Non-human Primate Model Of Endometriosis Research Articles

Dissecting the miR-451a-Mif Pathway in Endometriosis Pathophysiology Using a Syngeneic Mouse Model: Temporal Expression of Lesion Mif Receptors, Cd74 and Cxcr4.

Endometriosis is an enigmatic disease characterized by pain and infertility in which endometrial tissue grows in ectopic locations, predominantly the pelvic cavity. The pathogenesis and pathophysiology of endometriosis is complex and postulated to involve alterations in inflammatory, cell proliferation and post-transcriptional regulatory pathways among others. Our understanding on the pathogenesis and pathophysiology of endometriosis is further complicated by the fact that endometriosis can only be diagnosed by laparoscopy only after the disease has manifested. This makes it difficult to understand the true pathogenesis as a cause-and-effect relationship is difficult to ascertain. To aid in our understanding on endometriosis pathogenesis and pathophysiology, numerous rodent models have been developed. In this case, we discuss further assessment of a miR-451a—macrophage migration inhibitory factor (Mif) pathway which contributes to lesion survival. Specifically, we evaluate the temporal expression of lesion Mif receptors, Cd74 and Cxcr4 using host mice which express wild-type or miR-451a deficient lesions. Similar to that observed in humans and a non-human primate model of endometriosis, Cd74 expression is elevated in lesion tissue in a temporal fashion while that of Cxcr4 shows minimal increase during initial lesion establishment but is reduced later during the lifespan. Absence of miR-451a during initial lesion establishment is associated with an augmentation of Cd74, but no Cxcr4 expression. The data obtained in this study provide further support for a role of Mif receptors, Cd74 and Cxcr4 in the pathophysiology of endometriosis.

Loss of MIG-6 results in endometrial progesterone resistance via ERBB2

Female subfertility is highly associated with endometriosis. Endometrial progesterone resistance is suggested as a crucial element in the development of endometrial diseases. We report that MIG-6 is downregulated in the endometrium of infertile women with endometriosis and in a non-human primate model of endometriosis. We find ERBB2 overexpression in the endometrium of uterine-specific Mig-6 knockout mice (Pgrcre/+Mig-6f/f; Mig-6d/d). To investigate the effect of ERBB2 targeting on endometrial progesterone resistance, fertility, and endometriosis, we introduce Erbb2 ablation in Mig-6d/d mice (Mig-6d/dErbb2d/d mice). The additional knockout of Erbb2 rescues all phenotypes seen in Mig-6d/d mice. Transcriptomic analysis shows that genes differentially expressed in Mig-6d/d mice revert to their normal expression in Mig-6d/dErbb2d/d mice. Together, our results demonstrate that ERBB2 overexpression in endometrium with MIG-6 deficiency causes endometrial progesterone resistance and a nonreceptive endometrium in endometriosis-related infertility, and ERBB2 targeting reverses these effects.

Serum MicroRNA Biomarkers Regulated by Simvastatin in a Primate Model of Endometriosis.

Endometriosis is a chronic inflammatory and estrogen-dependent disease that causes pain and infertility in reproductive-aged women. Due to the delay in diagnosis, there is a pressing need for accurate biomarkers. Detection of serum noncoding RNA molecules such as microRNAs (miRNAs) shows promise as a noninvasive diagnostic strategy; we previously identified miRNAs that are highly sensitive and specific biomarkers for the disease. In this study, we investigate the expression of these miRNAs in a nonhuman primate model of endometriosis. As part of a pilot study evaluating simvastatin for the treatment of endometriosis, the disease was induced in 16 baboons by induction laparoscopy and the animals were divided into 2 groups. One group was treated with simvastatin for 90 days, while the second group received vehicle only. Endometriosis was evaluated after 3 months by laparoscopy. Serum samples were analyzed for 9 circulating miRNAs using quantitative real time-polymerase chain reaction, focusing on the miRNAs we found to be dysregulated in human endometriosis. In the simvastatin-treated endometriosis group, levels of miR-150-5p and miR-451a were decreased, while miR-3613-5p levels were increased compared to the untreated endometriosis group. The changes in circulating miRNA expression patterns parallel our previous results in human patients and show that specific miRNAs correlate with endometriosis severity and reverted toward control expression levels after simvastatin treatment. This is the first report showing serum miRNA expression normalized in response to endometriosis treatment, supporting the potential for this class of biomarkers to be used both to diagnose endometriosis and to monitor its progression and response to therapy.

Nerve fibre infiltration and expression in peritoneal lesions of endometriosis in a nonhuman primate model of endometriosis

Baboon ( Papio anubis) models of endometriosis are thought to mimic the early stages of spontaneous human peritoneal endometriotic disease. The objective of this study was to investigate the presence and course of nerve fibre ingrowth during peritoneal lesion formation in specimens collected at 3 months (early stage of lesion development) and 15 months (late stage of lesion development) after disease initiation compared to pelvic peritoneum (control). Five-micron sections of paraffin-embedded peritoneal lesions were obtained from normally cycling baboons with 3-month (n = 12), 15-month (n = 12) induced endometriosis and pelvic peritoneum (n = 10) from baboons with no endometriosis. Immunohistochemical staining was performed with specific antibodies: protein gene product 9.5 – broad marker of nerve fibres and neurones, neuropeptide Y – sympathetic neurones, substance P – sensory neurones, vasoactive intestinal peptide – parasympathetic neurones, nerve growth factor – development of new neurones and high-affinity receptor for nerve growth factor (tropomyosin receptor kinase A) – neuronal differential. Significantly, more nerve fibres were identified in peritoneal endometriotic lesions collected 15 months after the initiation of experimental protocols compared with 3-month and control samples (p < 0.001). Nerve fibres were immunoreactive for all the tested markers – protein gene product 9.5, neuropeptide Y, substance P, vasoactive intestinal peptide, nerve growth factor and tropomyosin receptor kinase A – indicating the presence of different types of nerve fibres. In conclusion, peritoneal lesions of endometriosis in a nonhuman primate model of endometriosis were found to be progressively and spontaneously innervated by both myelinated and unmyelinated sensory nerve fibres, parasympathetic and sympathetic neurones. These nerve fibres may play an important role in the mechanisms of pain generation in this condition.

Systemic Iron Deficiency in a Nonhuman Primate Model of Endometriosis.

Endometriosis is characterized by endometrial tissue development outside the uterus. Anemia and iron depletion do not commonly accompany endometriosis in women, despite chronic abdominal inflammation and heavy menstrual bleeding. The objective of this study was to examine iron kinetics associated with endometriosis by using a NHP model, to better understand the underlying mechanism of abnormal hematogram values in women with endometriosis. Hematologic data from 46 macaques with endometriosis were examined for signs of iron depletion. Bone marrow, liver, and serum were used to elucidate whether iron loss or inflammation best explained the hematologic findings. Additional serum markers and intestinal biopsies from NHP with and without endometriosis were evaluated for patterns in iron kinetics across the menstrual cycle and for relative dietary iron-absorbing capacity. Almost half of the NHP with endometriosis were anemic. Overall, NHP had decreased RBC counts, increased MCV, increased percentage of reticulocytes, decreased serum hepcidin, and decreased hepatic and bone marrow iron. Intestinal expression of ferroportin 1, a mediator of iron absorption, was increased, indicating that despite high dietary iron, intestinal iron absorption did not compensate for iron losses. We concluded that use of oral iron supplementation alone does not replenish iron stores in endometriosis. Consequently, iron stores should be evaluated in women with endometriosis, even without overt clinical signs of anemia.

Lesion kinetics in a non-human primate model of endometriosis

Endometriosis is a common cause of pelvic pain and infertility in women of reproductive age. It is characterized by the presence of endometrial tissue outside the normal location, predominantly in the pelvic peritoneum causing severe abdominal pain. However, the severity of the symptoms of endometriosis does not always correlate with the anatomic severity of the disease. This lack of correlation may be due to morphological lesion variation during disease progression. This study examined lesion kinetics in a non-human primate model of endometriosis to better understand lesion dynamics. Endometriosis was experimentally induced in nine normal cycling female adult olive baboons (Papio anubis) by i.p. inoculation of autologous menstrual endometrium on Day 2 of menses for two consecutive menstrual cycles. Diagnostic laparoscopies were performed between Day 8-12 post-ovulation at 1, 3, 6, 9 and 12 months, followed by a necropsy at 15 months, after the second inoculation. In two animals, lesions were excised/ablated at 6 months and they were monitored for lesion recurrence and morphological changes by serial laparoscopy. Furthermore, five control animals underwent surgeries conducted at the same time points but without inoculation. A total of 542 endometriotic lesions were observed. The location, macroscopic (different colours) and microscopic appearance confirmed distinct endometriosis pathology in line with human disease. The majority of the lesions found 1 month after tissue inoculation were red lesions, which frequently changed colour during the disease progression. In contrast, blue lesions remained consistently blue while white lesions were evident at the later stages of the disease process and often regressed. There were significantly lower numbers of powder burn, blister and multicoloured lesions observed per animal in comparison to black and blue lesions (P-value≤0.05). New lesions were continually arising and persisted up to 15 months post-inoculation. Lesions reoccurred as early as 3 months after removal and 69% of lesions excised/ablated had reoccurred 9 months later. Interestingly, endometriotic lesions were also found in the non-inoculated animals, starting at the 6-month time point following multiple surgeries. Documentation of lesion turnover in baboons indicated that lesions changed their colour from red to white over time. Different lesion types underwent metamorphosis at different rates. A classification of lesions based on morphological appearance may help disease prognosis and examination of the effect of the lesion on disease symptoms, and provide new opportunities for targeted therapies in order to prevent or treat endometriosis. Surgical removal of endometriotic lesions resulted in a high incidence of recurrence. Spontaneous endometriosis developed in control baboons in the absence of inoculation suggesting that repetitive surgical procedures alone can induce the spontaneous evolution of the chronic disease. Although lesion excision/ablation may have short-term benefits (e.g. prior to an IVF cycle in subfertile women), for long-term relief of symptoms perhaps medical therapy is more effective than surgical therapy.

Endometrial Stromal Cells and Immune Cell Populations Within Lymph Nodes in a Nonhuman Primate Model of Endometriosis

Mounting evidence suggests that immunological responses may be altered in endometriosis. The baboon (Papio anubis) is generally considered the best model of endometriosis pathogenesis. The objective of the current study was to investigate for the first time immunological changes within uterine and peritoneal draining lymph nodes in a nonhuman primate baboon model of endometriosis. Paraffin-embedded femoral lymph nodes were obtained from 22 normally cycling female baboons (induced endometriosis n = 11; control n = 11). Immunohistochemical staining was performed with antibodies for endometrial stromal cells, T cells, immature and mature dendritic cells, and B cells. Lymph nodes were evaluated using an automated cellular imaging system. Endometrial stromal cells were significantly increased in lymph nodes from animals with induced endometriosis, compared to control animals (P = .033). In animals with induced endometriosis, some lymph node immune cell populations including T cells, dendritic cells and B cells were increased, suggesting an efficient early response or peritoneal drainage.

The Endometrial Response to Chorionic Gonadotropin Is Blunted in a Baboon Model of Endometriosis

Endometriosis-associated infertility has a multifactorial etiology. We tested the hypothesis that the endometrial response to the early embryonic signal, human chorionic gonadotropin (hCG), alters over time in a nonhuman primate model of endometriosis. Animals with experimental or spontaneous endometriosis were treated with hCG (30 IU/d), from d 6 after ovulation for 5 d, via an oviductal cannula. Microarray analysis of endometrial transcripts from baboons treated with hCG at 3 and 6 months of disease (n=6) identified 22 and 165 genes, respectively, whose levels differed more than 2-fold compared with disease-free (DF) animals treated with hCG (P<0.01). Quantitative RT-PCR confirmed abnormal responses of known hCG-regulated genes. APOA1, SFRP4, and PAPPA, which are normally down-regulated by hCG were up-regulated by hCG in animals with endometriosis. In contrast, the ability of hCG to induce SERPINA3 was lost. Immunohistochemistry demonstrated dysregulation of C3 and superoxide dismutase 2 proteins. We demonstrate that this abnormal response to hCG persists for up to 15 months after disease induction and that the nature of the abnormal response changes as the disease progresses. Immunohistochemistry showed that this aberrant gene expression was not a consequence of altered LH/choriogonadotropin receptor distribution in the endometrium of animals with endometriosis. We have shown that endometriosis induces complex changes in the response of eutopic endometrium to hCG, which may prevent the acquisition of the full endometrial molecular repertoire necessary for decidualization and tolerance of the fetal allograft. This may in part explain endometriosis-associated implantation failure.

Proteomic analysis of endometrium from fertile and infertile patients suggests a role for apolipoprotein A-I in embryo implantation failure and endometriosis

Pregnancy is dependent upon the endometrium acquiring a receptive phenotype that facilitates apposition, adhesion and invasion of a developmentally competent embryo. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry of mid-secretory endometrial biopsies revealed a 28 kDa protein peak that discriminated highly between samples obtained from women with recurrent implantation failure and fertile controls. Subsequent tandem mass spectroscopy unambiguously identified this peak as apolipoprotein A-I (apoA-I), a potent anti-inflammatory molecule. Total endometrial apoA-I levels were, however, comparable between the study and control group. Moreover, endometrial apoA-I mRNA expression was not cycle-dependent although there was partial loss of apoA-I immunoreactivity in luminal and glandular epithelium in mid-secretory compared with proliferative endometrial samples. Because of its putative anti-implantation properties, we examined whether endometrial apoA-I expression is regulated by embryonic signals. Human chorionic gonadotrophin (hCG) strongly inhibited apoA-I expression in differentiating explant cultures but not when established from eutopic endometrium from patients with endometriosis. Pelvic endometriosis was associated with elevated apoA-I mRNA levels, increased secretion by differentiating eutopic endometrial explant cultures and lack of hCG-dependent down-regulation. To corroborate these observations, we examined endometrial apoA-I expression and its regulation by hCG in a non-human primate model of endometriosis. As in humans, hCG strongly inhibited endometrial apoA-I mRNA expression in disease-free baboons, but this response was entirely lost upon induction of pelvic endometriosis. Together, these observations indicate that perturbations in endometrial apoA-I expression, modification or regulation by paracrine embryonic signals play a major role in implantation failure and infertility.

The non-human primate model of endometriosis: research and implications for fecundity

The development of an animal model of endometriosis is crucial for the investigation of disease pathogenesis and therapeutic intervention. These models will enhance our ability to evaluate the causes for the subfertility associated with disease and provide a first-line validation of treatment modulators. Currently rodents and non-human primate models have been developed, but each model has their limitations. The aim of this manuscript is to summarize the current findings and theories on the development of endometriosis and disease progression and the effectiveness of therapeutic targets using the experimental induced model of endometriosis in the baboon (Papio anubis).