1 IVF Centre, Maternity Hospital, Kuwait, 2 Faculty of Medicine, Kuwait University, Kuwait and 3 Department of Obstetrics and Gynaecology, St Bartholomew's and The Royal London School of Medicine and Dentistry, Whitechapel, London E1 1BB, UK
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Abstract |
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Key words: antiphospholipid antibodies/IVF/two consecutive miscarriages
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Introduction |
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Available evidence suggests that abnormal auto-immune antibodies are statistically correlated with recurrent miscarriage in ~1015% of patients (Rai et al., 1995; Hatasaka et al., 1997
) and an increased antibody prevalence has been uniformly demonstrated by a number of studies in an infertile population seeking IVF treatment (Gleicher et al., 1994
; Fisch et al., 1995
; Nip et al., 1995
). Whereas antiphospholipid antibodies (APA), namely the lupus anticoagulant (LA) and anti-cardiolipin (aCL) antibodies have become routine in the evaluation of women with recurrent miscarriage (Rai et al., 1996
), there is no agreement in the literature that such tests should be performed in all IVF patients as they are probably a poor predictor of IVF cycle outcome (Gleicher et al., 1994
; Birdsall et al., 1996
; Denis et al., 1997
). Nevertheless, perhaps consideration should be given to performing tests for APA in all patients in whom IVF treatment cycles end in one or two clinical miscarriages (Kowalik et al., 1997
; Balasch et al., 1998
).
This study investigates the prevalence of APA in subfertile couples who conceived after IVF or ICSI and embryo transfer but miscarried on two consecutive occasions. These data are compared with two control groups of women: fertile women with recurrent miscarriage (three or more consecutive miscarriages of natural conceptions), and women with primary infertility undergoing their first IVF or ICSI treatment cycle.
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Material and methods |
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Sixteen women (group 1) with two consecutive miscarriages of pregnancies conceived after repeat conventional IVF or ICSI and embryo transfer were evaluated for APA in the period of 36 months after the last miscarriage. Clinical pregnancies were diagnosed by the presence of a regular gestation sac with embryonic heart activity, 45 weeks after embryo transfer. The details of the ovarian stimulation and embryology procedures for the IVF or ICSI and embryo transfer have been previously described (Egbase et al., 1996). Clinical miscarriage was defined as miscarriage before 20 completed weeks of gestation. The control populations consisted of: (i) 42 consecutive fertile women with recurrent miscarriage (three or more consecutive miscarriages) who registered at the recurrent miscarriage clinic (group 2); and (ii) 60 consecutive womenwith primary infertility (group 3) undergoing their first IVF or ICSI treatment cycle during the 36 months when the APA was being evaluated in group 1 (the study group).
All women had a complete medical history taken and physical examination performed, and patients with chronic medical diseases (diabetic mellitus, renal disease, etc.) were excluded from the study. In addition, only women with a normal uterine cavity as confirmed by pelvic transvaginal ultrasonography, hysterosalpingography and/or hysteroscopy were included in all three groups. Patients in groups 1 and 2 were evaluated for APA (LA and aCL) 36 months after the last pregnancy loss while patients in group 3 were evaluated within 3 months prior to ovarian stimulation for their first IVF or ICSI treatment cycle.
Blood samples were taken according to approved protocols and evaluated for the presence of APA using the enzyme-linked immunosorbent assay (ELISA) method as previously described by Harris (1990) and Kutteh et al. (1997). Individual ELISA plates (Immulon-2; Dynatech Labs, Chantilly, VA, USA) were coated with 30 µl of cardiolipin (Sigma Chemical Co, Poole, Dorset, UK) at a concentration of 45 µl/ml in ethanol. The plates were air-dried overnight at 4°C, blocked with 200 µl of 10% fetal calf serum (FCS; Gibco, Grand Island, NY, USA) in x1 phosphate-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 triplicate. The plates were then washed to remove unbound antibody and proteins, and a secondary antibody, alkaline phosphatase-conjugated antihuman immunoglobulin G (IgG) (Caltag Labs, San Francisco, CA, USA) or IgM (Biosource; Tago Immunologicals, Camarillo, CA, USA) was added to the plate.
After incubation and washing, p-nitrophenyl phosphate substrate (Sigma 104) was added and used to measure indirectly the level of aCL antibodies in a patient serum. The optical density of the samples, caused by the cleavage of the substrate by the enzyme, was determined at 405 nm by a BioRad Microplate Reader Model 450 (BioRad Laboratories, CA, USA) and was used to quantify the amount of aCL in the sera. Every assay plate also included a known high positive aCL sample [>100 GPL (phospholipid units for IgG)] run in triplicate. Plates were incubated until the high positive wells achieved an optical density of >1.0. Referenced standard sets for cardiolipin (Louisville APL Diagnostics, Louisville, KY, USA) and known negative sera were used on every plate. All results were defined in GPL and the corresponding phospholipid units for IgM (MPL) as follows: <10 units, negative; 1019 units, borderline; >20 units, positive. Samples with borderline values were re-tested on two further occasions.
The following tests were carried out in all patients to detect LA: prothrombin time, activated partial thromboplastin time, kaolin clotting time, diluted Russell's viper venom time and tissue thromboplastin inhibition test. Each assay was performed with a mixture of patient and control plasma (1:1, v/v). Patients were considered to be APA seropositive when the aCL (IgG and/or IgM), LA or both were shown to be positive on two occasions at least 68 weeks apart.
The statistical differences in seropositivity in the three groups of patients were compared using the 2-test and Fisher's exact test when appropriate.
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Results |
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Table I summarizes the mean age, cause and duration of infertility (groups 1 and 3) and the live births and miscarriages (groups 1 and 2) which were similar in relevant groups. The peripheral blood karyotype was normal in all patients in the three groups. The number of APA seropositive patients with two consecutive miscarriages after repeat IVF and ICSI in group 1 (4/16) was not significantly different from that in group 2 fertile women with three or more recurrent miscarriages (9/42) (i.e. 25.0 versus 21.4%). The difference was statistically significant by the
2-test when women in either group 1 or group 2 were compared with the women with primary infertility (group 3) being treated with first IVF or ICSI cycle [25.0 versus 6.6%, P = 0.033; 21.42 versus 6.6% (4/60), P = 0.027 respectively] (Table II
). However, because the number of patients, in particular in group 1, was small (making the
2-test of limited validity), Fisher's exact test was also used to compare the groups. When Fisher's exact test was used, the difference seen between groups 1 and 3 was not statistically significant (P = 0.11).
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Discussion |
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In this study, we observed that 6.6% of women with primary infertility being treated in their first IVF or ICSI cycle were positive for APA. This rate of seropositivity is slightly lower than is commonly reported in the literature in the infertile population and is probably related to the precise definition of our population, i.e. women with primary infertility without previous IVF or ICSI treatment. This information is generally not provided in many of the reported studies (Birdsall et al., 1996; Denis et al., 1997
; Kutteh et al., 1997
; Branch, 1988). Notwithstanding this, our study is in agreement with the notion that APA seropositivity in the infertile population in general is higher than that in normal pregnant and non-pregnant women who have not experienced miscarriages, where the prevalence is reported as 24% (Lockwood et al., 1989
; Pattison et al., 1991
).
The implication of the high APA seropositivity on the outcome of IVF or ICSI treatment cycle is contentious. While studies demonstrate that the prevalence of APA is higher in women with repeated failure of embryo transfer than in women who successfully conceive after IVF (Birkenfeld et al., 1994; Geva et al., 1995
; Kaider et al., 1996
), and that IVF patients with positive APA experience a higher miscarriage rate compared with APA negative women (Kowalik et al., 1997
), many reports also conclude that APA is probably a poor predictor of outcome in the general IVF population (Gleicher et al., 1994
; Fisch et al., 1995
; Nip et al., 1995
; Kowalik et al., 1997
; Balasch et al., 1998
). A consideration to carry out routine APA evaluation in the general IVF population does not seem to be justified at present (Balasch et al., 1996), and the suggestion that APA measurements should be performed after one clinical miscarriage (Kowalik et al., 1997
) could not be supported by the findings in a recent study (Balasch et al., 1998
) where the incidence of APA seropositivity was similar in patients who had one miscarriage compared to those with ongoing pregnancies in an IVF programme.
This report describes for the first time APA seropositivity in women with two consecutive miscarriages after successive IVF or ICSI treatment cycles. Whereas the efficacy of commencing investigations after two consecutive losses of spontaneous pregnancies has not been established in the low-risk general obstetrics population (RCOG, 1998), this might not be the case in the women who suffer two consecutive miscarriages after successive repeat IVF or ICSI treatment cycles, given the background of higher prevalence of APA in the IVF population. Our study has shown that the number of women with APA seropositivity was similar (25.0 versus 21.4%) in patients after two consecutive miscarriages of pregnancies conceived following repeated IVF or ICSI treatment cycles when compared to fertile women with three or more consecutive miscarriages. There was however an apparent difference when compared with the number of APA seropositive women with primary infertility being treated by first IVF or ICSI (25.0 versus 6.6%, P = 0.033), which was significant using the 2-test but not when Fisher's exact test was used. This discordance in statistical significance may be explained by the small sample size in group 1. Although the high prevalence of APA in the general IVF population compared to normal non-pregnant women does not justify the routine evaluation for autoimmune antibodies in the infertile population, the occurrence of two consecutive miscarriages (rather than three or more pregnancy losses) after repeat IVF or ICSI suggests a subset of women in whom routine APA screening prior to further assisted reproductive treatment may be advised. In the event of positive results on two occasions, medical treatment with low-dose aspirin and subcutaneous heparin should be considered (RCOG, 1998).
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Notes |
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References |
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Submitted on October 22, 1998; accepted on February 19, 1999.