laparoscopic-induction-endometriosis-preclinical-swine-model

Laparoscopic Induction of Endometriosis in a Preclinical Swine Model by Autologous Endometrial Transplantation

Pigs are increasingly used as models of human diseases, largely because their anatomy, physiology, and genetics more closely resemble those of humans. ...

University of Missouri

National Swine Resource and Research Center

Aromatase catalyzes the synthesis of estrogens and has been shown to have an important role during the establishment of pregnancy in the pig. This study confirmed the differential expression of the three aromatase isoforms.

Porcine-specific expression of the three functional CYP19 paralogs in early conceptus, placenta, and gonads.

Maternal recognition of pregnancy (MRP) is a term utilized in mammals to describe pathways in which the conceptus alters the endometrial environment to prevent regression of corpora lutea to ensure continued production of progesterone (P4) required for establishment and maintenance of pregnancy. For nearly 40 years after publication of the endocrine/exocrine theory, conceptus estrogen (E2) was considered the primary maternal recognition signal in the pig. Conceptus production of prostaglandin E2 (PGE2) was also considered to be a major factor in preventing luteolysis. An addition to E2 and PGE2, pig conceptuses produce interleukin 1B2 (IL1B2) and interferons (IFN) delta (IFND) and gamma (IFNG). The present review provides brief history of the discovery of E2, PGs and IFNS which led to research investigating the role of these conceptus secreted factors in establishing and maintaining pregnancy in the pig. The recent utilization of gene editing technology allowed a more direct approach to investigate the in vivo roles of IL1B2, E2, PGE2, AND IFNG for establishment of pregnancy. These studies revealed unknown functions for IFNG and ILB2 in addition to PGE2 and E2. Thus, pregnancy recognition signal is via a servomechanism in requiring sequential effects of P4, E2, IL1B2, PGE2 and IFNG. Results indicate that the original established dogma for the role of conceptus E2 and PGs in MRP is a far too simplified model that involves the interplay of numerous mechanisms for inhibiting luteolysis, inducing critical elongation of the conceptuses and resolution of inflammation in pigs.

Maternal recognition of pregnancy in the pig: A servomechanism involving sex steroids, cytokines and prostaglandins.

Conceptus estrogens and prostaglandins have long been considered the primary signals for maternal recognition of pregnancy (MRP) in the pig. However, loss-of-function studies targeting conceptus aromatase genes (CYP19A1 and CYP19A2) and prostaglandin-endoperoxide synthase 2 (PTGS2) indicated that conceptuses can not only signal MRP without estrogens or prostaglandins but can maintain early pregnancy. However, complete loss of estrogen production leads to abortion after day 25 of gestation. Although neither conceptus estrogens nor prostaglandins had a significant effect on early maintenance of corpora lutea (CL) function alone, the two conceptus factors have a biological relationship. To investigate the role that both conceptus estrogens and prostaglandins have on MRP and maintenance of pregnancy, a triple loss-of function model (TKO) was generated for conceptus CYP19A1, CYP19A2, and PTGS2. In addition, a conceptus CYP19A2-/- model (A2KO) was established to determine the role of placental estrogen during later pregnancy. Estrogen and prostaglandin synthesis were greatly reduced in TKO concept uses which resulted in a failure to inhibit luteolysis after day 15 of pregnancy despite the presence of conceptuses in the uterine lumen. However, A2KO placentae not only maintained functional CL but were able to maintain pregnancy to day 32 of gestation. Despite the loss of placental CYP19A2 expression, the allantois fluid content of estrogen was not affected as the placenta compensated by expressing CYP19A1 and CYP19A3, which are normally absent in controls. Results suggest conceptuses can signal MRP through production of conceptus PGE or stimulating PGE synthesis from the endometrium through conceptus estrogen. Failure of conceptuses to produce both factors results in failure of MRP and loss of pregnancy.

Conceptus estrogen and prostaglandins provide the maternal recognition of pregnancy signal to prevent luteolysis during early pregnancy in the pig†.

Base editing could correct nonsense mutations that cause cystic fibrosis (CF), but clinical development is limited by the lack of delivery methods that efficiently breach the barriers presented by airway epithelia. Here, we present a novel amphiphilic shuttle peptide based on the previously reported S10 peptide that substantially improved base editor ribonucleoprotein (RNP) delivery. Studies of the S10 secondary structure revealed that the alpha-helix formed by the endosomal leakage domain (ELD), but not the cell penetrating peptide (CPP), was functionally important for delivery. By isolating and extending the ELD, we created a novel shuttle peptide, termed S237. While S237 achieved lower delivery of green fluorescent protein, it outperformed S10 at Cas9 RNP delivery to cultured human airway epithelial cells and to pig airway epithelia in vivo, possibly due to its lower net charge. In well-differentiated primary human airway epithelial cell cultures, S237 achieved a 4.6-fold increase in base editor RNP delivery, correcting up to 9.4% of the cystic fibrosis transmembrane conductance regulator (CFTR) R553X allele and restoring CFTR channel function close to non-CF levels. These findings deepen the understanding of peptide-mediated delivery and offer a translational approach for base editor RNP delivery for CF airway disease.

Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells.

In cattle, oviductal function is controlled by the ovarian sex-steroids estradiol and progesterone. Here, we tested the hypothesis that the exposure to contrasting sex-steroid milieus differentially impacts the oviductal fluid composition. Estrous cycles of non-lactating, multiparous Nelore cows were pre-synchronized and then synchronized with a protocol designed two induce ovulation of large or small follciles. Larger preovulatory follicle (day 0) and corpora lutea (day 4) and greater estradiol (day 0) and progesterone (day 4) concentrations were observed in the large follciles group. Four days after induced ovulation, oviductal fluid was collected post-mortem. Quantitative mass spectrometry was used to determine the concentration of amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, lysophosphatidylcholines, sphingomyelins, hexoses, prostaglandins, and related compounds. Multivariate analyses (orthogonal projections to latent structures discriminant analysis) were conducted to compare the metabolomic signatures of oviductal fluids. Correlation network analysis was conducted to measure the strength and hierarchy of associations among metabolites. Of the 205 metabolites quantified, 171 were detected in at least 50% of the samples and were included in further data analysis. After orthogonal projections to latent structures discriminant analysis analysis, samples of the large follciles and small follciles were divided clearly into two non-overlapping clusters. Twenty metabolites had different or tended to have different concentrations in the oviductal fluid when comparing groups. Seven of these 20 analytes had greater concentrations in large follciles cows. Moreover, total sum of biogenic amines, phosphatidylcholines, and prostaglandins were higher in the small follciles group. The correlation network showed that the large follciles group metabolites' concentrations were highly intercorrelated, which was not observed in the small follciles group. We concluded that the periovulatory endocrine milieu regulates the composition of the oviductal fluid.

The metabolomic composition of the oviductal fluid is controlled by the periovulatory hormonal context in Bos indicus cows†.

The eutherian placenta is highly complex, evolving to regulate the inflammatory phase of pregnancy during conceptus attachment and placental tissue development. Tripartite motif family-like (TRIMLs) proteins are implicated in downregulating inflammation. In mammals, TRIML1 and TRIML2 show preferential expression in gonads, preimplantation embryos and placenta. TRIML1 domains differ between eutherians and marsupials, while TRIML2 is absent in marsupials, suggesting it may play a unique role in regulating the inflammatory phase during conceptus attachment, critical for establishing and maintaining pregnancy to term. This study aimed to investigate the expression pattern of TRIML1 and TRIML2 in various tissues, as well as during embryo development, conceptus attachment, and placental formation in pigs. Transcripts for TRIML2 were detected in embryos, conceptuses, extraembryonic membranes, ovary and testis but not in any of the other tissues examined. In contrast, TRIML1 expression was only observed in testis. In situ hybridization of TRIML1 and TRIML2 confirmed these results. The specific expression of TRIML2 in immune privileged sites is consistent with it serving as an anti-inflammatory factor to provide immunological protection of the eutherian placenta. To further investigate the role of TRIML2, CRISPR/Cas9 gene editing was employed to knock out either TRIML1 (control) or TRIML2. TRIML1 -/- and TRIML2 -/- porcine fetal fibroblasts were used for somatic cell nuclear transfer, and the resulting embryos were transferred into surrogate gilts. Early conceptus and placental development were not affected by the loss of conceptus TRIML2. Although a tissue-specific expression pattern was found, TRIML1 or TRIML2 are not required for pregnancy establishment in the pig.

Expression of tripartite motif family-like protein 1 and 2 in early conceptus development and placentation in the pig.

Endometriosis is a benign gynecological condition in which endometrial-like tissue grows outside of the uterus, and the mechanisms of its pathogenesis are not fully understood. The anatomical and physiological parallels between pigs and humans have made pigs a good model for investigating other human diseases. Here, we report on the development of a swine model for endometriosis in which the peritoneal cavity was inoculated with autologous endometrial tissue fragments labeled with fluorescein isothiocyanate (FITC) dye-doped silica nanoparticles. After 6 weeks of endometriosis induction, FITC positive endometriotic lesions were observed on the peritoneal surface of the abdominal wall as well as on the serosal surfaces of the uterus and small intestine. Histological analysis of the endometriotic lesions revealed endometrial-like epithelial and stromal cells that were morphologically comparable to human endometriotic lesions. The identification of epithelial and stromal cells in these lesions was confirmed by immunostaining of e-cadherin, an endometrial epithelial marker, and Chicken ovalbumin upstream promoter-transcription factor II, an endometrial stromal marker. Our results illustrate successful induction of endometriosis with fluorescent-labeled endometriotic lesions to identify in situ endometriotic lesions. According to the size and physiological similarity of pigs to humans, this is an important first step toward the development of a non-invasive tool to diagnose endometriosis. Furthermore, the availability of a new large animal model for translational endometriosis research provides a novel preclinical animal model for laparoscopic monitoring and retrieval of experimental endometriotic lesions with ample serum for serial analyses.

The development of a preclinical swine model for endometriosis†.

Release of interferon-gamma (IFNG), a pro-inflammatory type II interferon, by the early conceptus is critical for pregnancy establishment in the pig. Yet, the cellular and molecular mechanisms underpinning conceptus-derived IFNG actions in maternal peripheral immune cells and at the embryo-maternal interface remain unclear. Here we show that pregnancy status up-regulates expression of IRF1, an IFNG target-gene, in peripheral blood mononuclear cells on day 15 of pregnancy in the pig. In a second study, loss-of-function IFNG (IFNG-/-) embryos were generated by using CRISPR/Cas9 gene editing and somatic cell nuclear transfer. Single-nuclei RNA sequencing of endometrium from gilts carrying wild-type (Control) or IFNG-/- conceptuses on day 15 of pregnancy revealed cell-type-specific signatures at the embryo-maternal interface in response to conceptus IFNG. Changes in the transcriptome of epithelial and IFNG receptor-expressing immune cells were evident between Control and IFNG-/- samples, along with a downregulation of IFNG target-genes involved in chemotaxis and immune cell differentiation in IFNG-/- recipient endometria. A notable reduction in monocytes and macrophages was observed in IFNG-/- samples, confirmed by immunohistochemistry for AIF1. Differential gene expression (DEG) analysis revealed 391 DEGs in monocytes, implicating IFNG in macrophage polarization, with evidence suggesting a shift toward an M2 phenotype. The study concludes that conceptus IFNG plays an important role in monocyte recruitment and macrophage polarization at the embryo-maternal interface for regulation of inflammation upon conceptus attachment. Insufficient numbers or skewed IFNG-activated macrophages within the endometrium may contribute to pregnancy failure in the IFNG-/- pig model.

Programming of the endometrial immune milieu by interferon gamma during early pregnancy in pigs†.

Precision genome editing technologies have transformed the generation and application of genetically engineered livestock, positioning domestic animals as powerful models for agriculture, biomedicine, and basic research. Historically, genetic engineering in livestock was constrained by the absence of embryonic stem cells, low efficiency of homologous recombination, long gestation periods, and extended generation intervals. The advent of programmable nucleases, including zinc finger nucleases, transcription activator-like effector nucleases, and CRISPR-Cas systems, has overcome many of these barriers by enabling efficient targeted genome modification. This review focuses on the application of genome editing technologies in mammalian livestock species, with pigs highlighted as a representative model. We discuss the historical development of livestock genetic engineering and summarise two main routes used to establish genome edited livestock models. Key technical considerations unique to domestic animals are also presented. Genome editing has accelerated the production of livestock models for human disease, xenotransplantation, reproductive biology, and agricultural improvement, including disease resistance, altered growth traits, and enhanced product composition. Emerging epigenome editing strategies further expand the toolkit by allowing locus-specific gene regulation without permanent DNA modification. Despite rapid technological progress, challenges remain, including mosaicism, off-target effects, limited ability to segregate unintended edits, and ethical and regulatory concerns surrounding food and biomedical applications. Continued refinement of genome editing strategies will be essential for maximizing the utility and responsible implementation of genome-edited livestock. Overall, precision genome editing represents a transformative platform with broad implications for sustainable agriculture, translational research, and global food security.

Article	Year
Porcine-specific expression of the three functional CYP19 paralogs in early conceptus, placenta, and gonads. Reproduction (Cambridge, England)\| PubMed ID: 37490351	2023
Maternal recognition of pregnancy in the pig: A servomechanism involving sex steroids, cytokines and prostaglandins. Animal reproduction science\| PubMed ID: 38522133	2024
Conceptus estrogen and prostaglandins provide the maternal recognition of pregnancy signal to prevent luteolysis during early pregnancy in the pig†. Biology of reproduction\| PubMed ID: 38904948	2024
Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells. Nucleic acids research\| PubMed ID: 39315713	2024
The metabolomic composition of the oviductal fluid is controlled by the periovulatory hormonal context in Bos indicus cows†. Biology of reproduction\| PubMed ID: 39418319	2024
Expression of tripartite motif family-like protein 1 and 2 in early conceptus development and placentation in the pig. Reproduction & fertility\| PubMed ID: 40146919	2025
The development of a preclinical swine model for endometriosis†. Biology of reproduction\| PubMed ID: 40445172	2025
Programming of the endometrial immune milieu by interferon gamma during early pregnancy in pigs†. Biology of reproduction\| PubMed ID: 41042227	2025
Precision Genome Editing in Domestic Animals: A New Era for Agriculture and Medicine. Reproduction in domestic animals = Zuchthygiene\| PubMed ID: 42816914	2026

Mariana Sponchiado

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