A retrospective study on IVF/ICSI outcome in patients with anti-nuclear antibodies: the effects of prednisone plus low-dose aspirin adjuvant treatment

A portion of infertile patients consistently fail to conceive despite recurrent transfers of high-quality embryos. The mechanism leading to this reproductive failure is still unclear. To investigate this failure, several researchers have focused on the association between autoimmune factors and IVF/ICSI outcome, and the focus is especially directed toward autoantibodies [1‐5]. It has been reported that ANA were relevant to adverse reproductive events, including recurrent spontaneous abortion, endometriosis, infertility, IVF failure, and ovarian dysfunction [6‐10].

In a broad sense, ANA are a group of autoantibodies that target the entire cell including DNA, RNA and proteins [11]. Most of the common nuclear antigens can be found on the surface structures of apoptotic cells, and these molecules with the phospholipids on the surface of the apoptotic cells can induce an autoimmune reaction, resulting in an abnormal release of anti-phospholipids antibodies (APA) or ANA [12, 13]. A basic cellular abnormality, such as increased apoptosis or decreased clearance of apoptotic cells might explain the increased APA and ANA levels in individuals with autoimmune diseases [14]. One iconoclastic theory that ANA such as an anti-double strand DNA (anti-dsDNA) antibody, anti-RNP antibody, or anti-ribosomal P protein antibody could penetrate into living cells has been confirmed in vivo and in vitro[15‐17].

It has been suggested that ANA could impair oocyte quality and embryo development, leading to reduced pregnancy and implantation rates [7, 10], and women with recurrent pregnancy loss had a significantly higher incidence of ANA than controls [9]. Shirota et al. [18] found that the presence of the anti-centromere antibody might interfere with the oocyte maturation from MI to MII and impair the embryo cleavage potential. Embryo development was severely impaired or failed when co-cultured with IgG from ANA + women [19].

In our study, we retrospectively analysed IVF/ICSI outcomes in ANA + women and compared them with ANA-controls. Differing from previous reports, we studied the effects of prednisone plus low-dose aspirin (P + A) adjuvant therapy in ANA + women before IVF/ICSI cycles. The aim of this study is to investigate the potential effect of ANA and different ANA titres on IVF/ICSI outcome, and to determine the role of adjuvant treatment in these ANA + patients.

For several years, detection of autoantibodies has been recommended in clinical practice for women with infertility including the detection of ANA, APA and ATA. A link of APA with recurrent pregnancy loss has been established, and treatment based on anticoagulation such as subcutaneous heparin is effective [3]. As an organ-specific autoantibody, ATA affect pregnancy outcome negatively by damaging the thyroid function, and the thyroxin replacement therapy is efficacious in preventing foetal loss for patients with recurrent miscarriage [1, 2]. However, the potential correlation between ANA and IVF/ICSI outcome and specific medication are less reported.

Previous reports showed that ANA were relevant to adverse reproductive events including recurrent spontaneous abortion, endometriosis, infertility, IVF failure and ovarian dysfunction [6‐10]. One recent study proposed that ANA might impair oocyte quality and embryo development, leading to reduced pregnancy and implantation rates [10]. A further exploration [20] similarly suggested that IVF outcomes were markedly poorer in ANA + women, and this effect became worse with an increased level of serum ANA.

In this study, we found that the presence of ANA in serum predicted an adverse IVF/ICSI outcome, primarily reflecting the lower rates of MII oocytes and normal fertilisation and the reduced rates of implantation and clinical pregnancy, as well as the increased rates of abnormal fertilisation and early miscarriage. The cleavage rate and the perfect and available embryo rates were not significantly different between the ANA + women and the controls, which was a finding that did not coincide with that of Ying Y et al. [10]. We found that ANA-titre ≤1:320 women had as poor IVF/ICSI outcomes as the ANA-titre > 1:320 women, and only the abnormal fertilisation rate in the IVF cycles in the ANA-titre > 1:320 women was significantly higher. These results did not suggest the titre-dependent effect of ANA on IVF/ICSI outcomes, in agreement with Hasegawa et al. [21] and Taniguchi et al. [22].

In general terms, abnormal autoimmune conditions may impair all stages of fertility, leading to ovarian and testicular failure, implantation failure or pregnancy loss through different putative mechanisms [5]. It has been proposed that APA and A-β2-GPI could result in thrombosis of placental blood vessels, dysfunctions of trophoblasts in the peri-implantation period or an imbalance of maternal hormones [23‐25]. Unlike APA and A-β2-GPI, the mechanism by which ANA determine reproductive failure remains speculative. Hasegawa et al. [21] held that ANA did not target specific organs and only presented as an abnormal degree of autoimmunity, based on their findings that the adverse IVF outcome was not ANA-titre dependent. One recent well designed study [26] demonstrated that the presence of APA, ANA or ATA in recipients using donor oocytes had no negative effect on pregnancy, implantation or miscarriage, which suggested that implantation failure might be because of poor-quality oocytes that may lead to a subsequent embryo development disorder. One in vivo test [27], in which anti-centromere antibodies were microinjected into mouse oocytes, showed the anti-centromere antibody could interfere with chromosome congression in the pro-metaphase. Shirota K et al. [18] considered that anti-centromere antibodies might infiltrate oocytes and lead to centromere dysfunction during meiosis and mitosis and impair the transition from MI to MII during oocyte maturation. Ying Y et al. [20] has confirmed that ANA exist in follicular fluid and embryos in ANA + patients, and serum and follicular fluid ANA negatively correlated with the number of high-quality embryos. The embryos co-cultured with IgG extracted from ANA + women were found to be severely impaired or even died [19].

Ando et al. [28] administered low-dose prednisolone (5 mg/d) or dexamethasone (0.5 mg/d) daily during the entire IVF cycle until the pregnancy test was performed in 51 lVF-ET cycles of patients positive for ANA, anti-DNA antibody, and/or lupus anti-coagulant (LAC), as well as 29 IVF-ET cycles of patients negative for any antibodies and discovered significant increases of pregnancy and implantation rates in the antibodies-positive patients with corticosteroid treatment but not in the antibody-negative patients. Hasegawa et al. [21] administered prednisolone (10 mg/d) plus low-dose aspirin (81 mg/d) to ANA + and/or APA + women from the first day of COH until pregnancy was confirmed by ultrasonography and discovered that the ANA + women with treatment had significantly better outcomes of IVF-ET (40.6% pregnancy rate and 20.3% implantation rate). Taniguchi et al. [22] administered prednisolone (15–60 mg/d) starting from the first day after OPU for 5 days in 56 IVF-ET cycles of 24 ANA + women and 167 IVF-ET cycles of 96 ANA-women and found the implantation rate and clinical pregnancy rate improved significantly in the ANA + woman but not in the ANA-women, which coincides with the findings of Ando et al. One previous prospective study administered prednisone plus aspirin for 4 weeks before IVF treatment to 52 women positive for ACA, ANA, anti-dsDNA antibody, rheumatoid factor, and/or LAC and ultimately obtained a satisfactory clinical pregnancy rate (32.7%) [29]. The present study pretreated ANA + women with prednisone (10 mg/d) plus low-dose aspirin (100 mg/d) (i.e., P + A) for three months before IVF treatment and observed that the ANA + cycles with P + A had markedly more 2PN, high-quality and available embryos, and an increased implantation rate. Our study also showed that the ANA titre was not relevant to the IVF/ICSI outcome and the adjuvant treatment was not closely related to the reduction of the serum ANA titre, which is in agreement with Hasegawa et al. [21]. In this study and that of Hasegawa et al. [21], women positive for autoantibodies were administered a 10-mg daily dose of prednisone or prednisolone, and this dosage is too low to reduce the autoantibody titres. Thus the corticosteroid effect may be derived from another mechanism such as an anti-inflammatory action or the regulation of immune cells as in the reduction of NK cells [21]. Low-dose aspirin for its anti-thrombotic effect may reduce uterine and intraovarian vascular resistance, improve blood perfusion and increase oocyte maturation, the high-quality embryos rate, and the implantation rate [21]. Although these five studies including the present work demonstrated a beneficial effect of corticosteroid or corticosteroid plus aspirin therapy, an ideal protocol for this adjuvant therapy (i.e., the indication of the patients, the drug selection and dosage, and the timing of commencement and end) requires further investigation.

In this study, we observed the detrimental effect of ANA on IVF/ICSI outcome and the beneficial effect of prednisone plus low-dose aspirin adjuvant treatment for ANA + patients. The comparison between the first IVF/ICSI cycle to a subsequent one would unavoidably yield bias, which is the limitation of our study. To minimise the bias, we rigorously selected the subjects, and there was a long interval between the first and the second IVF/ICSI cycles of at least 3 months. In our future work, we will attempt to perform a placebo-controlled, double blind, and prospective study.

These observations suggest that ANA could exert a detrimental effect on the IVF/ICSI outcome that may not be titre-dependent, and prednisone plus low-dose aspirin adjuvant treatment could be useful in ANA + patients. This hypothesis should be verified in placebo-controlled, double blind, and prospective studies.