Serum anti-thyroid peroxidase antibody in infertile Nepalese women: a hospital-based study

Anti-thyroid peroxidase; Autoimmune; Infertility; Thyroid disease; Background: Infertility is a critical component of reproductive health that can precipitate agony for a couple. Antibodies against thyroid peroxidase can lead to not only autoimmune thyroiditis which is highly prevalent in women, but may also represent a generalized autoimmune milieu in our body that can be associated with infertility. This study aims to assess anti-thyroid peroxidase antibodies in female patients with infertility.


INTRODUCTION
Infertility is a disease of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse . 1 Globally, infertility affects approximately 10 -15 % of couples. 2 According to a global survey report conducted in 2010, Nepal showed a prevalence of primary infertility at 1.9% and secondary infertility at 7.9%. 3 ,done by Subedi et al in the eastern region of Nepal, showed an incidence of infertility cases as 5.45 % which was dominated by secondary infertility and malefactor. The incidence of primary infertility cases was 1.7%. 4 Over the last decade, thyroid screening has been a routine procedure in all pregnant and infertile females. Among thyroid disorders and autoimmune diseases, thyroid autoimmunity is the most prevalent autoimmune condition affecting up to 5-20% of women in the reproductive life phase. 5 Thyroid autoimmunity may present with or without clinical or subclinical thyroid dysfunction. It has been associated with endometriosis, polycystic ovarian disease (PCOD), and premature ovarian failure cause of infertility. 5,6 Anti-thyroid peroxidase (anti-TPO) level is investigated only in clinical or subclinical thyroid disorder. Anti-TPO antibodies represent a generalized autoimmune milieu in our body. 7 The prevalence of anti-TPO antibodies have been observed to be significantly higher among infertile women as compared to the healthy, fertile women of the same age group. [7][8][9] This study was done to compare the status of anti -TPO antibodies between infertile females who were undergoing workup for conceiving against the values in the pregnant females in their first trimester (unassisted pregnancy) and also with the values seen in non-pregnant reproductive age group females with at least parity one to see the primary role of anti-TPO in the achievement of medical pregnancy.

MATERIALS AND METHODS
This was a cross-sectional observational study carried out in the Department of Biochemistry at Tribhuvan University Teaching Hospital over three months from November 2018 to January 2019. All infertile women undergoing work up in the infertility clinic between the age group of 20 to 45 years were recruited in the infertile female (IF) group. The thyroid and other reproductive hormones of these patients were within the normal range. Ultrasonography of these participants had ruled out polycystic ovarian disease (PCOD) and endometriosis. The malefactor was ruled out to recruit the cases. Age-matched pregnant females (PG) in the first trimester and non-pregnant females (NP) in the reproductive age, having at least one child, were chosen as controls. The controls were recruited from the obstetric outpatient department and general health clinic respectively.
Ethical clearance was obtained from the Institutional Review Board and written consent was taken from the candidates. For the analysis, a 3ml blood sample was collected from all the participants, the serum was separated and anti-TPO antibodies were analyzed following the protocol of Euroimmu anti-TPO enzyme-linked immunosorbent assay (ELISA) kit. The anti-TPO values above 50 IU/ml were taken as positive as per the instruction manual. Descriptive and statistical analysis was done by SPSS version 17. Oneway ANOVA was carried out to compare the mean anti-TPO values between the three groups and Fisher's exact test was used to compare the occurrence of positive cases among these groups.

RESULTS
During the study period, 87 females were enrolled in the study. This consisted of 54 cases in the IF group, with a mean age of 27.17 ± 4.12 years. There were 16 cases in the PG group with the mean age of 26.79 ± 4.14 years, and 17 participants NP group with the mean age of 26.38 ± 4.61years.
In total, there were 6 anti-TPO positive cases. This value accounted for 6.9% of the whole study population. Four positive cases of anti-TPO were detected in the IF group (7.4% within the IF group), 2 in the NP group (11.76% within the NP group) while no positive cases were detected in the PG group. Fisher's exact test showed the occurrence of positive cases in different groups to be insignificant (p=0.4). The anti-TPO values of the positive cases were winsorized and the results obtained are given in table 1. The mean anti-TPO was higher in pregnant females than in the other two groups. However, the statistical significance of this mean difference could not be established (p=0.066).

DISCUSSION
To be able to conceive is a desire of any couple in a marital relationship and infertility can lead to not only distress and depression but also discrimination and ostracism. 3  In the IF group, the occurrence of anti-TPO positive cases was only 7.4 % which is less than other studies. [5][6][7][8][9] These studies showed elevated anti-TPO values in infertile females suffering from PCOD and endometriosis. The current study excluded the females with a diagnosis of PCOD and endometriosis, which might be the reason for the low occurrence of positive cases.
This study did not find elevated anti-TPO in the PG group although various literature shows prevalence ranging from 4 to 19%. 7,11 This might be due to the small sample size which is the major limitation in this study. However, we detected a higher occurrence of anti-TPO cases in the general nonpregnant female population in the reproductive age group (11.76% within group NP). In total, elevated anti-TPO cases consisted of 6.9%, which lies within the reported prevalence of elevated anti-TPO cases (5-24%) in the general female population. 5,12,13 This study did not find any significant difference in the occurrence of elevated anti-TPO between IF and NP group, as shown by Pompe et al which again could be the result of the exclusion of the infertility cases associated with endometriosis and PCOD. 9 The same literature that shows a higher prevalence of infertility associated with higher anti-TPO levels have also concluded that the elevated anti-TPO levels alone do not reduce the chance of pregnancy. 7,8 Instead, studies have shown elevated anti-TPO value to be associated with miscarriage or preterm delivery, highlighting its role in healthy and sustained implantation 7,9,11,14 As indicated by literature, altered anti-TPO does not have a role in fertilization and implantation. This is also highlighted in our study, as we did not find any significant difference in the anti-TPO values between IF and PG groups. Since we did not follow up on the outcome of assisted pregnancy in anti-TPO positive infertile females and our PG control were in the first trimester of pregnancy, we could not comment on the outcome of pregnancy. Thus, although our study has a lower occurrence of elevated anti-TPO cases in the IF group and a small sample size of the PG group, our result is similar to the literature confirming elevated anti-TPO not to be associated with infertility due to implantation failure.
The mean anti-TPO levels between the three groups were compared. Even with a limited sample size, we found a higher trend of mean anti-TPO level in pregnant control in the first trimester among the three groups which was also seen in the study by Meena et al. 7 This might be the result of the altered physiological status of pregnancy.

CONCLUSIONS
Functional autoimmune antibodies, like anti-TPO, does not have a significant primary association with infertility.

Limitations of The Study
The sample size is the main limitation of the study as the study period was short. This is a hospital-based study, so it may not represent the whole population of the country. Finally, the outcome of the assisted pregnancy in infertile patients was not followed.