Antiphospholipid antibodies among pregnant women with recurrent fetal wastage in a tertiary hospital in Northern Nigeria

Abstract

Context:

The association between antiphospholipid antibodies (APAs) and pregnancy loss has been established and now considered as a treatable cause of pregnancy loss. Data on the prevalence of APA in patients with recurrent pregnancy loss are scarce in our environment.

Aims:

To determine the prevalence of APA in pregnant women with and without recurrent fetal wastage.

Settings and Design:

Antenatal clinic of Ahmadu Bello University Teaching Hospital, Zaria, Nigeria. A cross-section analytical study.

Subjects and Methods:

Eighty-five antenatal patients with recurrent fetal loss (cases) and an equal number of antenatal patients without recurrent fetal loss (control) matched for age were studied. Their sociodemographic data obtained and blood samples analyzed for lupus anticoagulant (LA) using activated partial thromboplastin time, direct Russel's viper venom time, hexagonal phospholipids, and IgG anticardiolipin antibody (ACA) using enzyme-linked immunosorbent assay.

Statistical Analysis Used:

Data were analyzed with Statistical Package for Social Sciences (version 17) by univariate analysis and Chi-square test.

Results:

The age range of the patients was 18–42 years with a median of 30 years. The prevalence of APA was 14.1% and 4.7% among the cases and controls, respectively. The prevalence of LA was 7.1% and 1.2% among the cases and controls, respectively, whereas ACA was 8.2% and 3.5%, respectively. However, one of the cases was positive for both APA and ACA, giving a prevalence of 1.2%.

Conclusions:

The prevalence of APA among antenatal patients with recurrent pregnancy loss was, at least, 3 times higher than that of normal antenatal clients. APA should be included in the investigation protocol of women with recurrent fetal wastages in our setting.

Introduction

Antiphospholipid antibodies (APAs) are a group of acquired heterogeneous autoantibodies directed against negatively charged phospholipids and phospholipid-binding proteins.[1,2] However, the most important clinically are the lupus anticoagulant (LA) and anticardiolipin antibodies (ACAs). Even though these antibodies have been found in individuals without any clinical consequences, approximately 15–20% of patients with these antibodies have obstetric complications and up to 50–75% of these pregnancies have poor outcomes.[1,3] Some of the obstetric complications are recurrent pregnancy loss, unexplained intrauterine fetal death, preterm deliveries, and severe pre-eclampsia.

Various prevalences of APA in Nigeria have been documented. In Benin City, the prevalence of lupus antibody among Nigerians with recurrent fetal loss was 4.35%,[4]15.4% in preeclampsia, 2% among apparently healthy pregnant women, and 8% among nonpregnant multiparous women.[4,5] Akinbami et al. found a prevalence of 9.3% and 2.3% of LA and IgG ACA, respectively among patients with complications in pregnancy in Lagos, Nigeria.[5] Another 5 cases of APA syndrome were reported in Sagamu.[6] It was found that pregnancy loss was the most frequent presentation, and IgG ACA was more commonly seen. Other studies have shown that APA is not a rare phenomenon, often overlooked and there is a paucity of knowledge on this disorder in Nigeria.[7,8]

Pregnancy loss is extremely traumatic emotionally and physically. This is even more so in the Nigerian cultural setting, where a lot of emphasis is placed on childbearing.[9]

Studies on the existence and prevalence of these antibodies in the Northern part of Nigeria have not been documented, even though the drugs used for the treatment of some of the diseases related to APA are readily available.

In addition, most of the studies carried out in Nigeria exclusively employed either the solid phase immunological assay or the liquid phase coagulation assay.[4,5,6,10] While both assays are effective screening methods, the former compromises sensitivity while the latter is more specific. It is also significant to note that most of the studies were on LAs.

The purpose of this study was to determine the prevalence of APA among pregnant women with or without fetal wastage in Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, one of the leading tertiary care centers in Northern Nigeria.

Subjects and Methods

This was a cross-sectional analytical study involving 85 patients with pregnancy loss defined as a history of ≥2 consecutive spontaneous abortions, and these pregnancies were confirmed by ultrasonography or human chorionic gonadotropin assay, history of previous unexplained intrauterine fetal death, and history of previous preterm delivery. Patients with disease conditions or on medications that are known to lead to the formation of APA, with history of thrombosis, cervical incompetence, or uterine fibroids were excluded from the study.

The same number of controls without history of pregnancy loss, not having any autoimmune or any medical disease, history of thromboembolic, and had at least one live birth were recruited. The cases and controls were matched for age and were recruited from the antenatal clinic of ABUTH.

After obtaining informed consent, sociodemographics data including age, educational level, marital status, and ethnicity were obtained and recorded in a pro forma designed for the study. Other data obtained included reproductive health profile, past medical and drug history including HIV and veneral disease research laboratory status. Thereafter, 10 ml of venous blood was obtained from both cases and controls for the LA and ACA tests. About 5.5 ml of the blood was put into a sample bottle and allowed to clot for 30 min. It was centrifuged at 4000 revolutions per minute (RPM) for 10 min to separate the serum from the cells. The serum was drawn into plain sample bottles and stored at −20°C, before being analyzed with the enzyme-linked immunosorbent assay (ELISA) kits for IgG ACA. The ELISA test kit used was cardiolipin antibody (IgG) test (Immuno Well product, San Diego, Califonia USA) that had a specificity of >99% and sensitivity of 10 GPL units. The Rato-2100C microplate reader was used to read the results at a wavelength of 405 nm and only those with medium to high titre units were selected as positive.

For the LA test, 4.5 ml of blood was put in a sample bottle containing 0.5 ml of 3.2% trisodium citrate and thoroughly mixed. It was centrifuged at 4000 RPM for 10 min to separate the plasma from the cells. The same amount of blood was drawn from 10 healthy volunteers, processed as mentioned above and were tested with the reagents that were intended to be used for the study. The mean and standard deviation were calculated and used as point of reference for the study. The activated partial thromboplastin time and direct Russel's viper venom time were carried out first as screening tests using Staclot PTT-LA (by Stago France) and LupoTek Detectin VL reagents, respectively (by R2 diagnostics USA). Any of the results that were >2 standard deviations above the mean was considered prolonged, and a confirmatory test was carried out using the Staclot LA hexagonal phase phospholipid. A prolongation of the clotting time of >8 s was considered positive as instructed by the manufacturer.

Approval for the study was given by the Ethical Review Board of ABUTH.

The data obtained was analyzed using Statistical Package for Social Sciences (version 17, Chicago SPSS Inc,). The prevalence of APA was calculated and compared between cases and control. The simple univariate analysis was used to analyze the sociodemographic data and reproductive profile of cases and controls. Chi-square test was used to determine any statistical significance and P value was considered statistically significant at <0.05.

Results

The total number of participants was 170, each group consisting of 85 subjects and all were suitable for analysis. The age range of the patients was 18–42 years with a median of 30 years, majority (61.2%) were in the age group 25–34 years.

The 16 participants that were positive for APA were characterized, 8 out of them were within the age group of 15–29 years. Eight of them had no tertiary education, and 2 of them were nulliparous. There was no statistically significant difference noted in the characteristics.

Nine cases with recurrent abortions were positive for APA, 8 had 2 abortions, and 1 had 3 abortions. None among those with >3 abortions was positive for APA.

Only three patients with intrauterine fetal death (IUFD) were positive for APA. None were positive among those with two or more IUFD. None among those with preterm deliveries was positive for APA.

Discussion

Recurrent fetal wastage is a heterogeneous condition associated with a lot of physical and emotional stress that has been challenging to the physician and patient. In a lot of cases, despite extensive investigations, the etiology remains unknown.[11]

A total of 16 patients were positive for APA in this study [Table 1], 12 cases and 4 controls, giving a prevalence of 14.1% for the cases and 4.7% for the controls. This is similar to findings from previous studies that were carried out in the developed and developing countries.[12,13,14] The prevalence of LA and IgG ACA among the cases were 7.1% and 8.2%, respectively. This was higher than what was found in studies done in Benin City by Awodu et al. and Lagos by Akinbami et al. where the prevalence of LA was 4.35% and IgG ACA 2.3%, respectively.[5,6] It was lower than that found in Senegal by Thiam et al. with a 21.1% prevalence for ACA,[15] Ibadan 24% LA,[16] Lagos 9.3% LA,[6] and India 11.79% IgG ACA.[17] This difference may be due to variations in the type of reagents and tests carried out with their different sensitivity and specificity and difference in patient selection criteria. The difference in the prevalence of APA between the cases and controls was found to be statistically significant (P = 0.032, odds ratio 3.329, 95% confidence interval 1.028–10.779). Only 1.2% of the patients were positive for both LA and ACA. A similar finding was obtained from a previous study.[18]

Table 1.

Characteristics of women who were positive for antiphospholipid antibodies (n=16)

Characteristics	n (%)	P	OR	95% CI
Total	16 (9.4)
Age (years)
15-29	8 (4.7)	0.285	0.238	0.019-3.011
≥30	8 (4.7)	0.285	0.332	0.332-53.123
Education
<Tertiary	8 (4.7)	0.715	1.000	0.104-9.614
Tertiary	8 (4.7)	0.715	1.000	0.104-9.614
Parity
0	2 (1.2)	0.550	1.4	1.005-1.950
>1	14 (8.2)	0.550	0.714	0.513-1.950
Abortions
Yes	9 (5.3)	0.019	2.333	0.992-5.489
No	7 (4.1)	0.019	0.429	0.182-1.008
IUFD
Yes	3 (1.8)	0.393	1.444	1.005-2.075
No	13 (7.7)	0.393	0.692	0.482-0.995
Preterm birth
Yes	0 (0)	0	0	0
No	16 (9.4)

IUFD=Intrauterine fetal death, OR=Odds ratio, CI=Confidence interval

It can be seen from this study that the number of subjects that were positive for the LA was similar to those positive for the ACA (7.1%, 8.2% among the cases and 1.2%, 3.5% among the controls, respectively). If only one of the antibodies was tested for, then about 50% of the subjects might have been missed. This shows the necessity of using both tests as means of investigation for APA. Similar findings and suggestions have been made in a previous study.[17]

Previous studies have shown an association between APA and other medical conditions such as hypertension and pre-eclampsia.[19,20] However, this was not seen in this study as none of the participants that were positive for APA had any significant medical or drug history.

It was also noticed that none of the patients with more than three abortions was positive for APA [Table 2]. This is different from the findings of Jawad et al.[21] that noticed a higher percentage of APA positive results among women with four abortions compared to three. This may be because the number of cases in this study with more than three abortions was too small for any meaningful deductions.

Table 2.

Antiphospholipid antibodies status among cases with abortions

Number of abortions	APA positive cases	APA negative cases
2	8	29
3	1	20
4	0	5
>4	0	1

APA=Antiphospholipid antibodies

Only three patients with IUFD were positive for APA [Table 3]. None were positive among those with two or more IUFD.

Table 3.

Antiphospholipid antibodies status among patients with intrauterine fetal death

IUFD	APA positive cases	APA negative cases
1	3	15
2	0	3
>2	0	4

IUFD=Intrauterine fetal death, APA=Antiphospholipid antibodies

The main limitation of this study was its small sample size and the fact that it was a hospital based study. A population-based study with a larger sample size is therefore recommended for future research.

Conclusion

This study has shown that APAs are an important cause of pregnancy wastage in Northern Nigeria. Therefore, assessing for the presence of these antibodies should become part of the investigative protocol for women with recurrent fetal wastage in our setting. Screening for both LA and ACA is better than just either of the antibodies, because this study has shown that both antibodies have a similar prevalence in our environment. It is important to note that the presence of APA does not preclude a successful pregnancy; it merely indicates a higher-risk pregnancy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.