1 Monash IVF, Epworth Hospital, Richmond 3121, 2 Centre for Early Human Development, Monash University, Clayton 3168, Victoria, Australia
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Abstract |
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Key words: antiphospholipid antibodies/assisted reproductive treatment/cumulative live birth rate/cumulative pregnancy rate/implantation failure
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Introduction |
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Women who present for in-vitro fertilization (IVF) have been shown to have a high prevalence of APA, especially those with pelvic pathology such as endometriosis and pelvic adhesions (El-Roeiy et al., 1987; Fisch et al., 1991
; Sher et al., 1994
; Dmowski et al., 1995
; Nip et al., 1995
; Schenk et al., 1996
). However, the relevance of the presence of APA to IVF outcome is still a matter of debate. A study by Sher et al. generated much interest because of its claim that IVF failure is positively correlated with APA seropositivity (Sher et al., 1994
). Further, Sher et al. (1994) claimed that high fecundity rates could be achieved in seropositive women treated with aspirin and heparin. Some studies have supported these findings (Birkenfeld et al., 1994
; Dmowski et al., 1995
; Geva et al., 1995
; Kaider et al., 1996
) but others have found no significant association between APA seropositivity and IVF failure (Gleicher et al., 1994
; Nip et al., 1995
; Birdsall et al., 1996
; Kowalik et al., 1997
; Kutteh et al., 1997
). The conclusions arrived at in these studies have been viewed with some scepticism because of the use of non-randomized small patient numbers, non-comparable controls, determining small number of APA epitopes or the inappropriate definition of positive results. More recently, Denis et al. published results of a large study where 793 patients have been tested for the presence of 21 APA epitopes (Denis et al., 1997
). Using various statistical methods, they demonstrated that elevated APA concentrations, at any cut-off point, were not associated with a change in IVF cycle outcome.
The aims of our study were to compare cumulative pregnancy and live birth rates for APA seronegative and seropositive patients and to determine whether there is a correlation between multiple IVF failure and the presence of any or a specific APA. To accomplish this, we tested sera from a population of IVF patients for the presence of IgG, IgM and IgA against six specific phospholipids: anticardiolipin, antiphosphoserine, antiphosphoethanolamine, antiphosphoinositol, antiphosphatidic acid, and antiphosphoglycerol. To avoid biases resulting from the many different methods available to determine APA status, the serological determinations were carried out by a laboratory specialized in reproductive immunology (Matzner et al., 1994). This group of investigators have published previously their APA results in a large group of infertile patients (Sher et al., 1994
). In addition to the above immunoassays, we used a bioassay to determine serum concentrations of LAC.
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Materials and methods |
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The study was approved by the research and ethics committee of Epworth Hospital, Richmond, Melbourne. Informed consent was obtained from all participants.
APA assay
Serum samples were tested for the presence of IgG, IgM and IgA against cardiolipin, phosphoserine, phosphoethanolamine, phosphoinositol, phosphatidic acid and phosphoglycerol, using an enzyme-linked immunosorbent assay (ELISA), as previously described (Matzner et al., 1994). For each APA epitope separately, seronegativity was defined as <2 SD above the mean and seropositivity was defined as >3 SD above the mean. All other results were defined as borderline.
Lupus anticoagulant was measured using dilute Russell's viper venom test (DRVVT) (LA-SCREEN and LA-CONFIRM kits, Gradipore, North Ride, NSW, Australia). Negative results were defined as 100120% of the internal control.
Treatment protocols
Ovarian stimulation protocols used in our institute have been previously described (MacLachlan et al., 1989). Briefly, patients were down-regulated using a gonadotrophin-releasing hormone agonist Nafarelin 0.5 mg/day (Synarel; Searle, Sydney, NSW, Australia), starting in the luteal phase (long protocol) or on day 2 of the follicular phase (flare or boost protocol). Multiple follicular development was initiated using individually adjusted doses of purified urinary follicle-stimulating hormone (Metrodin; Serono, Melbourne, Victoria, Australia). The ovarian response was monitored with serum oestradiol concentrations and transvaginal ultrasonography. Patients were given 5000 IU human chorionic gonadotrophin (HCG) i.m. (Profasi; Serono) when at least three follicles of >17 mm were present and serum oestradiol was appropriately rising. Oocytes were retrieved transvaginally 36 h later under general anaesthesia. In GIFT cycles, one to four oocytes were transferred laparoscopically to one tube together with 1.5x105 motile spermatozoa purified on a Percoll density gradient. Routine IVF or intracytoplasmic sperm injection (ICSI) and embryo culture were used as indicated and one to four embryos were transferred 4872 h after egg retrieval.
Treatment outcome
Failed cycles were categorized as blood ßHCG < 25 mIU/ml 16 days after oocyte recovery. Pregnancy was categorized as blood ßHCG > 25 mIU/ml 16 days after oocyte recovery with at least one increased value at 5 days later. Biochemical pregnancy was defined as ßHCG <1000 mIU/ml with no gestational sac visible on vaginal ultrasound. Early spontaneous abortion was defined as fetal loss during the first trimester, and late abortion as fetal loss during the second trimester.
Statistical analysis
Data analysis was carried out using several different methods. Cumulative pregnancy and live-birth rates in seropositive and seronegative patients were estimated by the KaplanMeier procedure. Differences between groups were evaluated using 2 test and MannWhitney U test were appropriate. Cox's regression procedure was used to estimate the `risk ratio' of pregnancy with the number of cycles as a dependent variable by each APA epitope individually and by the number of positive epitopes. Chi-square tables and logistic regression models were used to calculate odds ratios of pregnancy for each APA epitope and for the number of positive epitopes. The same methods were used to analyse the association between APA status and probability of biochemical pregnancy, early and late abortions and live birth.
Analysis of the results were performed using the SAS version 6.2 computer analysis program. P < 0.05 was considered as statistically significant.
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Results |
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The cumulative pregnancy and live birth rates per transfer for APA seropositive and seronegative patients are presented in Tables I and II respectively. No statistically significant difference could be found between the two groups. We further analysed the cumulative pregnancy and live birth rates for patients with different numbers of positive APA. For patients with
2 positive epitopes the cumulative pregnancy rate per transfer was 18.5, 35.5, 41.4, 48, 48, and 48% for cycles 16 respectively. For patients with
3 positive epitopes the cumulative pregnancy rate per transfer was 21.4, 29.3, 43.3, 57.5, and 57.5% for cycles 16 respectively. The number of positive or positive and borderline results (maximum 7 positive APA epitopes) was not associated with decreased cumulative pregnancy or live birth rates.
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Demographic and clinical data for the control group, group I and group II are presented in Table IV. No statistically significant difference between the groups was found with respect to patient's age and the number of eggs/embryos transferred per cycle. The diagnosis of tubal infertility was more frequent in group I compared to the control group (P < 0.05). The proportions of patients who had the flare versus the long down-regulation protocol for ovarian stimulation were equally distributed in all groups.
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Association between the presence of APA and of pelvic pathology (endometriosis or tubal disease) was assessed. Of the 79 patients with pelvic pathology 28 (34.4%) were seronegative and 37 (46.8%) were seropositive. Of the 91 patients without pelvic pathology 53 (58.2%) were seronegative and 27 (29.7%) were seropositive (P < 0.01, 2 test).
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Discussion |
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All of the APA studies to date have compared pregnancy and implantation rates in a cohort of assisted reproductive treatment patients with various APA results. However, live table analyses, namely cumulative pregnancy and life birth rates, have been recognized as the most appropriate method available to present success rates for assisted reproductive treatment (Guzick and Bross, 1992; Hull, 1992
). This is the first study to use life table analyses to evaluate a possible correlation between APA status and assisted reproductive treatment outcome.
In the study by Sher et al. all serum samples in our study were tested by the Reproductive Immunology Associates Laboratory for the presence of 18 APA and the same normal controls were used (Sher et al., 1994). In contrast with previous findings (Sher et al., 1994
), we found no correlation between the presence of these APA and assisted reproductive treatment outcome. Our results support recent works (Denis et al., 1997
; Kowalik et al., 1997
; Kutteh et al., 1997
). By using life table analyses we add strength to the conclusion that there is no relationship between circulating APA and the early process of implantation or maintenance of pregnancy among assisted reproductive treatment patients.
Our results are perhaps not surprising, given that the ability to achieve a pregnancy is not impaired in women with the full-blown antiphospholipid antibody syndrome (recurrent miscarriage, thrombosis and thrombocytopenia). If the presence of APA could prevent or damage the implantation process, we would expect the frequency of infertility to be higher in this group of patients than in the normal population. To the best of our knowledge there is no evidence in the literature supporting this association.
The association between recurrent miscarriage and APA is a consistently reported feature of primary antiphospholipid syndrome (Asherson et al., 1989; Rai et al., 1995
). APA, especially IgG anticardiolipin and LAC, are found in 15% of women with recurrent miscarriage. Our study demonstrated that the rate of first trimester miscarriage was higher in patients having positive and/or borderline APA (45 and 36.6% respectively), compared with APA-negative patients (27%). This difference was not statistically significant. Similar results have been found previously (Kowalik et al., 1997
). Other investigators found no relationship between APA concentrations and pregnancy loss rates in IVF patients (Birdsall et al., 1996
; Denis et al., 1997
; Balasch et al., 1998
). In our study only one patient had recurrent (three consecutive) miscarriages and no patients were found to be positive for LAC. These findings suggest that although LAC may be important in recurrent fetal loss, it appears to have no significance in recurrent implantation failure.
Transitory positive APA tests are a common occurrence and are often due to viral or other infections present at the time of testing (Rai et al., 1995). In a study of 500 consecutive women with recurrent miscarriage, only 66% of those with an initial positive test for LAC, 37% of those who were IgG anticardiolipin positive and 36% who were IgM anticardiolipin positive had a repeat positive test (Rai et al., 1995
). In our study and in all others referred to, serum APA concentrations were measured once only. In order to exclude those with only transitory positive tests, repeat testing at least 8 weeks after the initial test is necessary to confirm the presence of APA in patients with an initially positive result. Until such a comprehensive study is conducted we cannot know the real importance of APA in infertility.
In agreement with Sher et al. (1994), we found that the prevalence of APA was significantly higher in patients with organic pelvic disease than in patients without pelvic pathology (respectively 46.8 and 29.7% in our study and 53 and 14% in Sher's study) (Sher et al., 1994). Other studies found no difference in the prevalence of anticardiolipin and antiphosphatidylserine between patients with tubal factor infertility, male factor infertility and idiopathic infertility (Birdsall et al., 1996
; Kowalik et al., 1997
). Enlarging the tested APA panel may account for the difference between the studies. It is possible that the tissue damage in pelvic diseases such as endometriosis or pelvic inflammatory disease predisposes the occurrence of autoantibodies such as APA. However, their occurrence does not predispose assisted reproductive treatment failure.
Five groups have performed non-randomized treatment of APA-positive women undergoing assisted reproductive treatment (Birkenfeld et al., 1994; Sher et al., 1994
; Schenk et al., 1996
; Kutteh et al., 1997
; Rubinstein et al., 1997
; Sher, 1997
). In a small group of patients no significant benefit of heparin and aspirin treatment in APA-positive women undergoing IVF was demonstrated (Kutteh et al., 1997
). The four other groups of investigators reported improved outcome after treatment with aspirin, heparin or glucocorticosteroids. It is still possible that there is a benefit to this empiric treatment; however, this benefit presumably would be achieved through other mechanisms not related to APA status. Aspirin, a cyclo-oxygenase synthesis inhibitor, might influence other systems involving prostaglandins. For example, previous studies (Nye et al., 1997
) found that aspirin could inhibit hypothalamicpituitaryadrenal response to some specific stimulations. Taking into account the well known severe side-effects of long lasting anticoagulation treatment, we cannot support routine treatment with different combinations of these agents at this time. Randomized prospective controlled trials to investigate the efficiency of these agents, regardless of APA status, are certainly needed.
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Acknowledgments |
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Notes |
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References |
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Submitted on October 30, 1998; accepted on February 10, 1999.