Alterations in endometrial immune cell populations affect endometrial cell functions

Endometriosis is a debilitating disease that is diagnosed in 0.5-5% fertile and 25-40% of infertile women, but the underlying mechanisms involved in progression of the disease as well as the associated infertility are yet unclear. Research now shows evidence that the immune system has a pivotal role in endometriosis development as well as the related infertility. Among other immune cells, the T-helper lymphocytes accumulate in the normal endometrium during the mid- to late-secretory phase of the menstrual cycle and are considered essential for endometrial receptivity. The goal of this project was to 1) study the relationship between anti-inflammatory T-regulatory (Treg) and pro-inflammatory T-helper-17 (Th17) lymphocytes in the eutopic endometrium of patients with the primary complaint of infertility; 2) explore the involvement of interleukin-17 (IL-17) in the promotion of a pro-inflammatory environment and the consequences of this altered microenvironment on eutopic endometrial and ectopic endometriotic cells; and 3) characterize EMMPRIN expression in the eutopic endometrium in women with endometriosis-related infertility and its correlation to elevated IL-17 expression. Endometrial biopsy samples collected from patients during the mid- to late-secretory phase of their menstrual cycles were evaluated for Treg and Th17 lymphocyte subsets and the Th17 specific cytokine, IL-17 expression. These data were compared to the fertility status in these patients. Overall, Treg cell counts were higher and Th17 cell counts were lower in patients who conceived compared to those that did not get pregnant. Conversely, patients who maintained their infertile status had a lower Treg cell count and higher Th17 cell count in their eutopic endometrium. The ratio of Treg:Th17 cell counts was significantly correlated to their fertility status. Patients with a ratio less than 3 failed to conceive in spite of medical or surgical intervention. Laparoscopic intervention for ectopic lesion excision had a boosting effect on the endometrial Treg cell population which was in turn correlated to a positive pregnancy outcome. The IL-17 expression was elevated in both the glandular and stromal compartments of the endometrium in patients with a low Treg:high Th17 cell ratio. IL-17 is associated with various inflammatory conditions including endometriosis. IL-17 treatment did not have any effect on cell proliferation in endometrial or endometriotic cell type. But IL-17 did positively affect cell migration and invasion in the endometriotic cells, though not in the endometrial cells. To understand this differential effect we assessed IL-17 receptor (IL-17R) expression in both cell types and observed that the receptor expression was fairly similar in both cell types. Thus we concluded that the differential effect was probably due to specific interaction processes of IL-17 with different cell types or another co-receptor expressed specifically on the endometriotic cells could mediate this effect. The inflammatory NFkB pathway is the hallmark of IL-17 activity, but our results showed that in our uterine cell lines, IL-17 did not induce the NFkB pathway. Instead IL-17 activated an alternate MAPK signal transduction pathway, but only in the endometriotic 12Z cells. Because it is known that cell motility requires MMP induction, we also evaluated changes in EMMPRIN expression in these cells when treated with IL-17. Endometriotic cells showed a transient increase in EMMPRIN expression post treatment which could partially explain their enhanced motility in the presence of IL-17. We also assessed the correlation between IL-17 and EMMPRIN expression in the eutopic endometrial samples from patients with endometriosis. We found that IL-17 expression positively correlated with EMMPRIN expression in about 90% of the samples tested. Our experiments using IL-17 may reveal the mechanisms involved with the creation of a pro-inflammatory environment and its consequence on eutopic endometrial cells as well as processes involved in the establishment of ectopic endometriotic lesions. We believe this project addresses questions that will greatly increase our understanding of how a specific immune cell niche may regulate endometrial receptivity. Moreover, a better understanding of the mechanisms of Epithelial to Mesenchymal Transition will aid research in the field of endometriosis as well as cancer

Physiological mechanisms through which heat stress compromises reproduction in pigs

Seasonal variations in environmental temperatures impose added stress on domestic species bred for economically important production traits. These heat‐mediated stressors vary on a seasonal, daily, or spatial scale, and negatively impact behavior and reduce feed intake and growth rate, which inevitably lead to reduced herd productivity. The seasonal infertility observed in domestic swine is primarily characterized by depressed reproductive performance, which manifests as delayed puberty onset, reduced farrowing rates, and extended weaning‐to‐estrus intervals. Understanding the effects of heat stress at the organismal, cellular, and molecular level is a prerequisite to identifying mitigation strategies that should reduce the economic burden of compromised reproduction. In this review, we discuss the effect of heat stress on an animal\u27s ability to maintain homeostasis in multiple systems via several hypothalamic‐pituitary‐end organ axes. Additionally, we discuss our understanding of epigenetic programming and how hyperthermia experienced in utero influences industry‐relevant postnatal phenotypes. Further, we highlight the recent recognized mechanisms by which distant tissues and organs may molecularly communicate via extracellular vesicles, a potentially novel mechanism contributing to the heat‐stress response

Human Ovarian Cancer Tumor Formation in Severe Combined Immunodeficient (SCID) Pigs

Ovarian cancer (OvCa) is the most lethal gynecologic malignancy, with two-thirds of patients having late-stage disease (II-IV) at diagnosis. Improved diagnosis and therapies are needed, yet preclinical animal models for ovarian cancer research have primarily been restricted to rodents, for data on which can fail to translate to the clinic. Thus, there is currently a need for a large animal OvCa model. Therefore, we sought to determine if pigs, being more similar to humans in terms of anatomy and physiology, would be a viable preclinical animal model for OvCa. We injected human OSPC-ARK1 cells, a chemotherapy-resistant primary ovarian serous papillary carcinoma cell line, into the neck muscle and ear tissue of four severe combined immune deficient (SCID) and two non-SCID pigs housed in novel biocontainment facilities to study the ability of human OvCa cells to form tumors in a xenotransplantation model. Tumors developed in ear tissue of three SCID pigs, while two SCID pigs developed tumors in neck tissue; no tumors were detected in non-SCID control pigs. All tumor masses were confirmed microscopically as ovarian carcinomas. The carcinomas in SCID pigs were morphologically similar to the original ovarian carcinoma and had the same immunohistochemical phenotype based on expression of Claudin 3, Claudin 4, Cytokeratin 7, p16, and EMA. Confirmation that OSPC-ARK1 cells form carcinomas in SCID pigs substantiates further development of orthotopic models of OvCa in pigs

MicroRNA21 inhibition affects porcine oocyte maturation and alters protein expression critical for metabolic pathway function

MicroRNA21 (MIR21) abundance in porcine oocytes and cumulus cells increases during in vitro maturation. The mechanism by which MIR21 regulates oocyte maturation and the effect on the developmental competence of subsequent embryos remains unclear. The objective of this study was to assess the function of MIR21 during porcine oocyte maturation and its effect on embryonic development. Treatment with peptide nucleic acid MIR21 inhibitor (MIR21-PNA), designed to specifically bind to and prevent MIR21 activity during in vitro oocyte maturation, decreased cumulus cell expansion, and the oocyte ability to achieve metaphase II maturation stage when compared to control groups. Following parthenogenetic activation, the cleavage rate at 48 h in the MIR21-PNA group was decreased (p ≤ 0.03) relative to the control groups. Additionally, liquid chromatography-mass spectrometry (LC-MS/MS) of oocyte and cumulus cell total protein following MIR21-PNA treatment during in vitro maturation identified changes in signaling pathways with primary involvement of glucose metabolism (GM) pathways. Furthermore, there was no difference (p = 0.21) in oocyte maturation of control and MIR21-PNA treated oocytes when cultured in pyruvate lacking medium. Finally, MIR21-PNA treatment decreased (p = 0.04) glutathione and increased (p = 0.07) reactive oxygen species production in the oocyte. These data suggest that MIR21 influences porcine oocyte maturation by regulating GM pathways in the cumulus–oocyte complex.This article is published as Li, Yunsheng, Malavika K. Adur, Steven M. Lonergan, Aileen F. Keating, and Jason W. Ross. "MicroRNA21 inhibition affects porcine oocyte maturation and alters protein expression critical for metabolic pathway function." Molecular Reproduction and Development (2022). doi:10.1002/mrd.23641. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made

Characterization of the effects of heat stress on autophagy induction in the pig oocyte

Background: Heat stress (HS) occurs when body heat accumulation exceeds heat dissipation and is associated with swine seasonal infertility. HS contributes to compromised oocyte integrity and reduced embryo development. Autophagy is a potential mechanism for the oocyte to mitigate the detrimental effects of HS by recycling damaged cellular components. Methods: To characterize the effect of HS on autophagy in oocyte maturation, we utilized an in vitro maturation (IVM) system where oocytes underwent thermal neutral (TN) conditions throughout the entire maturation period (TN/TN), HS conditions during the first half of IVM (HS/TN), or HS conditions during the second half of IVM (TN/HS). Results: To determine the effect of HS on autophagy induction within the oocyte, we compared the relative abundance and localization of autophagy-related proteins. Heat stress treatment affected the abundance of two well described markers of autophagy induction: autophagy related gene 12 (ATG12) in complex with ATG5 and the cleaved form of microtubule-associated protein 1 light chain 3 beta (LC3B-II). The HS/TN IVM treatment increased the abundance of the ATG12-ATG5 complex and exacerbated the loss of LC3B-II in oocytes. The B-cell lymphoma 2 like 1 protein (BCL2L1) can inhibit autophagy or apoptosis through its interaction with either beclin1 (BECN1) or BCL2 associated X, apoptosis regulator (BAX), respectively. We detected colocalization of BCL2L1 with BAX but not BCL2L1 with BECN1, suggesting that apoptosis is inhibited under the HS/TN treatment but not autophagy. Interestingly, low doses of the autophagy inducer, rapamycin, increased oocyte maturation. Conclusions: Our results here suggest that HS increases autophagy induction in the oocyte during IVM, and that artificial induction of autophagy increases the maturation rate of oocytes during IVM. These data support autophagy as a potential mechanism activated in the oocyte during HS to recycle damaged cellular components and maintain developmental competence.This article is published as Hale, B.J., Li, Y., Adur, M.K. et al. Characterization of the effects of heat stress on autophagy induction in the pig oocyte. Reprod Biol Endocrinol 19, 107 (2021). doi:10.1186/s12958-021-00791-4.</p

Physiological mechanisms through which heat stress compromises reproduction in pigs

Seasonal variations in environmental temperatures impose added stress on domestic species bred for economically important production traits. These heat‐mediated stressors vary on a seasonal, daily, or spatial scale, and negatively impact behavior and reduce feed intake and growth rate, which inevitably lead to reduced herd productivity. The seasonal infertility observed in domestic swine is primarily characterized by depressed reproductive performance, which manifests as delayed puberty onset, reduced farrowing rates, and extended weaning‐to‐estrus intervals. Understanding the effects of heat stress at the organismal, cellular, and molecular level is a prerequisite to identifying mitigation strategies that should reduce the economic burden of compromised reproduction. In this review, we discuss the effect of heat stress on an animal's ability to maintain homeostasis in multiple systems via several hypothalamic‐pituitary‐end organ axes. Additionally, we discuss our understanding of epigenetic programming and how hyperthermia experienced in utero influences industry‐relevant postnatal phenotypes. Further, we highlight the recent recognized mechanisms by which distant tissues and organs may molecularly communicate via extracellular vesicles, a potentially novel mechanism contributing to the heat‐stress response.This is the peer reviewed version of the following article: Ross, Jason W., Benjamin J. Hale, Jacob T. Seibert, Matthew R. Romoser, Malavika K. Adur, Aileen F. Keating, and Lance H. Baumgard. "Physiological mechanisms through which heat stress compromises reproduction in pigs." Molecular reproduction and development 84, no. 9 (2017): 934-945, which has been published in final form at doi: 10.1002/mrd.22859. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.</p

Abstract LB-042: Successful tumor formation following xenotransplantation of primary human ovarian cancer cells into severe combined immunodeficient (SCID) pigs

Abstract Ovarian cancer (OvCa) is the most lethal gynecologic malignancy, with 2/3 of patients having late-stage disease (stages II-IV) at diagnosis. Preclinical animal models for cancer research have primarily been restricted to rodents, but many preclinical cancer drug trials that succeed in mice fail in humans, potentially due to vast differences in physiology and size. Therefore, we sought to determine if pigs, which are more similar to humans in terms of anatomy and physiology, would be a viable preclinical animal model for OvCa. Our group has pioneered the development of severe combined immunodeficiency (SCID) pigs for biomedical research, a key technological advancement for xenotransplantation of human ovarian cancer cells to form tumors. In this study, we injected human OSPC-ARK-1 cells, a chemotherapy-resistant primary ovarian serous papillary carcinoma cell line, into the neck muscle and ear tissue of four SCID and two non-SCID pigs. Tumors developed in the ears of three SCID pigs, while two SCID pigs developed tumors in neck tissue, all of which were confirmed at pathology as true neoplasms. Non-SCID animals did not develop tumors in any location. Confirmation that OSPC-ARK1 cells can form tumors in SCID pigs substantiates further development of orthotopic models of OvCa in pigs for researching improved OvCa therapeutic and diagnostic methods. Citation Format: Adeline N. Boettcher, Matti Kiupel, Malavika Adur, Emiliano Cocco, Alessandro Santin, Sara Charley, John Risinger, Christopher Tuggle, Erik Shapiro. Successful tumor formation following xenotransplantation of primary human ovarian cancer cells into severe combined immunodeficient (SCID) pigs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-042

Circulating biomarkers associated with pelvic organ prolapse risk in late gestation sows

Sow mortality, as the result of pelvic organ prolapse (POP), has been increasing in the last decade in the U.S. swine industry. The objective of this study was to identify potential biological markers associated with risk of POP in sows. We hypothesized that sows differing in perineal score (PS) from PS1–PS3 (PS1—a presumed low POP risk; PS2—a presumed moderate POP risk; and PS3—a presumed high POP risk) would differ in circulatory biomarkers of inflammation and hormonal profiles. On gestation week 15, 2,864 individual sows were assigned a PS, and subsequently, 1.0%, 2.7%, and 23.4% of PS1, PS2, or PS3 sows, respectively, experienced POP. During PS assignment at days 107–116 of gestation, blood samples were collected from sows on two farms of similar genetics, feed sources, and health status. Whole blood was subjected to complete blood count (CBC) analysis (n = 212) and steroid hormones were measured in serum from a subset (n = 110) of animals assigned PS3 parity matched to PS1. Lipopolysaccharide-binding protein (LBP), tumor necrosis factor-alpha (TNF-α), haptoglobin, C-reactive protein (CRP), and creatine kinase (CK) levels were also evaluated. Complete blood count analysis revealed decreased (P ≤ 0.05) mean platelet volume (3.9%), lymphocytes (6.5%), and monocytes (7.5%) in PS3 compared to PS1 sows. Increased (P ≤ 0.02) abundance of androstenedione (13.4%), androsterone (18.2%), estrone (24.8%), and 17β-estradiol (26.2%) was observed in PS3 compared to PS1 sows. Additionally, a 25.8% increase (P = 0.04) in LBP in PS3 compared to PS1 sows was observed. Many dynamic physiological changes occur in sows during late gestation as they approach farrowing. The data presented herein demonstrate that distinct differences in concentrations of circulating biomarkers exist between late gestation sows at high or low risk for POP and may serve as a useful tool for understanding the etiology of POP and evaluation of mitigation strategies.This article is published as Kiefer, Zoë E., Jamie M. Studer, Amanda L. Chipman, Malavika K. Adur, Christine Mainquist-Whigham, Nicholas K. Gabler, Aileen F. Keating, and Jason W. Ross. "Circulating biomarkers associated with pelvic organ prolapse risk in late gestation sows." Journal of Animal Science 99, no. 8 (2021): skab207. doi:10.1093/jas/skab207.</p

Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period

Heat stress (HS) deleteriously affects multiple components of porcine reproduction and is causal to seasonal infertility. Environment-induced hyperthermia causes a HS response (HSR) typically characterized by increased abundance of intracellular heat shock proteins (HSP). Gilts exposed to HS during the peri-implantation period have compromised embryo survival, however if (or how) HS disrupts the porcine endometrium is not understood. Study objectives were to evaluate the endometrial HSP abundance in response to HS during this period and assess the effect of oral progestin (altrenogest; ALT) supplementation. Postpubertal gilts (n = 42) were artificially inseminated during behavioral estrus (n = 28) or were kept cyclic (n = 14), and randomly assigned to thermal neutral (TN; 21 ± 1 °C) or diurnal HS (35 ± 1 °C for 12 h/31.6 ± 1 °C for 12 h) conditions from day 3 to 12 postestrus (dpe). Seven of the inseminated gilts from each thermal treatment group received ALT (15 mg/d) during this period. Using quantitative PCR, transcript abundance of HSP family A (Hsp70) member 1A (HSPA1A, P = 0.001) and member 6 (HSPA6, P < 0.001), and HSP family B (small) member 8 (HSB8, P = 0.001) were increased while HSP family D (Hsp60) member 1 (HSPD1, P = 0.01) was decreased in the endometrium of pregnant gilts compared to the cyclic gilts. Protein abundance of HSPA1A decreased (P = 0.03) in pregnant gilt endometrium due to HS, while HSP family B (small) member 1 (HSPB1) increased (P = 0.01) due to HS. Oral ALT supplementation during HS reduced the transcript abundance of HSP90α family class B member 1 (HSP90AB1, P = 0.04); but HS increased HSP90AB1 (P = 0.001), HSPA1A (P = 0.02), and HSPA6 (P = 0.04) transcript abundance irrespective of ALT. ALT supplementation decreased HSP90α family class A member 1 (HSP90AA1, P = 0.001) protein abundance, irrespective of thermal environment, whereas ALT only decreased HSPA6 (P = 0.02) protein abundance in TN gilts. These results indicate a notable shift of HSP in the porcine endometrium during the peri-implantation period in response to pregnancy status and heat stress.This article is published as Adur, Malavika K., Jacob T. Seibert, Matthew R. Romoser, Katie L. Bidne, Lance H. Baumgard, Aileen F. Keating, and Jason W. Ross. "Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period." Journal of Animal Science 100, no. 7 (2022): skac129. doi:10.1093/jas/skac129. Posted with permission. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited

Interleukin 16- (IL-16-) Targeted Ultrasound Imaging Agent Improves Detection of Ovarian Tumors in Laying Hens, a Preclinical Model of Spontaneous Ovarian Cancer

Limited resolution of transvaginal ultrasound (TVUS) scanning is a significant barrier to early detection of ovarian cancer (OVCA). Contrast agents have been suggested to improve the resolution of TVUS scanning. Emerging evidence suggests that expression of interleukin 16 (IL-16) by the tumor epithelium and microvessels increases in association with OVCA development and offers a potential target for early OVCA detection. The goal of this study was to examine the feasibility of IL-16-targeted contrast agents in enhancing the intensity of ultrasound imaging from ovarian tumors in hens, a model of spontaneous OVCA. Contrast agents were developed by conjugating biotinylated anti-IL-16 antibodies with streptavidin coated microbubbles. Enhancement of ultrasound signal intensity was determined before and after injection of contrast agents. Following scanning, ovarian tissues were processed for the detection of IL-16 expressing cells and microvessels. Compared with precontrast, contrast imaging enhanced ultrasound signal intensity significantly in OVCA hens at early (P<0.05) and late stages (P<0.001). Higher intensities of ultrasound signals in OVCA hens were associated with increased frequencies of IL-16 expressing cells and microvessels. These results suggest that IL-16-targeted contrast agents improve the visualization of ovarian tumors. The laying hen may be a suitable model to test new imaging agents and develop targeted anti-OVCA therapeutics