The advent of tissue engineering has revolutionized the development of organs and tissues, enabling the creation of three-dimensional structures that support cell proliferation and differentiation. This process, essential for organ transplantation, ensures biocompatibility, immunogenicity, and structural fidelity by preserving the extracellular matrix and its components, such as growth factors and signaling molecules. In the context of ovarian tissue engineering, the restoration of ovarian function presents a promising avenue for addressing conditions like ovarian insufficiency, which arises from hormonal, genetic, autoimmune factors, or treatments like chemotherapy. Current challenges in ovarian transplantation, including the need for revascularization, highlight the limitations of existing techniques such as oocyte preservation. Tissue engineering approaches, particularly ovarian recellularization through decellularization, show promise in restoring both reproductive and endocrine functions by mimicking the ovarian microenvironment. Emerging technologies, such as 3D printing and CRISPR/Cas9 gene editing, although facing their own limitations, offer complementary methods to enhance ovarian tissue engineering. This review examines the state of the art in artificial ovarian reconstruction, with a focus on restoring endocrine and reproductive functions, ultimately advancing women's health by developing bioengineered ovaries.

BACKGROUND:Sperm acquire fertility ability during epididymal maturation mainly in the epididymal caput and corpus, and once matured, are stored in the epididymal cauda. During storage, interactions with cauda epididymosomes (epEVs) may influence sperm fertility potential; however, the role of such interactions on sperm fertility remains elusive. OBJECTIVES:To investigate the effect of epEVs on sperm fertility potential using the bovine model. MATERIALS AND METHODS:A pool of epEVs from cauda epididymal fluid of five bovine males was characterized regarding size, concentration, morphology, and specific markers. To determine the sperm-epEVs interaction protocol, green-labeled epEVs (PKH67) were incubated with post-thawed cauda epididymal sperm from three bovine males at ratios of 500, 1000, or 2000 epEVs/sperm, and for incubation periods of 1.5, 3, or 6 h. After that, Hoechst-stained sperm were analyzed by flow cytometer. Green fluorescence percentage and intensity of 5000 positive Hoechst events were considered as sperm-epEVs interacting. Controls were performed by incubating sperm with PKH67 in PBS. A total of 49 microRNAs (out of 380 investigated) were found detected in the epEVs and the top five were investigated in sperm by quantitative polymerase chain reaction (qPCR) following sperm-epEVs interaction. Subsequently, sperm that interacted with epEVs were used to produce embryos by in vitro fertilization. RESULTS:epEVs displayed 114.20 ± 3.60 nm, 3.48 × 109 ± 1.84 × 108 particles/mL, a cup-shaped morphology, and positivity for ALIX, CD-81, and CD-63 markers. Following determination of the protocol (1000 epEVs/sperm for 3 h), two microRNAs (miR-935 and -421) previously detected in epEVs, were found in sperm. Finally, sperm incubated with epEVs resulted in higher (p = 0.04) blastocyst rates (epEVs: 38.9% ± 7.3%; 58/149; control: 26.6% ± 5.6%; 40/145). DISCUSSION AND CONCLUSION:Incubation of sperm with 1000 epEVs/sperm for 3 h promoted sperm-epEVs in vitro crosstalk that enhanced blastocyst rates. These findings suggest that epEVs modulate paternal contribution to development and provide valuable insights to promote a fast and dynamic control of male fertility.

Oocyte in vitro maturation (IVM) represents a crucial phase in embryo production, often compromised by reactive oxygen species (ROS). Strategies to mitigate ROS are essential to improve oocyte quality. This study investigated the effects of liposome-encapsulated α-pinene (Lip-α-pinene) on bovine oocyte IVM and embryonic development following parthenogenetic activation. Cumulus-oocyte complexes (COCs) were matured in vitro for 22-24 h in control medium or supplemented with Lip-blank, or 0.01, 1.0 or 100.0 µg/mL Lip-α-pinene. Lip-α-pinene had a size of 75.86 ± 0.95 nm, low polydispersity (0.26 ± 0.00), and zeta potential of -31.55 ± 2.23 mV. Nuclear maturation was not affected. However, COCs matured with 1.0 µg/mL Lip-α-pinene showed well-preserved ultrastructure of oocyte and cumulus cells, reduced ROS levels and lipid accumulation compared to control, Lip-blank, and 100.0 µg/mL Lip-α-pinene. This was accompanied by relative abundance of NRF2, SOD, and PRDX6. Furthermore, 1.0 and 100.0 µg/mL Lip-α-pinene increased cleavage rates and cells per blastocyst compared to control, while blastocyst rates and lipid content were not affected. In conclusion, 1.0 µg/mL Lip-α-pinene enhances antioxidant capacity of bovine oocytes by reducing ROS and lipid accumulation, associated with abundance of NRF2, SOD, and PRDX6 transcripts, thereby improving oocyte competence and quality of parthenogenetic embryos.

While molecular signature contributes to sperm quality and fertility potential, sperm may lost key molecules during cryopreservation. Since sperm cells are transcriptionally inert and rely on interactions with epididymosomes (epEVs) to acquire molecules, herein, we investigated the effects of the addition of epEVs to the cryopreservation extender on post-thaw sperm quality and fertility potential. Epididymal sperm were obtained from six bulls (n = 6) and a pool of epEVs was formed from the epididymal fluid of 15 bulls. After, a total of 30 × 106 sperm was cryopreserved in the presence (epEVs) or not (Control) of PKH67 green-labeled epEVs. Post-thaw, sperm were evaluated for interaction with epEVs by flow cytometry (FC) and fluorescence microscopy (FM); motility, vigor, and morphology by microscopy; membrane integrity by FC and FM; microRNA levels by qPCR; and fertility potential by in vitro embryo production (IVEP). EpEVs-sperm showed interaction with green-epEVs, and higher motility and plasma membrane integrity compared to Control-sperm. Levels of 28 microRNAs were higher or uniquely detected in epEVs-sperm. IVEP with epEVs-sperm resulted in a higher rate of hatched blastocyst. Thus, epEVs-supplemented to the sperm cryopreservation extender improved post-thaw sperm quality and embryo developmental potential. These findings provide new insights into the molecular modulation of sperm fertility.

During pregnancy, changes in the uterus-placenta-fetus complex are modulated by the presence of the conceptus and may determine pregnancy success. Many pathological changes in the uterus-placenta-fetus complex throughout the pregnancy of bovine embryos cloned by somatic cell nuclear transfer (SCNT) have been described. We hypothesized that maternal blood and allantoic-fluid biochemical profiles would differ during late gestation according to pregnancy origin (artificial insemination or SCNT) and subsequent outcome (live birth or pregnancy loss). Twenty-two healthy multiparous pregnant Nellore cows were used for blood and allantoic fluid collection every fortnight starting at 150 days of gestation until natural birth or abortion. Data were normalized to birth or abortion (day 0), and the three preceding fortnights were analyzed. A comprehensive description of reference values for various biochemical and blood gas parameters using serum and total blood samples, and a few blood gas parameters in allantoic fluid of naturally-produced (control; n = 6) and SCNT clone (clone; n = 16) pregnancies in Nellore cows is the most important result of this study. Serum cholesterol was higher in SCNT than control pregnancies; however, no diagnostic threshold or predictive-performance measure was evaluated. The central hypothesis of this study in which gestational changes in the uterus-placenta-fetus complex can be differentially detected earlier by biochemical and hemogasometric analyzes in maternal blood and/or in the allantois fluid of SCNT and control pregnancies was partially supported. Potassium, phosphorus, calcium, glucose, and urea levels differed between AI- and SCNT-derived pregnancies, indicating distinct gestational metabolism. Only cholesterol was higher in SCNT than control pregnancies and showed different temporal trajectories among outcome subgroups. Its metabolism could be related to abortion in SCNT pregnancies, but more investigations should be done.

The mammary gland is a modified sweat gland responsible for milk production. It is affected by diseases that reduce animals’ quality of life, consequently leading to economic losses in livestock. With advancements in tissue bioengineering and regenerative medicine, studying the extracellular matrix (ECM) of the bovine mammary gland can improve our understanding of its physiology and the processes that affect it. This knowledge could also enable the development of sustainable therapeutic alternatives for both the dairy production chain and human oncology research. A common approach in regenerative medicine is decellularization, a process that removes all cells from tissue while preserving its architecture and ECM components for subsequent recellularization. The success of recellularization depends on obtaining immunologically compatible scaffolds and using appropriate cell culture sources and methods to ensure tissue functionality. However, tissue culture technology still faces challenges due to specific requirements and high costs. Here, we review the literature on biomaterials and tissue engineering, providing an overview of the ECM of the bovine mammary gland and advances in its bioengineering, with a focus on regenerative medicine for bovine species. The methodology employed consists of a structured search of scientific databases, including PubMed, Google Scholar, and SciELO, using specific keywords related to tissue engineering and the bovine mammary gland. The selection criteria prioritized peer-reviewed articles published between 2002 and 2025 that demonstrated scientific relevance and contributed to the understanding of bovine mammary gland bioengineering. Although research on this topic has advanced, vascularization, tissue maturation, and scalability remain key barriers to widespread application and economic viability.

The development of liposomal formulations tailored for interaction with granulosa cells holds significant promise for applications in reproductive biotechnology. In this study, both anionic and cationic liposomes were formulated using purified and nonpurified phosphatidylcholines and produced using probe sonication followed by extrusion. Liposomes were characterized for size, polydispersity index (PDI), and surface charge, and evaluated for morphology, cytotoxicity, internalization efficiency, and effects on gene expression. Dynamic light scattering revealed nanometric sizes for both formulations (122.1 ± 2.1 nm for anionic and 119.4 ± 2.7 nm for cationic, respectively), with low PDI values (< 0.2), and stable zeta potentials (-31.7 ± 1.4 mV and 48.3 ± 2.6 mV, respectively). Morphology analyses confirmed spherical/near-spherical morphology. Cytotoxicity assays demonstrated that anionic liposomes were biocompatible across all tested concentrations, whereas cationic liposomes induced a dose-dependent effect. Fluorescence microscopy revealed efficient uptake of both liposomal types by granulosa cells, with cationic liposomes exhibiting intracellular fluorescence up to 12 h. Gene expression analysis targeting lipid metabolism, oxidative stress response, steroidogenesis, and apoptosis regulation genes indicated that neither liposomal formulation induced detrimental shifts in cellular homeostasis in the analyzed period. In conclusion, the findings support the use of differently charged liposomal formulations as promising platforms for intracellular delivery in cell-based applications, offering potential for future strategies involving the delivery of genetic material and subsequent modulation of cellular functions.

The first interactions among the embryo, endometrium, and corpus luteum are essential for pregnancy success. Small extracellular vesicles (sEVs) are part of these interactions. We previously demonstrated that small extracellular vesicles from in vivo- or in vitro-produced bovine embryos contain different miRNA cargos. Herein we show: (1) the presence and origin (in vivo or in vitro) of the blastocyst differentially reprograms endometrial transcriptional profiles; (2) the endometrial explant (EE) cultured with in vivo or in vitro embryos release small extracellular vesicles with different miRNA contents, and (3) the luteal explant (CLE) exposed to these small extracellular vesicles have distinct mRNA and miRNA profiles. To elucidate this, the endometrial explant were cultured in the presence or absence of a single Day-7 in vivo (EE-artificial insemination; EE-AI) or in vitro (EE-in vitro fertilization; EE-IVF) embryo. After of culture we found, in the endometrial explant, 45 and 211 differentially expressed genes associated with embryo presence and origin, respectively. Small extracellular vesicles were recovered from the conditioned media (CM) in which endometrial explant and embryos were co-cultured. Four miRNAs were differentially expressed between small extracellular vesicles from CC-EE-AI and CC-EE-IVF. Luteal explants exposed in culture to these small extracellular vesicles showed 1360 transcripts and 15 miRNAs differentially expressed. The differentially expressed genes associated with embryo presence and origin, modulating cells' proliferation, and survival. These results demonstrate that in vivo- or in vitro-produced bovine embryos induce molecular alterations in the endometrium; and that the embryo and endometrium release small extracellular vesicles capable of modifying the messenger RNA (mRNA) and miRNA profile in the corpus luteum. Therefore, the small extracellular vesicles-mediated embryo-endometrium-corpus luteum interactions possibly regulate the corpus luteum viability to ensure pregnancy success.

Major histocompatibility complex class I (MHC-I) gene expression in the placenta is modulated to tailor the maternal immune response to fetal antigens during pregnancy. This study evaluated MHC-I expression through immunohistochemistry (IHC) using an anti-mouse preimplantation embryo development (PED) clone Qa-2 and anti-bovine leukocyte antigen I (BoLA) monoclonal antibody clone IL-A88 (n = 23), as well as RT-qPCR (n = 17) for classical and non-classical (BoLA-NC) genes in control and cloned bovine placentomes during early and near-term gestation. Control samples showed minimal Qa-2 protein expression in early gestation, with intense labeling in trophoblasts and the maternal uterine epithelium near term. In contrast, cloned samples exhibited intense Qa-2 labeling in both maternal and trophoblastic epithelia at both stages, while trophoblast giant cells (TGCs), located apposed to the maternal epithelium, showed no labeling. Control samples exhibited intense IL-A88 labeling in the maternal epithelium at both stages. In cloned samples, weak to no labeling was observed in early gestation, with intense labeling in maternal and fetal epithelium near term. RT-qPCR revealed significant upregulation of BoLA-NC3 in early gestation, with sustained elevated expression in cloned samples in the near term. These findings suggest that altered BoLA protein expression and gene regulation in cloned pregnancies may contribute to pregnancy complications and increased losses.

Epigenetic alterations play a crucial role in the pathogenesis of cancer, as changes in the expression of DNA-associated proteins can affect gene expression. However, these changes may be reversible following treatment. This study aimed to evaluate the expression of acetylated histones H3 and H4 and the deacetylase enzymes HDACs 1, 2, and 6 in canine mammary tissues in order to identify potential alterations due to aberrant protein expression in neoplastic tissues. For this purpose, mammary tissue samples from 91 canine patients were divided into four groups: G1, control group composed of mammary tissues with no histopathological changes (n = 11); G2, simple mammary adenomas (n = 19); G3, simple mammary carcinomas without metastasis (n = 46); and G4, simple mammary carcinomas with lymph node metastasis (n = 15). The tissues were subjected to immunohistochemical analysis to assess protein expression. Antibody validation was performed by Western blot. The antibody expression results were evaluated semiquantitatively, considering the staining intensity and the percentage of marked cells. Univariate and multivariate analyses with a 5% significance level revealed differences in the expression of acetylated histones and deacetylase enzymes among the experimental groups (p < 0.05). Reduced acetylation of H3 (H3K9Ac) was observed in both nonmetastatic and metastatic simple mammary carcinomas compared to normal mammary tissue. Additionally, lower expression of HDAC1 and HDAC2 was found in neoplastic mammary tissues compared to normal tissue (p < 0.05). Conversely, HDAC6 exhibited higher expression in neoplastic mammary tissues (p < 0.05). There was no difference in the expression of acetylated H4 (H4K12Ac) among the groups (p > 0.05). Multivariate analysis showed a positive association between the expression of HDAC1 and HDAC2 and a negative association between H3K9Ac and HDAC6. These associations highlighted aberrant expression in mammary carcinomas compared to normal mammary tissues, indicating that epigenetic alterations exist in canine mammary neoplasms and that high HDAC6 expression may explain the observed hypoacetylation of H3 in neoplastic tissues. Collectively, these findings suggest that such alterations could potentially be therapeutic targets for the treatment of mammary cancer in dogs.

To analyze the effects of high body energy reserve (BER) within the oviductal environment and its composition, Nellore cows were fed two different nutritional plans to obtain animals with moderate BER (MBER) and high BER (HBER). After obtaining the groups with different BERs, all animals were subjected to oestrus synchronization and artificial insemination, and 120 hours after ovulation induction, the cows were slaughtered, the reproductive tract was removed, and the ipsilateral oviduct to the corpus luteum was collected and dissected. Analyses were performed only for animals that had an 8-cell embryo in the isthmus. After embryo identification, we evaluated the molecular profiles of extracellular vesicles from oviductal flushing (OF-EVs) and luminal epithelial cells (OV-Cell) and performed histomorphological analysis of oviductal tissue from the ampullary and isthmic oviductal regions. The HBER group presented higher concentrations of ampullary extracellular vesicles (AMP-EVs) and larger sizers of isthmic extracellular vesicles (IST-EVs). The miRNA profile of AMP-EVs showed that the differentially expressed miRNAs were predicted to regulate pathways associated with cell growth, migration, differentiation and metabolism, with the HBER group being more susceptible to insulin modulation. The MBER animals showed greater ampullary vascularization than the HBER animals did. Additionally, the miRNA profile and differential gene expression (DEG) data obtained for ampullary (AMP-Cell) and isthmic (IST-Cell) luminal epithelial cells revealed pathways related to insulin metabolism. Thus, elevated BER may lead to oviductal insulin resistance, affecting normal functioning and, probably, embryo metabolism during early development, thus impacting gestational rates in these animals.

Body energy reserves influence reproductive performance in cattle. Previous findings from our laboratory showed that cows with high body energy reserves (HBER) have lower ovulation and embryo recovery rates compared to cows with moderate reserves (MBER). To investigate whether these reproductive differences are associated with changes in the uterine environment, Nelore cows from the same herd were assigned to MBER or HBER groups through nutritional management. Following estrous synchronization and artificial insemination, animals were slaughtered ∼120 h after ovulation induction. Samples from the uterotubal junction (UTJ) and anterior uterine horn (ANT) were collected. Extracellular vesicles (EVs) were isolated from uterine fluid by flushing, and endometrial tissue was sampled for molecular analysis. Nanoparticle tracking analysis revealed no differences in EV concentration or size between groups. However, when comparing MBER and HBER groups, miRNA profiling identified 8 and 9 differentially expressed miRNAs between MBER and HBER in EVs from the UTJ and ANT, respectively, and 2 differentially expressed miRNAs in endometrial cells from the UTJ, suggesting potential differences in molecular profiles. Transcriptomic analysis of endometrial cells revealed 430 and 35 differentially expressed genes (DEGs) in the UTJ and ANT, respectively, between MBER and HBER groups. The higher number of DEGs in the UTJ may suggest a greater molecular response, which is reflected by more extensive pathway enrichment compared to the ANT. miRNA-mRNA integration, performed by intersecting predicted miRNA targets with the differentially expressed mRNAs from our RNA-seq data, suggests that differentially expressed genes may be regulated by miRNAs altered between groups, indicating miRNA-mediated effects of metabolic condition on the uterine transcriptome. These findings suggest that high body energy reserves are associated with enrichment of immune and metabolism related pathways in the uterine environment, especially in the UTJ, which may reflect a pro-inflammatory, metabolically altered state potentially impairing early embryo development and maternal-embryonic communication.

Assisted reproductive technology (ART) enhances livestock fertility and genetic quality while reducing disease transmission. In vitro embryo production (IVEP) is a crucial technique that requires optimization. Microfluidics has emerged as a promising platform for IVEP by offering a controlled environment with a continuous nutrient supply. This study introduces a novel non-toxic and reusable microdevice with reversible sealing designed for the in vitro maturation (IVM) of bovine cumulus oocyte complexes (COCs). The microdevice is constructed from PMMA and PDMSLAM on a glass slide, coated with Pluronic 167 F.The use of the microdevice for bovine IVM resulted in similar maturation rates (polar body extrusion rates) compared to the control (4-well-plate) (p = 0.73). Oocytes matured in the microdevices showed reduced lipid droplet accumulation (p = 0.0001), increased reactive oxygen species (ROS) levels (p = 0.0024), and higher glutathione (GSH) levels (p = 0.000002) compared to control. The expression of genes related to lipid metabolism and cellular stress was assessed in cumulus cells from COCs matured in microdevices or control plates, revealing the downregulation of SREPB1 in microdevice-matured COCs. Control and microdevice-matured oocytes were parthenogenetically activated to evaluate embryo development. Although no differences were found in cleavage and blastocyst rates, blastocysts from microdevice-matured COCs had a higher total cell count than controls (p = 0.0031). These findings suggest that microfluidics influence oocytes’ lipid metabolism, potentially enhancing early embryo development and increasing blastocyst cell numbers.

Dairy cows often experience a period of negative energy balance (NEB) during the post-calving period, which can significantly impact economic outcomes due to extended calving-to-conception intervals and overall reduced fertility. This reduction is due, in part, to the impact on uterine biology by high nonesterified fatty acids (NEFA) and beta-hydroxybutyrate concentration. The uterine fluid (UF) contains small extracellular vesicles (UF-EVs) that, through their cargo, including microRNAs (miRNAs), respond to metabolic stress, affecting the uterine environment. This study aimed to assess the long-term impact of NEB intensity on the uterine environment of dairy cows. Post-partum dairy cows were classified based on NEFA concentrations in their blood during the 3 weeks post-calving as having either Low or High NEB. At 30 and 60 DPC, the synchronization protocol was started, and UF samples were collected (corresponding to ~15 days after initiation of the synchronization protocol) to isolate UF-EVs and uterine epithelial cells for miRNA and transcriptome profiling. We also investigated whether UF-EVs could modulate epithelial uterine naïve cells. Our results indicate that the uterine environment of dairy cows experiencing a High NEB post-calving is unfavorable for embryo development at 60-day post-calving. Importantly, we show that UF-EVs can reproduce this phenotype in epithelial uterine naïve cells, suggesting that UF-EVs may act as modulators of the uterine response to metabolic challenges.

Developing embryos are susceptible to fluctuations in the nutrients and metabolites concentrations within the reproductive tract, which can lead to alterations in their developmental trajectory. Ketotic dairy cows have diminished fertility, and elevated levels of the ketone body beta-hydroxybutyrate (BHB) have been associated with poor embryonic development. We used an in vitro model based on either in vitro fertilization (IVF) or parthenogenesis to investigate the effects of BHB on the preimplantation bovine embryo development, epigenome, and transcriptome. Embryo culture medium was supplemented with BHB at a similar concentration to that present in the blood of cows suffering with severe ketosis, followed by analysis of blastocysts formation rate, diameter, total number of cells, levels of H3K9 beta-hydroxybutyrylation (H3K9bhb), apoptosis, and transcriptional alterations. As a result, we observed that BHB reduced the blastocysts rates, the diameter and the total number of cells in both parthenotes and IVF embryos. Exposure to BHB for either 3 or 7 days greatly increased the H3K9bhb levels in parthenotes at the 8-cells and blastocyst stages, and affected the expression of HDAC1, TET1, DNMT1, KDM6B, NANOG, and MTHFD2 genes. Additionally, culture of IVF embryos with BHB for 7 days dramatically increased H3K9bhb and reduced NANOG in blastocysts. RNA-seq analysis of IVF blastocysts revealed that BHB modulated the expression of 118 genes, which were involved with biological processes such as embryonic development, implantation, reproduction, proliferation, and metabolism. These findings provided valuable insights into the mechanisms through which BHB disrupts preimplantation embryonic development and affects the fertility in dairy cows.

In brief:Oocytes with subtle differences in chromatin configuration and nuclear lamina characteristics, detectable by a refined germinal vesicle (GV) classification system here described, respond differently to meiotic maturation systems leading to different in vitro maturation (IVM) outcomes. Abstract:The nuclear, cytoplasmic and molecular maturation of the mammalian oocyte is a finely orchestrated sequence of events that relies on proper cumulus-oocyte communication. Bovine oocytes enter the IVM systems at the GV stage exhibiting four different chromatin configurations (GV0-GV3). Herein, we associate the oocyte chromatin and nuclear lamina configurations to propose a refined GV classification (GV0, GV1.1-GV1.3, GV2.1-GV2.3 and GV3.1-GV3.3). This refined GV classification system was correlated with oocyte meiosis resumption and transzonal projections (TZPs) density of cumulus-oocyte complexes (COCs) submitted to three IVM systems (control IVM and a modified IVM preceded or not by a pre-IVM step). Pre-IVM resulted in lower polar body extrusion rates at 19 h IVM, albeit ∼24% of the oocytes extruded their first polar body at 9 h IVM. Pre-IVM sustained 80% of oocytes meiotically arrested but altered GV distribution, reducing GV2 and increasing GV1.3 and GV3.3 categories. Pre-IVM reduced TZP densities predominantly in pre-matured GV3 and GVBD COCs. At 9 h of IVM, both groups matured in modified IVM showed lower TZP densities compared to immature and IVM control. Gene expression supports the TZP density differences, with ERK2 and PRKACA upregulation in pre-matured cumulus and in modified IVM groups at 9 h of IVM. GDF9 and BMP15 levels were similar between treated and control groups at all time points. Our findings indicate that despite the IVM system, the initial oocyte GV stage influences pre-IVM and IVM outcomes. The refined GV classification system is a useful tool to oocyte biologists.

Despite >100 million years of mammal diversification, similar placental morphologies have independently evolved multiple times, presenting a long-standing evolutionary puzzle: what genetic mechanisms lead to convergent forms? MicroRNAs (miRNAs), despite their diversity in mammals and known roles as developmental regulators, remain under-examined as drivers of morphological evolution. We identified presence-absence patterns for 429 miRNA gene families across 300 eutherian genomes and discovered that placental phenotype is highly predictable from genomic miRNA repertoires (classification accuracy >94-97%). We identified 30 miRNA gene families associated with placentation type, whose gene targets are enriched for developmental processes. Notably, convergent placental morphologies consistently involve identical miRNA families, revealing that evolution is constrained to predictable genetic pathways. We demonstrate evidence of mechanism through MIR-11968, associated with cotyledonary placentation and displaying tissue-restricted expression in cows. MiRNA-mediated regulation therefore constrains placental morphological diversification into reproducible programs, offering insights into how genetic architecture shapes the predictability of convergent evolution. Significance Statement The genetic architecture that repeatedly guides distinct mammal lineages towards similar complex placental phenotypes has, until now, remained elusive. We leverage an extensive comparative genomic dataset of 429 microRNA gene families across 300 eutherian genomes and identify 30 specific microRNA gene families whose presence or absence patterns are strongly associated with different placentation types. We discover that placental phenotype is highly predictable from genomic microRNA repertoires, and that convergent placental morphologies consistently involve identical microRNA families. We provide evidence of the role MiR11968 in the cotyledonary placental phenotype, exhibiting tissue-restricted expression in cows. This striking pattern reveals a fundamental principle of evolution: that the genetic pathways available for complex morphological innovation are far more constrained and predictable than previously appreciated. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, BB/X007367/1, BB/R017522/1 São Paulo Research Foundation, 2016/22790-1, 2017/50438-3, 2018/14137-1 Leverhulme Trust, RF-2024-492

As observed in other species during pregnancy, bovine placentomes are assumed to suppress classical MHC I antigens, whereas overexpress non-classical MHC I to be recognized but not rejected by maternal immune system. However, in cloned bovine classical antigens were overexpress early in the pregnancy instead of non-classical ones. Then, MHC I antigens were investigated by immunohistochemistry (N=23) and RT-qPCR (N=16) in control and cloned bovine placentomes in early gestation and near term. In controls, IL-A88 staining was present in all stages, majorly in maternal tissues, whereas Qa-2 reacted only near term. Clones had an opposite pattern. Trophoblast giant cells near the uterine epithelium were weakly stained. By PCR, especially the non-classical isoform BoLA-NC3 resulted in significant higher expression in early gestation and down-regulation near term in controls compared to clones, as stain produced by Qa-2 antibody. The presence of MHC I in the placentomes indicated that bovine epitheliochorial placentas followed similar principles than that known for other species. Controls and clones reacted differently to the applied antibodies and differ partly in gene expression, which may explain pregnancy problems.

Somatic cell nuclear transfer (SCNT), or cloning, is used to reprogram cells and generate genetically identical embryos and animals. However, the cloning process is inefficient, limiting its application to producing valuable animals. In swine, cloning is mainly utilized to produce genetically modified animals. Indeed, recombinant DNA technologies have evolved considerably in recent years, with homologous recombination and gene editing technologies becoming more efficient and capable of recombining both alleles in a single cell. The selection of appropriate cells and their use as nuclear donors for SCNT is the most common method for generating edited and genetically modified animals for commercial and research purposes. This article reviews current applications of swine cloning and shares our personal experiences with the procedure in this species.