Evaluation of Ovarian Reserve among Newly Diagnosed Hashimoto’s Thyroiditis Related Subclinical and Overt Hypothyroid Reproductive Women: A Cross-sectional Study

1. Associate Professor, Department of Biochemistry, Government Medical College Nalgonda, Telangana, India. 2. Associate Professor, Department of Biochemistry, ESIC Medical College and PGIMSR, Chennai, Tamil Nadu, India.

Abstract

Introduction: Thyroid autoimmunity is a common autoimmune disorder among reproductive women. It is proposed that, thyroid peroxidase antibodies pass through the blood follicle barrier during follicular development which damages the growing follicles and oocytes. Previous studies have demonstrated the association between thyroid autoimmunity and ovarian failure. Serum Anti-Müllerian Hormone (AMH) levels are used as a marker of ovarian reserve in clinical practice.

Aim: To evaluate ovarian reserve among newly diagnosed Hashimoto’s Thyroiditis (HT) related subclinical and overt hypothyroid women.

Materials and Methods: A cross-sectional study was conducted at the Department of Biochemistry, Government Medical College Nalgonda, Telangana, India, from August 2020 to July 2021. The study included 180 subjects, 60 in each Overt Hypothyroid (EU group) (OH group), Subclinical Hypothyroid (SCH group) and Euthyroid subjects. Socio-demographic details and other parameters age, Body Mass Index (BMI), AMH, Thyroid Stimulating Hormone (TSH), free T4 (fT4), Thyroid Peroxidase Antibodies (TPOAb), Follicular Stimulating Hormone (FSH), Luteinizing Hormone (LH), Estradiol (E2), Antral Follicular Count (AFC) were evaluated and compared in all the three groups. The above data were recorded and analysed using Statistical Package for Social Sciences (SPSS) version 20.0 and the Analysis of Variance (ANOVA) test, Chi-square test, and Spearman’s correlation test were performed for statistical analysis.

Results: The median age, BMI, and total AFC were not statistically significant (p-value >0.05). Serum TPOAb levels were significantly (p-value <0.001) high in the OH group and SCH group when compared to the EU group. The AMH levels were significantly (p-value=0.015) high in the EU group compared to the OH group, but there were no statistically significant differences among other groups. The AMH values were negatively correlated with age among all the three groups. There was no significant correlation between AMH and other parameters analysed among different groups.

Conclusion: The AMH levels were significantly low in the OH group when compared to the EU group. There were no statistically significant differences among other groups. Age was found to be an independent factor for low AMH levels among all the three groups.

Anti-müllerian hormone, Thyroid autoimmunity, Thyroid stimulating hormone, Thyroid peroxidase antibodies

Autoimmune Thyroid Disease (AITD) is the most common endocrine disease among women of reproductive age. Its prevalence was reported to range from 5% to 15% (1). Hypothyroidism is associated with menstrual irregularities and infertility.Severe hypothyroidism is commonly associated with ovulatory dysfunction due to numerous interactions of thyroid hormones with the female reproductive system. Thyroid responsivity by the ovaries could be explained by the presence of thyroid hormone receptors in human oocytes (2). The pathophysiology of how Autoimmune Thyroid Disease (AITD) reduces ovarian reserve has not been well established. Given that thyroxin discovered in human follicular fluid is important for oocyte development and AITD decreases thyroid function by disturbing the hypothalamus pituitary gonadal axis, AITD may decrease ovarian reserve by affecting endocrinology (3). Secondly, antithyroid peroxidase antibodies may activate an immune reaction to damage the ovarian tissue because antithyroid antibodies are also detected in ovarian follicles (4),(5).

It is well known that a prolonged reduction in thyroid hormone concentration results in a broad spectrum of reproductive alteration, including abnormal folliculogenesis, alterations in the ovulation and fertilisation rate, and ovarian failure [6-8]. In clinical practice, hormonal markers like Follicle Stimulating Hormone (FSH), Estradiol (E2), Anti-Müllerian Hormone (AMH), and ultrasound parameter Antral Follicle Count (AFC) are used for evaluating ovarian reserve. Anti-müllerian hormone is produced by the granulosa cells of ovarian antral follicles. AMH concentrations show a significant correlation with oocyte count after superovulation and can be used as a good marker for evaluating ovarian reserve (9). Previous studies showed varying results on the association between TSH, Thyroid Peroxidase Antibodies (TPOAb), and AMH (ovarian reserve). A study conducted by Ozturkunsal I et al., showed no significant correlation between AMH concentrations with the serum levels of TPOAb and TSH (10). Chen CW et al., showed positive TPOAb was associated with low ovarian reserve (11). Another study by Osuka S et al., showed that TPOAb is not likely to affect ovarian reserve in euthyroid women with normal TSH, but elevated TSH is associated with decreased AMH levels (12). Studies conducted by Polyzos NP et al., Korevaar TIM et al., Kucukler FK et al., and Morales Martínez FA et al., showed no relationship between thyroid hormone levels and AMH levels (13),(14),(15),(16).

As the earlier studies showed varying results on how ovarian reserve was affected by thyroid autoimmunity, the present study aimed to evaluate ovarian reserve among newly diagnosed Hashimoto’s thyroiditis related subclinical and overt hypothyroid women using serum AMH levels and other relevant markers. The authors also aimed to find out the possible correlation between thyroid hormone status, thyroid autoantibodies, and AMH levels in the study subjects.

Material and Methods

A cross-sectional study was conducted at the Department of Biochemistry, Government Medical College Nalgonda, Telangana, India, from August 2020 to July 2021. The study was approved by Institutional Ethical Committee (No: IEC/GMC/06-2020). Written Informed consent was obtained from all the subjects.

Sample size calculation: From a previous study with an alpha error of 5% and power of 80%, required sample size was 168 (15). The sample size was calculated by using MedCalc statistical software version 20.0. Total of 180 women aged between 20-40 years were enrolled in the present study, with 60 subjects in each group.

• Newly diagnosed Hashimoto’s Thyroiditis (HT) patients with Overt Hypothyroid (OH) (n=60).
• Newly diagnosed HT patients with Subclinical Hypothyroid (SCH) (n=60).
• Healthy subjects with Euthyroid (EU) (n=60).

Inclusion criteria: Newly diagnosed Hashimoto’s Thyroiditis patients with OH and SCH who were attending General Medicine and Gynaecology Departments were included in the study, and healthy subjects with euthyroid were involved as the control group.

Exclusion criteria: Patients with thyroidectomy, thyroid treatment, polycystic ovarian syndrome, endometriosis, and history of ovarian surgery, patients on radiotherapy, chemotherapy, and diabetic subjects were excluded from the study.

Diagnosis of Hashimoto’s thyroiditis was made when patients have high levels of antithyroid antibodies and parenchymal heterogeneity on ultrasonography of the thyroid gland (15). Increased TSH levels >10 μIU/mL together with low free T4 (fT4) were considered overt hypothyroidism and mildly increased TSH levels (4.2-10 μIU/mL) in the presence of normal free T3 (fT3) and fT4 values were regarded as subclinical hypothyroidism. Euthyroid was defined as patients with normal thyroid function tests (15).

Study Procedure

Venous blood samples were collected between the third to sixth days of the menstrual cycle from all the subjects. Serum was separated from whole blood and samples were stored at -20°C until an assay was performed. The TSH, fT4, TPOAb, FSH, Luteinizing Hormone (LH), E2, and AMH were measured on the Beckman Coulter – Access 2 Immunoassay System using the Chemiluminescence Immunoassay (CLIA) method with Beckman Coulter commercial kits. The AMH concentration <1 ng/mL was considered as low. Reference ranges for TSH (0.34-5.6μIU/mL), fT4 (0.58-2.19 ng/dL), TPOAb (0.0-9.0 IU/mL), FSH (1.85-8.78 mIU/mL), LH (2.12-10.89 mIU/mL), E2 (26.5-161.3 pg/mL). These reference values were as given in the kit inserts used in the laboratory for assay. On the same morning as the blood tests, transvaginal ultrasonography evaluation of the Antral Follicle Count (AFC) was performed with a MINDRAY DC-7 model ultrasonography device in the lithotomy position. Antral follicles with a diameter of 2-10 mm were counted. Thyroid gland ultrasonography was performed using Philips ClearVue 650 ultrasound machine by an endocrinologist.

Statistical Analysis

Kolmogorov-Smirnov test was used to check the normal distribution of the quantitative variables. Descriptive statistics were presented as median and interquartile ranges (25th-75th percentile) for non normally distributed parameters. The Analysis of Variance (ANOVA) test or Kruskal-Wallis test were used to evaluate the significance of the mean difference between more than two groups where applicable. To find out the correlation between two continuous variables, Spearman’s correlation test was used. Linear regression analysis was performed to test the influence of independent variables on AMH levels (dependent variable). Statistical Package for the Social Sciences (SPSS) for windows version 21.0; (SPSS Inc., Chicago, IL, USA) was used for statistical estimations. The statistical significance level was considered at p-value<0.05.

Results

The age, BMI, and hormonal parameters including AMH and AFC of the three groups were represented in (Table/Fig 1). Comparisons were made among different groups (OH group vs EU group, SCH group vs EU group, OH group vs SCH group) (Table/Fig 1). Median age, BMI and total AFC were similar among the different groups and the differences between the median age, BMI and total AFC were not statistically significant (p-value>0.05). Serum TPOAb levels were high in OH group 366.1 IU/mL (IQR: 305.9-410) and SCH group 277.1 IU/mL (IQR: 222.3-320) when compared to EU group 6.05 IU/mL (IQR: 3.7-8.6). There were statistically significant differences (p-value<0.001) among the different groups (OH vs EU, SCH vs EU, OH vs SCH) (Table/Fig 1).

Serum TSH values were high in the OH group and SCH group, when compared to the EU group. Differences between serum TSH values among the three groups was a statistically significant (p-value <0.001). Serum fT4 values were low at 0.5 ng/dL (IQR;0.32-0.67) among Hashimoto’s thyroiditis when compared to EU group 1.3 ng/dL (IQR;1.1-1.42) the differences between the serum fT4 values among OH vs EU, SCH vs EU, OH vs SCH groups was statistically significant, p-value<0.001. Serum FSH, LH, and E2 values were low in the OH group and SCH group when compared to the EU group. Differences between serum TSH values among the three groups was a statistically significant (p-value<0.05). Serum AMH values were low at 1.1 ng/mL (IQR: 0.27-1.3) in the OH group when compared to the EU group 1.45 ng/mL (IQR: 1.17-2.30), the difference was statistically significant (p-value <0.05). But the difference in AMH values among the SCH group vs EU group, OH group vs SCH group was not statistically significant (Table/Fig 1).

Correlation analysis of AMH with other parameters showed that the AMH values were negatively correlated with age among all the three groups, (OH group: r-value=-0.718; p-value <0.001, SCH group: r-value=-0.402; p-value=0.028, and EU group: r-value=-0.701; p-value <0.001). There was no significant correlation between the AMH values and BMI, TPOAb, fT4, FSH, LH, E2, and total AFC among all the three groups (Table/Fig 2). Linear regression analysis was performed on total Hashimoto’s cases (OH group+SCH group) to test the influence of independent variables on AMH levels (dependent variable). Linear regression analysis in total Hashimoto’s Thyroiditis cases showed AMH levels were independently correlated with only age t-value=-3.03 and p-value=0.004 (Table/Fig 3).

Discussion

Thyroid autoimmunity is the most common autoimmune disorder in women of reproductive age, the effect of the autoimmune system on the female reproductive system was demonstrated by many studies (17),(18),(19). It has been shown that autoantibodies were detected in women with premature ovarian failure, thyroid dysfunction or thyroid autoimmunity can affect the ovarian reserve (12). Although underlying mechanisms of how thyroid antibodies affect ovarian reserve are not clearly understood, the possible mechanism may be that anti-TPO passes through follicle barrier during follicular evolution and that may result in the destruction and damaging of growing the follicles and oocytes. Thyroid antibodies seem to have a direct impact on ovarian tissue (20),(21).

AMH has gained importance as a reliable marker of ovarian reserve. Several studies were conducted earlier to find out the association between ovarian reserve, AMH as a marker and thyroid autoantibodies, and TSH. A study by Polyzos NP et al., assessed the association between serum AMH levels, TSH, and thyroid autoantibodies levels and found that serum AMH levels were not associated with TPOAb or TSH levels (13). Another study by Saglam F et al., showed lower serum AMH levels in patients with autoimmune thyroid disease than in controls (22). However, in the multivariate analysis, they found that TSH was not the factor influencing AMH levels. Kuroda K et al., demonstrated that serum AMH levels showed an inverse correlation with serum TSH levels. But they did not include thyroid antibodies in their analysis (23).

In the present study, the authors have estimated the AMH, TSH, fT4, TPOAb, FSH, LH, E2, and AFC values among newly diagnosed Hashimoto’s Thyroiditis patients with overt and subclinical hypothyroidism. The current study found that, serum AMH levels were low in the OH group, when compared to the EU group (controls). AMH values were negatively correlated with age among all the three groups. Age was found to be an independent risk factor affecting the AMH levels. There was no significant correlation between the AMH values and BMI, TPOAb, fT4, FSH, LH, E2, and total AFC among all the three groups. Present study results showed that, there was a negative correlation between age and AMH levels among all three groups. Age was found to be an independent risk factor affecting the AMH levels. There was no significant correlation between AMH values and TPOAb and TSH values in the present study, this was similar to a study conducted by ÖztürkÜnsal İ et al., (10).

Chen CW et al., (11) reported that positive TPOAb are associated with low ovarian reserve. The current study, found high TPOAb among Hashimoto’s disease patients when compared to the euthyroid group but there was no significant correlation between AMH levels and TPOAb. Another study by Osuka S et al., (12) reported that TPOAb are not likely to affect ovarian reserve in euthyroid women with normal TSH, but elevated TSH was associated with decreased AMH levels. Studies by Polyzos NP et al., Korevar TIM et al., and Kucukler FK et al., reported that there was no relationship between thyroid hormone levels and AMH values (13),(14),(15). The present study findings are similar to these studies. The current study could help to assess ovarian reserve among overt and subclinical Hashimoto’s thyroid patients simultaneously and compared with the euthyroid subjects.

Limitation(s)

Newly diagnosed Hashimoto’s thyroiditis patients were included in the present study; the effect of thyroid autoimmunity on ovarian reserve might have been inadequately assessed as authors did not measure thyroglobulin antibodies.

Conclusion

The present study concluded that, there was significantly decreased ovarian reserve among overt hypothyroid individuals. Also there was no significant decrease of ovarian reserve among subclinical hypothyroid subjects. Age was found to be independent marker for decline in AMH levels irrespective of thyroid status. Thus, it was inferred that newly diagnosed Hashimoto’s thyroiditis related overt hypothyroidism but not the subclinical hypothyroidism was associated with decreased ovarian reserve.

Acknowledgement

The authors would like to thank Dr Jyothi, Assistant Professor, Department of Obstetrics and Gynaecology for her help in the data collection and support throughout the study.

DOI and Others

DOI: 10.7860/JCDR/2022/58369.17072

Date of Submission: Jun 10, 2022
Date of Peer Review: Jul 16, 2022
Date of Acceptance: Aug 27, 2022
Date of Publishing: Oct 01, 2022

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

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