin and aspirin therapy and/or aspirin alone therapy in women with positive CL, PS and/or LAC antibodies (Cowchock et al., 1992; Kutteh, 1996
; Yetman and Kutteh, 1996
; Rai et al., 1997
; Empson et al., 2002
). As expected, women in group 1 (RPL with common APA) treated with heparin and aspirin had a successful outcome in the majority of cases. The women in group 2 (RPL with other APA) had a live birth rate that was not significantly different from group 1 (P = 0.28). Successful deliveries occurred significantly more often when comparing group 1 with group 3 (P = 0.03), but not when comparing group 2 with group 3 (P = 0.20). While it is inconclusive from this study which treatment should be considered for those women with RPL and antibodies to PI, PG and/or PE, there was a trend toward improved outcome in the group treated with heparin and aspirin. A larger, prospective, randomized, controlled trial is necessary to elucidate this question.
The significance of a panel of APA to diagnose APS is an ongoing debate with many complex questions that can only be addressed with larger APA study groups. We were able to identify 54 women with a history of RPL and positive APA other than CL, PS or LAC, who had an otherwise normal evaluation. This required screening of 1400 couples over 7 years and suggested a possible diagnosis in 4% of the women tested. Although there are different views on the necessity for clinical testing of PG, PI, and PE for the diagnosis of APS, it appears that, in 810% of women with RPL, identification of these other APA may suggest a direction for treatment (Yetman and Kutteh, 1996
; Branch et al., 1997
). This study is limited to a small but significant study group of 54 women (groups 2 and 3) with RPL and other APA who may benefit from additional treatment. A larger group of women treated prospectively in a randomized study is necessary to ascertain the clinical significance of the individual phospholipids PI, PG and PE.
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Notes |
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* A preliminary abstract of this work was presented at the Second International Conference on Experimental and Clinical Reproductive Immunobiology on November 16, 2000, Amsterdam, The Netherlands.
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References |
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Branch, D.W., Silver, R.M., Pierangeli, S.S., van Leeuwen, I. and Harris, E.N. (1997) Antiphospholipid antibodies in women with recurrent pregnancy loss, fertile controls, and antiphospholipid syndrome. Obstet. Gynecol., 89, 549555.
Carreras, L.O., Forastiero, R.R. and Martinuzzo, M.E. (2000) Which are the best biological markers of the antiphospholipid syndrome? J. Autoimmun., 15, 163172.[ISI][Medline]
Carson, S.A. and Branch, D.W. (2001) Management of recurrent early pregnancy loss. 2002 Compendium of Selected Publications. American College of Obstetricians and Gynecologists, Washington, DC, USA, pp. 372383.
Chamley, L.W., McKay, E.J. and Pattison, N.S. (1993) Inhibition of heparin/antithrombin III cofactor activity by anticardiolipin antibodies: a mechanism for thrombosis. Thromb. Res., 71, 103111.[ISI][Medline]
Cowchock, F.S., Reece, E.A., Balaban, D., Branch, D.W. and Plouffe, L. (1992) Repeated fetal losses associated with antiphospholipid antibodies: a collaborative randomized trial comparing prednisone to low-dose heparin treatment. Am. J. Obstet. Gynecol., 166, 13181327.[ISI][Medline]
Dahlman, T., Sjöberg, H.E. and Ringertz, H. (1994) Bone mineral density during long-term prophylaxis with heparin in pregnancy. Am. J. Obstet. Gynecol., 170, 12131220.[ISI][Medline]
Empson, M., Lassere, M., Craig, J.C. and Scott, J.R. (2002) Recurrent pregnancy loss with antiphospholipid antibody: a systematic review of therapeutic trials. Obstet. Gynecol., 99, 135144.
Ermel, L.D., Marshburn, P.B. and Kutteh, W.H. (1995) Interaction of heparin with antiphospholipid antibodies (APA) from the sera of women with recurrent pregnancy loss (RPL). Am. J. Reprod. Immunol., 33, 1420.[ISI][Medline]
Harris, E.N. (1990) Annotation: antiphospholipid antibodies. Br. J. Haematol., 74, 19.[ISI][Medline]
Hauth, J.L. (1995) Low-dose aspirin: lack of association with an increase in abruptio placentae or perinatal mortality. Obstet. Gynecol., 85, 10551058.
Kutteh, W.H. (1996) Antiphospholipid antibody-associated recurrent pregnancy loss: treatment with heparin and low-dose aspirin is superior to low-dose aspirin alone. Am. J. Obstet. Gynecol., 174, 15841589.[ISI][Medline]
Kutteh, W.H., Wester, R. and Kutteh, C.C. (1994) Multiples of the median: alternate methods for reporting antiphospholipid antibodies in women with recurrent pregnancy loss. Obstet. Gynecol., 84. 811815.[Abstract]
Kutteh, W.H., Rote, N.S. and Silver, R. (1997) Antiphospholipid antibodies and reproduction: the antiphospholipid antibody syndrome. Am. J. Reprod. Immunol., 41, 133152.
Lockshin, M.D. (1997) Antiphospholipid antibody. J. Am. Med. Assoc., 277, 15491551.[ISI][Medline]
Patrono, C. (1994) Aspirin as an antiplatelet drug. N. Engl. J. Med., 330, 12871294.
Peaceman, A.M., Silver R.K., MacGregor, S.N. and Socol, M.L. (1993) Interlaboratory variation in antiphospholipid antibody testing. Am. J. Obstet. Gynecol., 169, 232233.[ISI][Medline]
Rai, R., Cohen, H., Dave, M. and Regan, L. (1997) Randomized, controlled trial of aspirin and aspirin plus heparin in pregnant women with recurrent miscarriage associated with phospholipid antibodies (or antiphospholipid antibodies). Br. Med. J., 314, 253257.
Rubinstein, M., Marazzi, A. and Polak de Fried, E. (1999) Low-dose aspirin treatment improves ovarian responsiveness, uterine, and ovarian blood flow velocity, implantation, and pregnancy rates in patients undergoing in vitro fertilization: a prospective, randomized, double-blinded placebo controlled assay. Fertil. Steril., 71, 825829.[ISI][Medline]
Shibata, S., Harpel, P., Bona, C. and Fillit, H. (1993) Monoclonal antibodies to heparin sulfate inhibit the formation of thrombinantithrombin III complexes. Clin. Immunol. Immunopathol., 67, 264272.[ISI][Medline]
Spinnato, J.A., Clark, A.L., Pierangeli, S.S. and Harris, E.N. (1995) Intravenous immunoglobulin therapy for the antiphospholipid syndrome in pregnancy. Am. J. Obstet. Gynecol., 172, 690694.[ISI][Medline]
Wilson, W.A., Gharavi, A.E. and Piette, J.C. (2001) International classification criteria for antiphospholipid syndrome: synopsis of a post-conference workshop held at the 9th International (Tours) APL Symposium. Lupus, 10, 457460.[Medline]
Yetman, D.L. and Kutteh, W.H. (1996) Antiphospholipid antibody panels and recurrent pregnancy loss: prevalence of anticardiolipin antibodies compared with other antiphospholipid antibodies. Fertil. Steril., 66, 540546.[ISI][Medline]
Submitted on March 6, 2002; resubmitted on June 10, 2002; accepted on July 22, 2002.