Background
Embryo implantation into the uterus is a critical step in the establishment of pregnancy and failure of this process is a major cause of infertility in women [
1].
Endometrium is receptive to the embryo for a short time-period, after exposure to 17-β-estradiol followed by progesterone [
2]. Embryo transfer studies in women and primates have identified the phase of uterine receptivity, the 'window of implantation', between post-ovulatory days 5–10 [
3] following the luteinizing hormone (LH) surge. Implantation of the embryo between post-ovulatory days 8–10 has a high chance of resulting in a successful pregnancy [
4]. Unexplained infertility accounts for approximately 30% of all infertility [
5,
6]. Defective uterine receptivity is thought to be a primary cause of unexplained infertility. Unexplained or primary infertility is likely due to multiple defects as it has been associated with numerous molecular and cellular disturbances in the endometrium [
7,
8].
Interleukin-11 (IL-11) and LIF belong to the interleukin-6 (IL-6) family of cytokines whose members exhibit pleiotropy and redundancy, with many overlapping functions [
9]. IL-11 binds to IL-11 receptor (IL-11 Rα) and initiates signalling whilst LIF signals by binding to the specific LIF receptor α (LIFR). Each ligand-receptor complex forms a hetero-dimer with gp130, the common transmembrane signal transducer. In the endometrium, intracellular signalling occurs via activation of the janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway [
10‐
12]. IL-11 and LIF are part of an exclusive group of genes that are essential for implantation in mice [
13]. Both endometrial IL-11 and LIF are obligatory for implantation in mice [
14‐
16]. STAT3 also has an important role in implantation in mice [
10,
17]. Blockade of phosphorylated (p) STAT3 at the time of embryo attachment reduces implantation rates in mice [
18,
19].
Although the role of LIF in human endometrial receptivity has been previously studied the results are conflicting [
8,
20]. There are very few studies examining the role of IL-11 and no studies for pSTAT3 in infertility in women [
21]. IL-11, LIF and pSTAT3 are expressed by endometrial, glandular and luminal epithelium in the mid-secretory phase of the menstrual cycle in women [
22‐
25]. Similarly, mid-secretory phase luminal and glandular epithelium express gp130, LIFRα and IL-11Rα [
22,
24,
26,
27]. Immunoreactive LIF is reduced in endometrial biopsies from infertile women [
28], while uterine flushings contain maximum levels of LIF protein during the mid to late secretory phase of the menstrual cycle [
29‐
31]. In addition, LIF is reduced in uterine flushings from women with primary infertility compared to fertile controls [
29‐
31]. Similarly, IL-11 is present in uterine fluid [
32]. These studies suggest IL-11 and LIF are secreted by the uterine glandular epithelium into the uterine lumen where they could act on the blastocyst or the endometrial luminal epithelium to facilitate blastocyst attachment and implantation.
In addition, in some women with endometriosis associated infertility, IL-11 and LIF immunoreactivity are reduced in glandular epithelium suggesting that both cytokines may also contribute to the pathology of infertility of unknown cause [
12]. While, IL-11 and LIF expression is spacially and temporally distinct in mice, they are both present in glandular epithelium and utilize similar signal transduction pathways at the time of implantation in women. It is important to determine whether they potentially act in a redundant manner. In a previous study, while LIF production was not different between women with unexplained infertility and normal fertile women, there was reduced secretion of soluble gp130 from endometrial explants. This indicates it is important to also examine IL-11 and LIF's signalling components together with the production of the ligands. pSTAT3 is of particular interest since it has an important role in implantation in mice. There are no studies examining the production of IL-11 and pSTAT3 in endometrium of women with primary unexplained infertility.
We hypothesized that IL-11, IL-11Rα, LIF and pSTAT3 are dysregulated in endometrium from women with unexplained infertility during the 'window of implantation'. Endometrial protein abundance for IL-11, pSTAT3, IL-11Rα and LIF was compared in women with primary unexplained infertility and normal fertile women.
Discussion
We demonstrated for the first time that IL-11 and pSTAT3 immunoreactivity in uterine glandular epithelium was decreased in women with unexplained infertility.
We observed a significant decrease in IL-11 and pSTAT3 in uterine glandular epithelium in some women with infertility compared to normal cycling fertile women. Unexplained infertility is likely caused by several defects therefore it is not surprising that we identified cohorts of infertile women with defective IL-11 and pSTAT3 production. IL-11 could be acting on the glandular epithelium to facilitate its secretory transformation. In addition, IL-11 has been identified in the uterine lumen of women [
32]. IL-11 present in the uterine lumen could act on the endometrial uterine epithelium to facilitate attachment or adhesion of the blastocyst.
Low glandular pSTAT3 may be due to either reduced STAT3 protein production and/or factors that stimulate STAT3 phosphorylation. IL-11 and LIF are known to signal via STAT3 in endometrium [
10,
25]. Interestingly, glandular epithelium from women with infertility that had low IL-11 staining did not consistently have low pSTAT3 staining. LIF protein levels did not correlate with pSTAT3 levels in glandular epithelium. This suggests factors other than these cytokines regulate STAT3 protein activation. It is also plausible that IL-11 and LIF stimulate pSTAT3 in glandular epithelium but the levels of STAT3 protein may be low in glandular epithelium of some infertile women. Numerous factors including progestins stimulate pSTAT3 and STAT3 [
9,
34]. The regulation of pSTAT3 and STAT3 in the uterine glandular epithelium remain to be elucidated.
Minimal pSTAT3 staining was found in eight of the ten infertile women. If the levels of pSTAT3 in some women was due to reduced STAT3 protein abundance this could result in inadequate LIF and IL-11 action. While LIF was not significantly reduced in the endometrial glands of women with infertility, LIF action could be impaired in women with low glandular pSTAT3. Similarly, some women had moderate to high IL-11 and low pSTAT3 in the glands. This could similarly result in abnormal IL-11 action. Further studies are required to determine the mechanisms of action of IL-11 and LIF in endometrial epithelium. In addition, LIF levels did not differ in uterine glandular epithelium between infertile and fertile women indicating IL-11 and LIF may have different roles in women with unexplained infertility.
In contrast to the present study IL-11, IL-11Rα and LIF staining were decreased in glandular epithelial cells in women with endometriosis associated infertility [
12]. This suggests the endometrial phenotype of women with primary infertility differs from that of women with endometriosis associated infertility. It is likely that different therapeutic strategies may be necessary to treat endometrial implantation failure depending upon aetiology.
IL-11, pSTAT3, IL-11Rα and LIF staining was overall low and patchy or absent in luminal epithelium of both infertile and fertile women. Few tissues contained luminal epithelium making it difficult to analyze the immunostaining in this cellular compartment.
IL-11 like LIF is present in the uterine secretome indicating both could act on the blastocyst or the surface luminal epithelium. The human blastocyst expresses gp130 and LIFRα [
35] but whether it also expresses IL-11Rα is not known. Although the function of IL-11 and pSTAT3 in endometrial glands needs to be elucidated, unexplained infertility in some women may be due to reduced levels of either IL-11 or pSTAT3.
Competing interests
The authors declare that they have no competing interests that would prejudice the impartiality of this scientific work.
Authors' contributions
All authors have read and approved the manuscript. ED designed the studies, performed immunohistochemistry for pSTAT3, analysed the data and drafted the manuscript. AS participated in designing the studies and collection of tissues. Y-LT performed immunohistochemistry for IL-11, IL-11Ralpha and LIF and participated in analysing the data. LAS participated in the design of the studies. JRAS participated in design of the studies, recruitment of subjects and collection of tissues.