Department of Obstetrics and Gynaecology, The University of Wuerzburg, Josef-Schneider-Str. 4, D-97080 Wuerzburg, Germany
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Abstract |
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Key words: infertility/in-vitro fertilization/recurrent IVF failure/thyroid antibodies
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Introduction |
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It has been suggested recently that autoimmune abnormalities are closely related to reproductive failure. The presence of thyroid antibodies, specifically thyroglobulin (TG) and thyroid peroxidase (TPO) antibodies, may play a significant role in pregnancy loss and adverse IVF outcome. These autoantibodies can persist in patients who have suffered from hyper- or hypothyroidism even after normalization of their thyroid function by appropriate pharmacological treatment. Furthermore, antithyroid autoantibodies can occur in asymptomatic, euthyroid women who have never suffered from a thyroid disease (Gerhard et al., 1991; Gleicher et al., 1993
).
Several authors (Stagnaro-Green et al., 1990; Glinoer et al., 1991
; Lejeunne et al., 1993
; Pratt et al., 1993a
; Kutteh et al., 1999
) suspected a link between the presence of thyroid antibodies and either isolated or recurrent pregnancy wastage. We have demonstrated a significantly increased incidence of thyroid antibodies in euthyroid, non-pregnant women with a history of recurrent spontaneous abortions compared to nulligravid and multigravid controls of childbearing age (Bussen and Steck, 1995
, 1997
; Bussen et al., 1999
).
If antithyroid autoantibodies are associated with pregnancy loss, they may affect fertilization, embryo development or implantation, and may be useful markers for predicting poor outcome in patients undergoing assisted reproductive technologies. A high incidence of thyroid autoantibodies was first described in patients who failed to conceive after undergoing at least three IVF and embryo transfer cycles (Geva et al., 1995). These results were confirmed in a consecutive study performed by the same authors (Geva et al., 1996
) and in recently published data (Kim et al., 1998
).
This study was undertaken to evaluate whether the presence of thyroid antibodies in euthyroid asymptomatic women may serve as a marker for an adverse outcome in couples with a history of recurrent IVF failure.
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Materials and methods |
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The control group included 24 consecutive patients (mean age ± SD: 30.3 ± 4.1 years) seeking infertility treatment in our department of assisted reproduction. Prior to enrolling them in our IVF programme due to andrological (n = 16) or tubal (n = 8) sterility, serum concentrations of thyroid autoantibodies and IgG and IgM isotypes of anticardiolipin were measured. All study and control group patients were determined to be euthyroid by demonstrating normal concentrations of thyroid-stimulating hormone (TSH). None suffered from endocrine dysfunctions. No woman had systemic lupus erythematosus according to the American Rheumatism Association or met any of the major or minor criteria for other autoimmune diseases (Tan et al., 1982). All women were nulligravid.
IVF protocol
Ovarian stimulation was induced by a human menopausal gonadotrophin (HMG) protocol (Humegon®; Organon, Oberschleissheim, Germany) starting on day 3 of the cycle after pituitary down-regulation by 3.75 mg triptorelin (Decapeptyl Depot®; Ferring, Kiel, Germany). Human chorionic gonadotrophin (HCG) 10 000 IU (Choragon 5000®; Ferring) was administered after the mean diameter of the dominant follicle reached 18 mm and after 67 days of steady rise of serum oestradiol concentrations. Oocyte recovery was performed by the vaginal route 3436 h after HCG administration. Oocytes were cultured in Ham's F10 medium supplemented with 10% heat inactivated maternal serum at 37°C in 5% O2, 5% CO2, 90% N2 at pH 7.4. At 4 h after recovery, ~100 000 motile spermatozoa were added to each oocyte. If the semen sample on the day of oocyte recovery contained fewer than 5x106 motile spermatozoa, intracytoplasmic sperm injection was performed as described previously (Bussen et al., 1997). After incubation for 1618 h, the oocytes were checked for the presence of pronuclei as evidence of fertilization. Fertilized oocytes were transferred to fresh medium and kept in culture for another 24 h. A maximum of three embryos was transferred 4446 h after oocyte recovery. After transfer, the luteal phase was routinely supported by vaginal progesterone suppositories (300 mg/day) and by at least one injection of 5000 IU of HCG.
A clinical pregnancy was diagnosed if HCG serum concentration >100 IU/ml was measured on two occasions more than 14 days after embryo transfer and if a gestational sac could be detected by ultrasound. These procedures have been reported previously (Bussen et al., 1997)
Laboratory evaluation
TSH was determined by using an automated fluorometric enzyme immunoassay (Chemolumineszenz IXM; Abbott, Wiesbaden, Germany). TPO antibodies and TG antibodies were assayed using enzyme-linked immunoassay (ELISA) kits (Pharmacia Upjohn Diagnostics GmbH, Freiburg, Germany). The concentration of antibody detection limit of these highly sensitive ELISAs is 1 IU/ml. A positive result in both assays was defined as titres >100 IU/ml according to the manufacturer`s instructions, which were based on investigations of >700 apparently healthy female blood donors.
The ELISA assay for anticardiolipin was performed using a previously published method (Loizou et al., 1985). IgG and IgM immunoglobulins were evaluated separately. Anticardiolipin concentrations were considered positive for IgG values >12 IU/ml and IgM values >6 IU/ml.
Statistics
The statistical analysis was performed using the SPSS/PC+ package. All values were expressed as mean ± SD. Spearman's test for correlation between two variables, MannWhitney non-parametric U-test and Fisher's exact test were used as appropriate. P < 0.05 was regarded as significant.
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Results |
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All 13 women of the study group with elevated thyroid antibody concentrations, but only one of the controls, were positive for TG antibodies (Fisher`s exact test: P = 0.001). The mean TG antibody concentrations were significantly higher in the study population compared with the control group (study group: 156 ± 167 IU/ml, range: 5600 IU/ml; control group: 33.5 ± 32.0 IU/ml, range: 5104; U-test: P = 0.009).
Four study patients and one control subject demonstrated positive serum concentrations of TPO antibodies (Fisher's exact test: not significant). Serum concentrations of TPO antibodies (study group: 54.8 ± 10.1 IU/ml, range: 1286 IU/ml; control group: 26.1 ± 58.0 IU/ml, range: 1436 IU/ml; U-test: not significant) were similar in patients with or without a history of three or more unsuccessful IVF attempts.
Four women with repeated IVF failure but no control subject were positive for both thyroid antibodies.
Among the 24 women of the study group, 13 patients were positive for anticardiolipin antibodies. Only IgM isotypes of anticardiolipin were found. No woman was positive for IgG autoantibodies. A comparable number of control subjects (15 out of 24) had positive test results for anticardiolipin antibodies (Fisher's exact test: not significant). In the serum of one of these women IgG as well as IgM antibodies were detectable. Serum concentrations of anticardiolipin antibodies did not differ significantly between study and control patients (respectively: IgG: 6.9 ± 4.8 IU/ml, range: 112; IgM: 8.2 ± 5.7 IU/ml, range: 121; and IgG: 7.8 ± 5.5 IU/ml, range: 119; U-test: not significant; IgM: 9.2 ± 5.7 IU/ml, range: 124; U-test: not significant).
No significant correlation between TG or TPO concentrations and anticardiolipin IgG or IgM serum concentrations was apparent (Table I).
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No evidence for a significant correlation between the cause of infertility and the presence of thyroid antibodies was demonstrated, either in the study or in the control patients.
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Discussion |
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Recently, significantly different pregnancy rates of 26 versus 40% were demonstrated in women with positive and negative titres of thyroid antibodies respectively (Kim et al., 1998). Neither Geva et al. (Geva et al., 1996
) nor Kim et al. (Kim et al., 1998
) found an association between the adverse IVF outcome and an increased number of retrieved oocytes, the fertilization rate or the number of embryos transferred. In both studies, only euthyroid, asymptomatic women with mechanical or unexplained infertility and no previous IVF attempts were investigated.
Kutteh et al. (Kutteh et al., 1999) failed to detect an increased prevalence of thyroid antibodies in women undergoing assisted reproduction compared to healthy reproductive-aged controls. Although 688 patients with a history of infertility were included in this recently published study, the investigated group was highly heterogeneous. Only 19% of the patients suffered from mechanical and none from andrological infertility, whereas in most of the patients no reason for the infertility was identified (unexplained infertility 38% of the patients). A considerable number of patients (19%) were diagnosed as having endocrinological disorders. The type of assisted reproduction technique the couples were scheduled for is not mentioned. It remains unclear if all patients were enrolled in an IVF programme or only received ovarian stimulation or other infertility treatment. Because the enrolment was not limited to euthyroid, asymptomatic women, 55 (8%) of the study patients had an abnormal thyroid function as demonstrated by elevated TSH concentrations. In contrast, others (Geva et al., 1996
; Kim et al., 1998
) as well as our own group, only included euthyroid patients to exclude the role of endocrine thyroid dysfunction as a cause for recurrent IVF failure.
Recent studies showed that women who carry thyroid antibodies are at increased risk of either isolated (Stagnaro-Green et al., 1990; Glinoer et al., 1991
; Lejeunne et al., 1993
) or recurrent (Pratt et al., 1993b
) pregnancy loss. In concordance, a significantly higher miscarriage rate has been reported in women who were positive for thyroid antibodies after successfully achieving a pregnancy by IVF (Kim et al., 1998
).
As previously published by Geva et al. (1995), we found no significant difference between the incidence of thyroid autoantibodies and the different subgroups of infertility (mechanical versus andrological infertility). These findings suggest that infertile women may suffer from subclinical autoimmune disorders in addition to their mechanical or other infertility problems.
It is possible that thyroid autoantibodies directly compromise trophoblastic development. However, it has been suggested that the relationship may be indirect and that the presence of such autoantibodies may represent a marker for increased T-cell activation and toxic cytokine production by T-lymphocytes (Stagnaro-Green et al., 1992; Gleicher, 1998).
Since it is still unknown whether thyroid antibodies are truly organ-specific, it is desirable to consider the possibility of cross-reactivity between these antibodies and other non-organ-specific autoantibodies. Neither our results nor previous publications (Pratt et al., 1993a; Bussen and Steck, 1997a) gave evidence of a correlation between the incidence of thyroid antibodies and non-organ specific autoantibodies. These findings suggest that thyroid antibodies may be an independent marker for autoimmune failure.
Considering the fetus as an allograft within the rules of classical transplantation immunity, it is still unclear why the fetus in successfully ongoing pregnancies is not rejected. Therefore, it needs further clarification which factors could interfere with this fetomaternal relationship. A recently published paper (Rinkevich, 1998) discussed the similarities between implantation in invertebrates compared to vertebrates, supporting the idea of evolutionary links between vertebrate and invertebrate transplantations (Loke and King, 1996
).
It has been demonstrated that immunotherapy with prednisolone improves pregnancy rates in patients with unexplained infertility who undergo ovulation induction with intrauterine insemination (Kim et al., 1996); however, the study group patients were not tested for the presence of autoantibodies. Recently, data were presented (Sher et al., 1998) showing a positive effect of intravenous immunoglobulin immunotherapy on the IVF outcome in patients who were positive for antithyroid antibodies. Considering the possible side-effects of these treatment schedules, further investigations are needed to understand the immunopathological mechanism of these organ-specific antibodies in relation to early implantation failure.
In conclusion, the incidence of thyroid antibodies in euthyroid women with recurrent IVF failure appears to be significantly increased. Although the important issue of cause and effect has not been fully clarified, this finding suggests that the presence of thyroid antibodies is significantly associated with a poor IVF outcome even in euthyroid women. Since these autoantibodies seem to be distinct and independent markers for reproductive failure, their identification provides the opportunity to identify women at risk for an adverse outcome in an IVFembryo transfer programme. Therefore, it is suggested to include the determination of thyroid antibodies in the evaluation of women with recurrent IVF failure.
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Notes |
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References |
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Submitted on June 16, 1999; accepted on October 19, 1999.