DISSECTING LOCAL AND SYSTEMIC INSULIN SIGNALING: IMPACTS ON THE FEMALE REPRODUCTIVE TRACT AND IMMUNE RESPONSE

Jenna C Douglas

Insulin signaling in the uterus is essential for establishing and maintaining pregnancy, by modulating metabolism, steroid hormones, and immunity. These processes critical to oocyte maturation, embryo implantation, endometrial decidualization, and placentation. Metabolic disorders affect approximately 11% of reproductive-aged women. Diagnoses are increasingly common in younger women, thereby affecting their lifelong reproductive health. Defective insulin signaling in pregnant women can lead to spontaneous abortion, miscarriage, and infertility. Insulin receptors (IRs), including insulin receptor (Insr) and insulin-like growth factor 1 receptor (Igf1r), are key tyrosine kinase transmembrane receptors that activate downstream pathways vital for cell differentiation, transcription, and polarity.To investigate insulin signaling, we use two methods: local ablation of insulin receptors in uterine endometrial stroma and ovarian granulosa cells (GCs), and systemic dysregulation induced by streptozotocin (STZ) injection to induce diabetes. The local ablation uses the Cre/LoxP system to target tissue-specific ablation of Insr and/or Igf1r in granulosa cells and endometrial stromal cells via the anti-Müllerian hormone receptor 2 (Amhr2) Cre and Forkhead box L2 (Foxl2) Cre. INSR and IGF1R ablation occurs in target tissues, while maintaining intact systemic insulin signaling. The STZ-induced diabetes model is used to evaluate local changes in the female reproductive tract with intact IR induced by systemic hyperglycemia. Ovarian function is unaltered in conditional knockouts (cKOs) driven by Amhr2-Cre and Foxl2-Cre mice. INSR and IGF1R granulosa cell receptor ablation in secondary and antral follicles is minimal in double-knockout (DKO) mice. Minimal receptor ablation resulted in normal hormone cycling, as assessed by estrous cycles and ovulatory capacity. DKO mice, however, showed increased GC apoptosis in secondary and antral follicles. STZ-induced diabetes results in increased expression of steroidogenic enzymes and potential halting of folliculogenesis. Thus, systemic insulin dysregulation has substantial effects on ovarian function. Local disruption of IRs in Igf1rd/d; Amhr2-Cre, Igf1rd/d; Foxl2-Cre, and DKOs results in subfertility. These mice exhibit normal uterine receptivity; however, implantation site spacing is altered at gestational day 5.5. This altered spacing is due to reduced prostaglandin synthesis, endometrial proliferation, and compromised decidualization. Systemic dysregulation impacts embryo implantation, as evidenced by reduced proliferation in endometrial stromal cells, and by avascularization in the uterine horns. Unlike in our cKO models, systemic dysregulation upregulated both IRs and glucose transporters in uterine tissue pre- and post-implantation. Spatial transcriptomics of control and single cKOs (Insrd/d; Amhr2-Cre, Insrd/d; Foxl2-Cre, Igf1rd/d; Amhr2-Cre, and Igf1rd/d; Foxl2-Cre) using nanoString GeoMx identified pathways altered during embryo implantation. Pathways include transforming growth factor beta (TGFβ), vascularization, and cellular organization. Immortalized human endometrial stromal cells with knocked-down INSR (siINSR), IGF1R (siIGF1R), or both (siDKD) showed reduced decidualization and expression of decidualization factors. Thus, these findings suggest that insulin receptors play a major role in regulating endometrial proliferation, decidualization, and cellular structure, and that systemic insulin dysregulation and/or hyperglycemia reduce uterine receptivity. Mid-term embryo reabsorption is observed in both models, cKOs and STZ, at GD 12.5. Local IR ablation reduces sustained proliferation and decidualization of the maternal decidua, thereby impacting placental and embryonic development. Trophoblast binding protein A (TPBPA) staining of reabsorption sites indicates that embryo reabsorption begins at or prior to GD 8.5 in DKOs. Changes in innate and adaptive immune populations in uterine tissue and amniotic fluid from STZ-induced diabetic mice were analyzed with flow cytometry at GD 12.5. In STZ mice, the total uterine immune population, natural killer cells, and T cells increased, indicating a sustained pro-inflammatory state. Therefore, insulin dysregulation alters the success of pregnancy by modulating immune populations, and local dysregulation impacts maternal decidua formation. INSR and IGF1R are key regulators of endometrial function, i.e., proliferation, decidualization, and cellular composition. Dysregulation of insulin production leads to similar effects on the female reproductive tract, ultimately impairing fertility. These findings highlight the intricacies of insulin signaling, both in local tissues and through systemic dysregulation by hyperglycemia.

DISSECTING LOCAL AND SYSTEMIC INSULIN SIGNALING: IMPACTS ON THE FEMALE REPRODUCTIVE TRACT AND IMMUNE RESPONSE

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