Altered eutopic endometrial T-regulatory and T-helper 17 lymphocyte ratio in women with unexplained subfertility

We first compared the profiles in patients with UI to those with RPL and observed no differences in age, BMI, normalized Treg and Th17 lymphocyte counts, Treg to Th17 lymphocyte ratio and IL-17 H-scores, irrespective of conception outcome ( Table 1 ). Hence, for all further comparisons as depicted in Figure 1 , patients with UI and RPL were grouped together as subfertile patients. Subfertile patients were then divided according to their conception status in subsequent cycles. Subfertile women who conceived successfully had a normalized Treg lymphocyte count similar to subfertile women who did not conceive ( p = 0.502; Table 2 ; Figure 3(a) ). Interestingly, subfertile women who did not conceive had a higher ( p = 0.002; Table 2 ; Figure 3(b) ) normalized Th17 lymphocyte count as compared to those who did conceive in subsequent cycles. A majority (65%) of the subfertile patients included in this study also had concurrent endometriosis, confirmed and scored mild to moderate disease via laparoscopic evaluation. In these subfertile patients with concurrent endometriosis, we observed that women who did not conceive had higher normalized Th17 lymphocyte counts ( p = 0.025; Table 3 ; Figure 4(b) ) than women who conceived. The normalized Treg lymphocyte counts were observed to be similar between the two groups of subfertile women with endometriosis ( p = 0.551; Table 3 ; Figure 4(a) ). We next evaluated the ratio between immune-tolerant Treg lymphocytes and pro-inflammatory Th17 lymphocytes for each patient’s sample. Subfertile patients who had a higher ( p = 0.004; Table 2 ; Figure 3(c) ) Treg:Th17 ratio conceived successfully in subsequent cycles, whereas patients who had a low Treg:Th17 ratio did not. Interestingly, the Treg:Th17 cell ratio was also a significant indicator ( p = 0.037; Table 3 ; Figure 4(c) ) of successful conception in patients with concurrent endometriosis. This assessment was noteworthy because it was consistently observed that a patient with a low Treg:Th17 ratio was less likely to conceive in subsequent cycles, irrespective of ART support and/or laparoscopic removal of endometriotic lesions. Interleukin-17 expression was not indicative of conception failure within the subfertile group ( p = 0.422; Table 2 ; Figure 3(d) ), irrespective of concurrent endometriosis ( p = 0.888; Table 3 ; Figure 4(d) ). Interestingly, we observed that patients with a low IL-17 H-Score had a high Treg:Th17 ratio and vice versa (data not shown), and this was observed to be consistent in both the glandular as well as the stromal compartments of eutopic endometrium.

All samples were taken with informed consent and in accordance with Institutional Review Board approved protocol at Greenville Health System (IRB00013885). Eutopic endometrial biopsy samples were collected from women with unexplained infertility ( n = 42) or recurrent pregnancy loss ( n = 22), as the primary complaint, and comparisons for T lymphocytes and IL-17 were conducted, as depicted in Figure 1 , based on conception status and concurrent presence of endometriotic lesions. Endometrial biopsy was obtained via pipelle collection during mid-secretory phase of the menstrual cycle (as judged by LH surge). Conception data included in these analyzes refer to conception (successful or not with assisted reproductive technology [ART] support) in the cycles subsequent to the endometrial biopsy collection. Presence of endometriotic lesions was confirmed in 43 of the 64 subfertile patients, via laparoscopy conducted after collection of the eutopic endometrial biopsy. Hence a further comparison was conducted to evaluate the potential correlation of concurrent endometriosis with the eutopic endometrial immune profiles and conception success with ART support after removal of endometriotic lesions. The remaining 21 patients did not undergo laparoscopic evaluation and hence were not included in this subset evaluated. Patients over the age of 40 years and/or with other underlying conditions for example polycystic ovarian syndrome or myasthenia gravis, were excluded from the study. Immunohistochemistry was performed on formalin fixed paraffin embedded eutopic endometrial tissue sections to detect FoxP3 (Treg transcription factor), RORγt (Th17 transcription factor), and IL-17 (Th17 specific cytokine). After deparaffinization and rehydration of sections, antigen retrieval was performed in 1X DAKO Target Retrieval solution (pH 9.0) (S2367; DAKO) at 97°C–99°C for 20 min. Endogenous peroxidase activity was quenched (for IL-17 only) in 0.3% hydrogen peroxide solution in methanol for 15 min at room temperature (RT). Blocking in 5% horse serum (for FoxP3), 5% rabbit serum (for RORγt), or 5% goat serum (for IL-17) in 1% bovine serum albumin (BSA) in phosphate buffered saline with Tween 20 (PBST) at RT for 30 min was followed by incubation in 10 μg/mL of mouse anti-human FoxP3 (236A/E7; ab20034; Abcam), 5 μg/mL of rat anti-human RORγt (AFKJS-9; 14–6988; eBioscience), or 2 μg/mL of rabbit anti-human IL-17 (H-132; sc-7927; Abcam) in 1% BSA/PBST at 4°C overnight. Sections were washed three times for 5 min each with PBST and then incubated with biotinylated horse anti-mouse IgG (Vectastain Elite ABC Kit Mouse IgG; Vector Labs), biotinylated rabbit anti-rat IgG (Vectastain Elite ABC Kit Rat IgG; Vector Labs) or biotinylated goat anti-rabbit IgG (Vectastain Elite ABC Kit Rabbit IgG; Vector Labs) diluted 1:200 with 1% BSA/PBST for 45 min at RT for FoxP3, RORγt and IL-17, respectively. Indirect detection was performed by incubation of sections with avidin-biotinylated peroxidase complex (Vectastain Elite ABC Kit), reacted with 0.6 mg/mL 3, 3'-Diaminobenzidine (DAB; Sigma) in Tris-HCl buffer and counterstained with Mayer’s hematoxylin nuclear stain. Negative control sections were treated with nonspecific IgG at similar concentration for each primary antibody. Slides with stained tissue sections were scanned using NanoZoomer 2.0HT (Hamamatsu) and digital images used for quantification of Treg and Th17 lymphocytes, as well as IL-17 expression, using NanoZoomer Digital Pathology (NDP.view) software. Quantification was done while blinded to the conception status of the patients. For T lymphocyte quantification and analysis, the area of the tissue section was measured in NDP. view, the number of individual cells positive for FoxP3 ( Figure 2(a) and (b) ) and RORγt ( Figure 2(c) and (d) ) were counted in the respectively stained tissue sections and normalized to cells per 10 mm 2 area of tissue section for each patient. This allowed for unbiased comparisons of Treg and Th17 lymphocyte numbers between patients. IL-17 immunostaining was analyzed semiquantitatively using H-score to evaluate mean staining intensity (MSI) in five fields each for glandular epithelium and stroma ( Figure 2(e) and (f) ) and allocated a score relative to positive and negative controls where 0 represented no stain, 1 represented faint, 2 represented moderate and 3 represented strong intensity. Percentage of cells at each MSI were assessed for each field, an average was calculated for each MSI score and used in the equation (1*Avg. MSI) + (2*Avg. MSI) + (3*Avg. MSI). A final score between 0 and 300 was assigned to each sample wherein 300 was equal to 100% cells stained strongly. The results obtained for Treg and Th17 lymphocytes as well as IL-17 immunostaining were then compared to the conception status for each patient to understand potential correlations. Statistical analyses were conducted using GraphPad Prism 8.4.3 (San Diego, CA) and results presented as mean ± SEM. Numerical data for age, body mass index (BMI), Treg and Th17 lymphocyte counts, Treg:Th17 ratio and IL-17 H-score were evaluated using non-parametric Mann-Whitney t -test. A value of p ⩽ 0.05 was considered statistically significant.

Aberrant immune responses are linked with occurrence of unexplained subfertility, 33 and atypical T-lymphocyte subpopulations are considered at least partly responsible for implantation failure. T-regulatory cells are known to accumulate in the normal endometrium during mid- to late-secretory phase and are considered essential for uterine receptivity. 21 On the other hand, Th17 lymphocytes play a critical role in autoimmunity, and thus may hinder the mechanisms mediating maternal tolerance toward the conceptus and impair maintenance of pregnancy. The purpose of this study was to examine intrauterine inflammation observed in subfertile women with UI or RPL, with a focus on T-helper lymphocyte subpopulations in the eutopic endometrium. Our goal was to evaluate the absolute and relative numbers of Treg and Th17 lymphocytes in the eutopic endometrium of subfertile women, during the mid-secretory phase of their menstrual cycles and elucidate an association of these immune profiles with the patient’s conception status and concurrent presence of endometriosis. Previous research has shown that peripheral blood 20 , 22 and decidual 20 CD4 + CD25 + Treg lymphocyte populations were highest during the first trimester of pregnancy. In fact, the CD4 + CD25 + Foxp3 + Treg cell population was enriched in the early normal pregnancy decidua as compared to the peripheral blood of the same pregnant woman. 23 , 24 This phenotype of increased Treg lymphocyte population is hence thought to contribute to the immunotolerant environment in the endometrium for the fetal semi-allogeneic cells. Its dysregulation in reproductive pathologies is often thought to contribute to the associated infertility, as seen in women with unexplained recurrent spontaneous abortion 29 , 34 and RPL. 28 Though Treg numbers were not indicative of successful conception in this study, we observed that patients with a higher pro-inflammatory Th17 lymphocyte count in their endometrium had a significantly lower chance of conceiving in subsequent cycles. This is supported by studies that have shown an increase in pro-inflammatory Th17 lymphocyte numbers in the peripheral circulation of patients with unexplained recurrent spontaneous abortion 29 and recurrent miscarriage. 30 A similar increase in Th17 lymphocytes was also reported in the decidua of patients with RPL 35 and recurrent spontaneous abortion. 30 , 35 Hence, a fine balance between these two cell types is crucial to maintain peripheral and local immune equilibrium. Peripheral circulation levels of Th17 to Treg cell ratios are increased in patients with RPL 28 and unexplained recurrent spontaneous abortion 29 as compared to normal fertile controls. Though the decidual populations of both T-cell types are inversely correlated in recurrent spontaneous abortion cases, 25 nothing specific has been studied with regards to this ratio and its correlation to unexplained subfertility, as it relates to the presence of endometriosis. Though peritoneal environment characterization studies have presented varying results, the overall hypothesis is that a defective T-cell response is associated with endometriosis establishment. Where one study reported a decrease in CD4 + Treg cells in stage I and II of the disease, 36 another has shown an increase in CD4 + CD25 high Foxp3 + cells 37 in the peritoneal cavity of women with endometriosis. Moreover, increased Th17 cells in the peritoneal fluid have been associated with more severe endometriotic lesions. 38 However very few studies have focused on the effect of endometriosis on the eutopic endometrial T-cell population. Though it was shown that total CD4 + T-cell numbers in the eutopic endometrium were lower than those seen in the ectopic lesions, 39 the T-cell subsets were not individually identified nor were they compared to the fertility status, as we have done in this study. Le et al. have recently reported an increased accumulation of Th17 cells and reduction in Treg cells in the peripheral circulation and ectopic endometrial tissue in patients with endometriosis, that seemed to be mitigated by hormone therapy but not by surgical removal of ectopic lesions. 40 All these previous studies support our findings that the low Treg and concurrent high Th17 cell phenotype is potentially responsible for the unsuccessful conception status in the subfertile patients with concurrent endometriosis, even after laparoscopic removal of ectopic lesions. Interleukin-17 (IL-17) is significantly higher in the peritoneal fluid of patients with minimal/mild stage endometriosis as compared to those with moderate/severe stage of disease. 41 , 42 Other studies have also shown abnormal serum 40 and peritoneal fluid 42 T-cell related cytokine levels in patients with endometriosis. IL-17 acts through the induction and expression of other pro-inflammatory cytokines and chemokines, 37 which mediate immune cell infiltration, angiogenesis and maintenance of endometriotic lesions. In this study we did not observe statistically significant differences in IL-17 expression profiles between the subfertile patient groups compared, but we did consistently note that patients with a low Treg:Th17 cell ratio had a higher endometrial IL-17 expression, and did not conceive in subsequent cycles regardless of ART support and removal of ectopic lesions. Extensive reviews 15 , 16 , 43 have presented the inflammatory nature of endometriosis elucidating among other factors the altered humoral and cell-mediated immune phenotypes that possibly alter eutopic endometrial characteristics during the crucial implantation window and thus affect endometrial receptivity in women with endometriosis. Even so, it has been noted that not all women with endometriosis suffer from the same state of subfertility, as we have shown with our results too. Moreover, studies have shown that removal of endometriotic lesions can benefit some women by altering their subsequent fertility status 7 , 44 by inducing a rapid change in the local and systemic inflammatory milieu but this change seems to be transient. 27 Overall, our experiments characterizing the eutopic endometrial T-lymphocyte populations in specific groups of patients have yielded promising results that support our hypothesis that an immune tolerant T-lymphocyte phenotype (high Treg and low Th17) in the eutopic endometrium is associated with a positive pregnancy outcome, whereas a pro-inflammatory T-lymphocyte phenotype (high Th17 and low Treg) in the eutopic endometrium is associated with subfertility and possible pregnancy loss, irrespective of concurrent endometriosis. This study has its limitations in that a control group of patients with normal fertility status and confirmed lack of endometriosis could not be included. Moreover, an endometrial biopsy taken post-laparoscopic removal of ectopic lesions could have helped discern a possible change in the immune profiles. But our data still strongly suggests that the fertility status of a woman with endometriosis potentially depends on the degree of inflammation in the eutopic endometrium during the window of implantation. Thus, supporting the use of the ratio of Treg:Th17 cell populations in the endometrium as a novel and reliable marker of uterine receptivity and potential predictor of successful conception in subfertile patients who are undergoing assisted reproduction treatments.

Subfertility is defined as the inability to conceive for over a year in the presence of apparently normal ovulation and sperm parameters, and patent fallopian tubes 1 , 2 and affects about 10%–15% of individuals in general. 1 Currently various less obvious etiologies are accepted for unexplained subfertility in women, such as ovulatory disorders, peritoneal adhesions, and uterine abnormalities, which would not be investigated as part of the routine fertility assessment, 3 , 4 and hence this condition is usually a diagnosis of exclusion. However, findings of concurrent disease conditions such as endometriosis are commonly associated with unexplained subfertility. 5 – 8 Endometriosis, a benign but highly inflammatory gynecological condition commonly encountered in women of reproductive age, is an estrogen dependent disorder characterized by the presence of endometrial cells outside the uterus, primarily on pelvic and/or abdominal organs 9 and in some cases in extra-pelvic regions. 10 Chronic pelvic pain, dysmenorrhea with or without abnormal bleeding are common symptoms of this condition. 11 , 12 Endometriosis accounts for 30%–50% of infertility 13 and may be present in up to 60 to 80% of unexplained infertility (UI) cases 6 – 8 in women. Endometriosis related infertility has numerous causes such as intraperitoneal inflammation, unovulated luteinized follicles and an increase in recurrent pregnancy loss (RPL). 14 – 16 Based on their inflammatory marker data, Fox et al. 5 have proposed that continued inflammation that occurs in women with endometriosis might account for the progression from RPL to infertility, over time. During the normal mid- to late-secretory phase of the menstrual cycle, specific populations of immune cells increase at the time of implantation to maintain a local immunotolerant environment in the uterus for the semi-allogeneic embryo. 17 Among these are the T-helper lymphocytes, which account for a significant 5%–10% of the endometrial leukocyte population. T-regulatory (Treg) cells are critical for localized immunotolerance at the implantation site and are seen to increase in the peripheral circulation 18 , 19 as well as the decidua 20 , 21 during early pregnancy, displaying a stable and highly tolerant phenotype. Hence, Treg lymphocytes are believed to be an essential factor for embryo tolerance, invasion and successful establishment of pregnancy. 22 , 23 However, it is proposed that the balance between Treg lymphocytes and their reciprocal cell types should be considered when evaluating local immune status, especially since the presence of Treg lymphocytes and their reciprocal pro-inflammatory subset known as T-helper 17 (Th17) lymphocytes are inversely related to each other in the decidua. 21 Chen et al. 24 reported that decreased ratios of serum Treg:Th17-related-cytokines may play a role in the pathogenesis of defects resulting in implantation failure, which was supported by Wang et al. 25 who detected significantly elevated Interleukin 17 (IL-17) cytokine levels in both peripheral blood and decidua of patients with unexplained recurrent abortion. Tayade and colleagues also demonstrated elevated peripheral and local IL-17, associated specifically with endometriosis. 26 , 27 Women with unexplained RPL have reduced peripheral levels of CD4 + CD25 + Tregs and significantly increased levels of Th17 lymphocytes, as well as increased ratios of Th17:Treg lymphocytes 28 , 29 when compared to normal fertile controls. Similar phenotypes have been reported in the decidual tissues of such patients. 30 , 31 Decreases in the endometrial Treg phenotype have also been implicated as a cause for unexplained implantation failure. 32 This accumulated evidence supports our hypothesis that the absolute numbers as well as relative proportions of T-lymphocytes found in the secretory stage endometrium may be a major determining factor for uterine receptivity. To study whether such a relationship exists we evaluated the Treg and Th17 lymphocyte profiles in the eutopic endometrium of subfertile women (UI or RPL as primary complaint), during the mid-secretory phase of the menstrual cycle. We also assessed the correlation between these immune cell profiles and proinflammatory cytokine IL-17 with the patients’ ability to conceive in subsequent cycles. A further comparison of these immune profiles was conducted in a subset of these subfertile patients who were confirmed to have endometriosis via laparoscopic evaluation, in an attempt to understand the potential role of endometriosis in the subfertile patient’s inability to conceive.