Regulatory T Cell and T Helper 17 Cell Imbalance in Patients with Unexplained Infertility

Introduction

Female infertility is a global reproductive health issue affecting individuals, families, and society.¹ Developed countries have a higher incidence of infertility.² Therefore, with economic development, the issue of infertility among Chinese women of childbearing age warrants continued attention. Research shows that the infertility rate among Chinese couples of childbearing age is between 15% and 25%.^3–5 Patients with unexplained infertility (UI) account for 10–17% of infertile women; UI is defined as a condition where individuals have not achieved clinical pregnancy after unprotected sexual intercourse for more than 12 months without any clear reason.⁶

Although the exact cause of UI is unclear, an imbalance in maternal immune mechanisms seems to play an important role. Previous studies have suggested that female infertility is an adaptive immune imbalance disease.^7–10 The fetus carries foreign (paternal) antigens, which serve as semi-allogeneic antigens and require the mother to establish immune tolerance. Adaptive immune imbalance may disrupt tolerance and lead to UI. T helper 17 cells (Th17) and regulatory T (Treg) cells are important subgroups of CD4⁺ T cells that differ in development and function.^11–13 Maintaining a balance between Th17 and Treg is crucial for controlling the immune status of the body. Previous studies have shown that an imbalance between Treg and Th17 cells is associated with immune disorders in patients with pregnancy disorders, such as recurrent spontaneous abortion (RSA) and polycystic ovary syndrome.^14,15 However, studies on Treg and Th17 cells in patients with UI are limited.

Currently, pre-pregnancy care and pregnancy-related counseling have garnered attention.^16,17 Providing an early diagnosis for patients with pregnancy disorders is the basis for subsequent active treatment. Recent studies have detected circulating immune cell subsets for the diagnosis of pregnancy disorders, including RSA and repeated implantation failures (RIF).^18,19 The aim of this study was to investigate the percentage and absolute count of Treg and Th17 cells in the peripheral blood of women with UI, as well as the Th17/Treg ratio. Additionally, we explored potential valuable diagnostic biomarkers for patients with UI and ascertained whether Treg and Th17 cells are associated with primary and secondary UI.

Materials and Methods

Study Population

All participants were enrolled from the Reproductive Center of the Affiliated Dongyang Hospital of Wenzhou Medical University between July 2021 and October 2023. The inclusion criteria for the UI group were as follows: (1) age between 21 and 40 years; (2) failure to achieve clinical pregnancy after unprotected sexual intercourse for more than one year; (3) regular menstrual cycle and normal levels of serum anti-Müllerian, luteinizing, and follicle stimulating hormones, as well as progesterone levels and thyroid function; (4) normal results of uterine ultrasound and hysterosalpingography; and (5) normal male partner’s semen analysis. The exclusion criteria were as follows: (1) clear infection, (2) assisted reproductive therapy within three months, and (3) other known diseases that may affect fertility. According to the clinical pregnancy conditions, UI is divided into primary and secondary UI. Primary UI refers to women who have never been diagnosed with a clinical pregnancy and meet the UI criteria, whereas secondary UI refers to women who have previously been diagnosed with a clinical pregnancy but are currently unable to establish a clinical pregnancy that meets the UI criteria.²⁰ The study also included healthy women who have given birth, as controls. The inclusion criteria for the control group were as follows: (1) age range from 21 to 40 years old; (2) given birth to one or more live babies within two years; (3) more than six months after the last delivery and in non-lactation period; and (4) no history of adverse pregnancy.

This study has been performed in accordance with the principles stated in the Declaration of Helsinki. This study was reviewed and approved by the Ethics Committee of the Affiliated Dongyang Hospital of Wenzhou Medical University. All participants provided written informed consent.

Sample Collection and Flow Cytometry

Fresh peripheral whole blood samples were collected from participants during the mid-luteal phase of their menstrual cycle. Using a ten color flow cytometer (Navios; Beckman Coulter), two staining plates were designed for monoclonal fluorescent antibody labeling of Treg and Th17 cells (Supplementary Table 1). The Kaluza software (version 2.0, Beckman Coulter) was used to analyze the dataset and obtain the proportion of Treg and Th17 cells to CD4⁺T cells as well as the proportion of CD4⁺T to total lymphocytes. Treg cells are defined as CD4⁺CD25⁺CD127^low/- lymphocytes, whereas Th17 cells are defined as CD4⁺CD45RO⁺CXCR5⁻CCR6⁺CXCR3⁻ lymphocytes.^21,22 The gate control strategy is shown in Supplementary Figure 1.

The total lymphocyte count was measured using a blood analyzer (XN-9000; SYSMEX, Japan), and the absolute count of Treg and Th17 cells was calculated based on the proportion.

Statistical Analysis

The STATA (version 14.0) and R (version 4.1.0) software were used for statistical analysis. The Shapiro–Wilk test was used for normality testing of continuous variables. The t-test and Wilcoxon rank-sum test were used to compare the intergroup differences in normally and non-normally distributed variables, respectively. The area under the receiver operating characteristic (ROC) curve (AUC) with a 95% confidence interval (CI) was used to assess the diagnostic performance. An AUC of > 0.800 indicated good diagnostic performance. Statistical significance was set at P-value < 0.05.

Results

This study included 37 patients with UI (30 with primary UI and 7 with secondary UI) and 26 age-matched healthy volunteers as the control group. The demographic and clinical data of the study population are shown in Supplemental Table 2.

Comparison Between the Control and UI Groups

Compared with that of the control group, the percentage of Treg in the UI group was significantly low (8.4 ± 1.8 vs 7.1 ± 1.4, P<0.01, Figure 1A), while there was no significant difference in the absolute count of Treg in the UI group (P>0.05, Figure 1B). However, the percentage and absolute count of Th17 cells (6.9 (5.3–7.5) vs 7.4 (6.1–8.8), P<0.05; 49.9 (34.8–58.8) vs 58.3 (46.5–72.4), P<0.05; Figure 1C and D, respectively), as well as the Th17/Treg ratio (0.86 (0.58–0.92) vs 1.08 (0.93–1.25), P<0.001, Figure 1E), were significantly higher than those in the control group.

Figure 1 Comparison of the percentage and absolute count of Treg and Th17 cells and the Th17/Treg ratio between the unexplained infertility (UI) group and the control group. ns, P > 0.05, * P < 0.05, **P < 0.01, ***P < 0.001. (A) Comparison of the percentage of Treg cells (P < 0.01). (B) Comparison of the absolute count of Treg cells (P > 0.05). (C) Comparison of the percentage of Th17 cells (P < 0.05). (D) Comparison of the absolute count of Th17 cells (P < 0.05). (E) Comparison of the ratio of Th17/Treg cells (P < 0.001).

Supplementary Figure 2 shows the typical performance of Treg and Th17 cells in flow cytometry analysis between the UI and control groups.

Diagnostic Value of Treg Cell Percentage, Th17 Cell Percentage, and Th17/Treg Ratio as Biomarkers

To identify potential biomarkers for UI diagnosis, ROC curves were used to evaluate the diagnostic value of Tregs percentage, Th17 cell percentage, absolute count of Th17 cells, and the Th17/Treg ratio, which showed significant differences between the UI and control groups (Figure 2). The results showed that the AUCs (95% CI) for Tregs, Th17 cell percentage, absolute count of Th17 cells, and Th17/Treg ratio were 0.717 (95% CI=0.596–0.848), 0.648 (95% CI=0.513–0.782), 0.681 (95% CI=0.550–0.812), and 0.813 (95% CI=0.709–0.917), respectively. The Th17/Treg ratio demonstrated remarkable diagnostic value and was found to be superior to the use of Treg and Th17 cell percentages or absolute counts of Th17 cells alone in distinguishing patients with UI from the control group.

Figure 2 ROC curve used to diagnose unexplained infertility using the percentage of Treg and Th17 cells, the absolute count of Th17 cells, and the Th17/Treg ratio. Abbreviation: ROC, receiver operating characteristic.

Comparison Between Primary and Secondary Infertilities

As shown in Figure 3, there was no significant difference between the percentages or absolute counts of Treg and Th17 cells, or Th17/Treg ratios in the peripheral blood of women with primary and secondary UI.

Figure 3 Comparison among the percentages and absolute counts of Treg and Th17 cells and the Th17/Treg ratios for primary and secondary unexplained infertility. (A) Comparison of the percentages of Treg cells; (B) Comparison of the absolute counts of Treg cells; (C) Comparison of the percentages of Th17 cells; (D) Comparison of the absolute counts of Th17 cells; (E) Comparison of the ratios of Th17/Treg cells.

Discussion

The establishment of pregnancy is a challenge for the maternal immune system, as it must retain the ability to resist pathogen immune responses and improve immune tolerance to help the fetus evade immune attacks.²³ This research explored the distribution percentages and absolute counts of Treg and Th17 cells in the peripheral blood of patients with UI from the perspective of peripheral circulatory immunity and reported, for the first time, changes in the Th17/Treg ratio in patients with UI.

Treg account for approximately 5–10% of peripheral CD4⁺T cells,²⁴ and primarily regulate in vivo immune responses, which are crucial for establishing maternal immune tolerance. Luo et al²⁵ observed a decrease in the percentage of Treg in the peripheral blood of patients with unexplained RSA, and their immune regulatory deficiency appeared to be mainly related to Treg impairment. Cai et al¹⁹ observed a significant reduction in Treg levels in the peripheral blood of patients with RIF. Previously, impairment of the Treg phenotype in the endometrial tissue was reported to affect female fertility in patients with UI.²⁶ In this study, flow cytometry was used to determine a reduction in the percentage of Treg in the peripheral blood of patients with UI; however, it was not related to secondary or primary UI.

Th17 cells are a subgroup of CD4⁺ T cells that mediate inflammatory responses and play an important role in acute and chronic allograft rejection reactions.^27–29 The inability of the mother to establish pregnancy may be caused by immune rejection of the fetus by semi-allogeneic antigens, where Th17 cells play an important role. Recent studies have shown that the percentage of Th17 cells increases in patients with unexplained RSA, which may be related to its pathogenesis.^30,31 In addition, Ghaebi et al³² detected a higher proportion of Th17 cells in women with RIF. The findings of the current study suggest a relationship between Th17 cells and UI and that the increased percentage and absolute count of Th17 cells may affect pregnancy outcomes of patients with UI. However, no significant differences were found in the distribution of Th17 cells between women with primary and secondary UI.

Both Treg and Th17 cells exhibit synergistic effects and mutual constraints.³³ The imbalanced distribution of Treg and Th17 cells, leading to an increase in the Th17/Treg ratio, is believed to cause various pregnancy-related diseases, including RSA, intrahepatic cholestasis of pregnancy, and preeclampsia.^34–36 To the best of our knowledge, this study is the first to investigate the Th17/Treg ratio in patients with UI and find that the Th17/Treg ratio in the UI group shifted toward a direction favorable for Th17 cells, disrupting the balance between Treg and Th17 cells. Moreover, as a diagnostic biomarker for UI, the Th17/Treg ratio was found to be superior to the use of Treg or Th17 cells alone.

In the past, research samples for unexplained pregnancy disorders were diverse and included endometrial and menstrual blood.^37,38 However, owing to its non-invasive sampling procedure and non-easily contaminated nature, peripheral blood samples have attracted increasing interest from researchers. In this study, we found that the peripheral blood Th17/Treg ratio has potential applicability in the diagnosis of UI; however, there are certain limitations. First, the patients participating in the study were from a single center and patient numbers were limited. Therefore, future multicenter studies with relatively large sample sizes are required to confirm this hypothesis. Second, combining subsequent treatment plans was difficult, thereby complicating follow up on outcomes. Moreover, the limitations of current diagnostic methods in detecting early adenomyosis and endometriosis make it difficult to rule out their potential causes of infertility. Finally, the study lacked an analysis of cellular functions, such as cytokine secretion, which constitutes scope for further research.

Conclusion

The distribution of Treg and Th17 cells is unbalanced in patients with UI. The percentage of Treg decreased significantly, whereas the percentage and absolute count of Th17 cells increased. Moreover, the Th17/Treg ratio in patients with UI increased significantly. Moreover, the Th17/Treg ratio may be a promising diagnostic biomarker of UI. However, the distribution of Treg and Th17 cells did not differ significantly between women with primary and secondary UI.