Antiphospholipid antibodies in infertile couples with two consecutive miscarriages after in-vitro fertilization and embryo transfer

P.E. Egbase1,3, M. Al Sharhan1, M. Diejomaoh2 and J.G. Grudzinskas3,4

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


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Of 682 women who had undergone in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) with embryo transfer, 84 were successful on two occasions, with 16 of these resulting in miscarriage before 20 completed weeks. Antiphospholipid antibodies (APA) were estimated by enzyme-linked immunosorbent assay in these women (group 1) and compared to two control groups: 42 fertile women with three or more miscarriages (group 2) and 60 women with primary infertility undergoing IVF or ICSI (group 3). An apparently higher prevalence of seropositivity was seen in group 1 women (25%) compared to the group 3 women (6.6%) and it was similar to that seen in group 2 women (21.4%). Therefore the recommendation that women with two consecutive miscarriages after IVF or ICSI should have APA estimations performed routinely may be justified.

Key words: antiphospholipid antibodies/IVF/two consecutive miscarriages


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Since the review of clinical and laboratory procedures at the in-vitro fertilization (IVF) Centre, Maternity Hospital, Kuwait in early 1994, more than 1000 treatment cycles for IVF and embryo transfer or intracytoplasmic sperm injection (ICSI) (Egbase et al., 1996Go, 1997Go) have been performed in 682 women. The clinical success of the IVF Centre has led the Kuwait Ministry of Public Health to review its policy concerning assisted reproductive technology treatment at public expense abroad, now resulting in almost all Kuwaiti citizens requiring this service to undergo these procedures in Kuwait. Given the comprehensive availability of these resources, it has been possible for couples to undergo assisted reproductive treatment procedures on more than one occasion. Consequently, it has been possible to have access to information concerning the clinical management of these couples with full outcome data including abortions (Makhseed et al., 1998Go).

Available evidence suggests that abnormal auto-immune antibodies are statistically correlated with recurrent miscarriage in ~10–15% of patients (Rai et al., 1995Go; Hatasaka et al., 1997Go) and an increased antibody prevalence has been uniformly demonstrated by a number of studies in an infertile population seeking IVF treatment (Gleicher et al., 1994Go; Fisch et al., 1995Go; Nip et al., 1995Go). 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., 1996Go), 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., 1994Go; Birdsall et al., 1996Go; Denis et al., 1997Go). 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., 1997Go; Balasch et al., 1998Go).

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.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
A retrospective analysis was undertaken of the case records of 1027 treatment cycles with oocyte retrieval procedures for conventional IVF or ICSI and embryo transfer from January 1995 to December 1997 at the IVF Centre, Maternity Hospital, Kuwait.

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 3–6 months after the last miscarriage. Clinical pregnancies were diagnosed by the presence of a regular gestation sac with embryonic heart activity, 4–5 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., 1996Go). 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 3–6 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) 3–6 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; 10–19 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 6–8 weeks apart.

The statistical differences in seropositivity in the three groups of patients were compared using the {chi}2-test and Fisher's exact test when appropriate.


    Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
A total of 1027 IVF or ICSI treatment cycles with oocyte retrieval was performed in 682 infertile women, of whom 153 had two or more cycles. Clinical pregnancy was diagnosed in 382 treatment cycles, 67 of which ended in miscarriage on one occasion and 11 in ectopic pregnancy. Two consecutive miscarriages occurred in 16 of 84 women who conceived on two occasion after successive IVF or ICSI treatment cycles.

Table IGo 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 {chi}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 IIGo). However, because the number of patients, in particular in group 1, was small (making the {chi}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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Table I. Clinical data
 

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Table II. Antiphospholipid (APA) seropositivity
 

    Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Recurrent miscarriage is commonly defined as the spontaneous loss of three or more consecutive pregnancies but opinion differs as to whether two pregnancy losses should be included in the definition, as the efficacy of commencing investigations after two losses has not been established [Drakeley et al., 1998; Royal College of Obstetricians and Gynecologists (RCOG), 1998]. There is a statistically significant correlation between abnormal antibodies and recurrent pregnancy loss in 10–15% of patients (Balasch et al., 1995Go; Rai et al., 1995Go; Hatasaka et al., 1997Go), and assessment of APA, LA and aCL of IgG or IgM class as markers has become routine in the evaluation of women with recurrent miscarriage (Rai et al., 1996Go). The same antibodies seemed to be implicated in the `reproductive autoimmune failure syndrome' described in infertile women (Gleicher et al., 1994Go; Sher et al., 1994Go; Fisch et al., 1995Go; Birdsall et al., 1996Go; Kutteh et al., 1997Go), the magnitude of the increase varying from 10 to 45%.

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., 1996Go; Denis et al., 1997Go; Kutteh et al., 1997Go; 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 2–4% (Lockwood et al., 1989Go; Pattison et al., 1991Go).

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., 1994Go; Geva et al., 1995Go; Kaider et al., 1996Go), and that IVF patients with positive APA experience a higher miscarriage rate compared with APA negative women (Kowalik et al., 1997Go), many reports also conclude that APA is probably a poor predictor of outcome in the general IVF population (Gleicher et al., 1994Go; Fisch et al., 1995Go; Nip et al., 1995Go; Kowalik et al., 1997Go; Balasch et al., 1998Go). 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., 1997Go) could not be supported by the findings in a recent study (Balasch et al., 1998Go) 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 {chi}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).


    Notes
 
4 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Balasch, J. (1995) Immunological factors in pregnancy wastage: truth or fiction? In Asch, R. and Studd, J.W.W. (eds), Progress in Reproductive Medicine. Parthenon, London, vol. 2, pp. 117–138.

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Submitted on October 22, 1998; accepted on February 19, 1999.