Yes Associated Transcriptional Regulator 1 (YAP1) and WW Domain Containing Transcription Regulator (WWTR1) are required for murine pregnancy initiation.

Endometrial stromal cell decidualization is required for pregnancy success. Although this process is integral to fertility, many of the intricate molecular mechanisms contributing to decidualization remain undefined. One pathway that has been implicated in endometrial stromal cell decidualization in humans in vitro is the Hippo signaling pathway. Two previously conducted studies showed that the effectors of the Hippo signaling pathway, YAP1 and WWTR1, were required for decidualization of primary stromal cells in culture. To investigate the in vivo role of YAP1 and WWTR1 in decidualization and pregnancy initiation, we generated a Progesterone Cre mediated partial double knockout (pdKO) of Yap1 and Wwtr1. Female pdKOs exhibited subfertility, a compromised decidualization response, partial interruption in embryo transport, blunted endometrial receptivity, delayed implantation and subsequent embryonic development, and a unique transcriptional profile. Bulk mRNA sequencing revealed aberrant maternal remodeling evidenced by significant alterations in extracellular matrix proteins at 7.5 days post-coitus in pdKO dams and enrichment for terms associated with fertility-compromising diseases like pre-eclampsia and endometriosis. Our results indicate a required role for YAP1 and WWTR1 for successful mammalian uterine function and pregnancy success.

Spatial Transcriptomic Analysis Identifies Epithelium-Macrophage Crosstalk in Endometriotic Lesions.

The mechanisms underlying the pathophysiology of endometriosis, characterized by the presence of endometrium-like tissue outside the uterus, remain poorly understood. This study aimed to identify cell type-specific gene expression changes in superficial peritoneal endometriotic lesions and elucidate the crosstalk among the stroma, epithelium, and macrophages compared to patient-matched eutopic endometrium. Surprisingly, comparison between lesions and eutopic endometrium revealed transcriptional similarities, indicating minimal alterations in the sub-epithelial stroma and epithelium of lesions. Spatial transcriptomics highlighted increased signaling between the lesion epithelium and macrophages, emphasizing the role of the epithelium in driving lesion inflammation. We propose that the superficial endometriotic lesion epithelium orchestrates inflammatory signaling and promotes a pro-repair phenotype in macrophages, providing a new role for Complement 3 in lesion pathobiology. This study underscores the significance of considering spatial context and cellular interactions in uncovering mechanisms governing disease in endometriotic lesions.

MicroRNA-210-3p Regulates Endometriotic Lesion Development by Targeting IGFBP3 in Baboons and Women with Endometriosis.

MicroRNAs (miRs) play an important role in the pathophysiology of endometriosis; however, the role of miR-210 in endometriosis remains unclear. This study explores the role of miR-210 and its targets, IGFBP3 and COL8A1, in ectopic lesion growth and development. Matched eutopic (EuE) and ectopic (EcE) endometrial samples were obtained for analysis from baboons and women with endometriosis. Immortalized human ectopic endometriotic epithelial cells (12Z cells) were utilized for functional assays. Endometriosis was experimentally induced in female baboons (n = 5). Human matched endometrial and endometriotic tissues were obtained from women (n = 9, 18-45 years old) with regular menstrual cycles. Quantitative reverse transcript polymerase chain reaction (RT-qPCR) analysis was performed for in vivo characterization of miR-210, IGFBP3, and COL8A1. In situ hybridization and immunohistochemical analysis were performed for cell-specific localization. Immortalized endometriotic epithelial cell lines (12Z) were utilized for in vitro functional assays. MiR-210 expression was decreased in EcE, while IGFBP3 and COL8A1 expression was increased in EcE. MiR-210 was expressed in the glandular epithelium of EuE but attenuated in those of EcE. IGFBP3 and COL8A1 were expressed in the glandular epithelium of EuE and were increased compared to EcE. MiR-210 overexpression in 12Z cells suppressed IGFBP3 expression and attenuated cell proliferation and migration. MiR-210 repression and subsequent unopposed IGFBP3 expression may contribute to endometriotic lesion development by increasing cell proliferation and migration.

Spheroids as a model for endometriotic lesions.

The development and progression of endometriotic lesions are poorly understood, but immune cell dysfunction and inflammation are closely associated with the pathophysiology of endometriosis. There is a need for 3D in vitro models to permit the study of interactions between cell types and the microenvironment. To address this, we developed endometriotic spheroids (ES) to explore the role of epithelial-stromal interactions and model peritoneal invasion associated with lesion development. Using a nonadherent microwell culture system, spheroids were generated with immortalized endometriotic epithelial cells (12Z) combined with endometriotic stromal (iEc-ESC) or uterine stromal (iHUF) cell lines. Transcriptomic analysis found 4,522 differentially expressed genes in ES compared with spheroids containing uterine stromal cells. The top increased gene sets were inflammation-related pathways, and an overlap with baboon endometriotic lesions was highly significant. Finally, to mimic invasion of endometrial tissue into the peritoneum, a model was developed with human peritoneal mesothelial cells in an extracellular matrix. Invasion was increased in the presence of estradiol or pro-inflammatory macrophages and suppressed by a progestin. Taken together, our results strongly support the concept that ES are an appropriate model for dissecting mechanisms that contribute to endometriotic lesion development.

Transcriptome and DNA methylome analyses reveal underlying mechanisms for the racial disparity in uterine fibroids.

Uterine fibroids (leiomyomas) affect Black women disproportionately compared with women of other races and ethnicities in terms of prevalence, incidence, and severity of symptoms. The causes of this racial disparity are essentially unknown. We hypothesized that myometria of Black women are more susceptible to developing fibroids, and we examined the transcriptomic and DNA methylation profiles of myometria and fibroids from Black and White women for comparison. Myometrial samples cluster by race in both their transcriptome and DNA methylation profiles, whereas fibroid samples only cluster by race in the latter. More differentially expressed genes (DEGs) were detected in the Black and White myometrial sample comparison than in the fibroid comparison. Leiomyoma gene set expression analysis identified 4 clusters of DEGs, including a cluster of 24 genes with higher expression in myometrial samples from Black women. One of the DEGs in this group, von Willibrands factor (VWF), was significantly hypomethylated in both myometrial samples from Black women and in all fibroids at 2 CpG probes that are near a putative enhancer site and that are correlated with VWF expression levels. These results suggest that the molecular basis for the disparity in fibroid disease between Black and White women could be found in the myometria before fibroid development and not in the fibroids themselves.

Obesity-induced follicular phase endometrial proteome dysregulation in a well-phenotyped population.

OBJECTIVE: Despite obesity's significant impact on reproduction, its influence on the physiology of the human endometrium is largely understudied. We hypothesized that endometrial proteomic differences exist between obese (OW; body mass index [BMI] ≥30 kg/m2) and normal-weight women (NWW; BMI, 18.5-24.9 kg/m2). DESIGN: Clinical cross-sectional study. SETTING: Academic Medical Center. PATIENT(S): Healthy, normally-cycling, 18 to 40-year-old women (n = 6 OW and n = 6 NWW). MAIN OUTCOME MEASURE(S): Participants underwent screening and midfollicular phase visits. Demographic and anthropometric characteristics, blood samples, ultrasounds, and follicular phase endometrial biopsies were collected. Proteomic analyses of endometrial samples (liquid chromatography-mass spectrometry) were performed. Proteins with ≥2-fold difference and a false discovery rate of <0.1 were considered statistically significant (Benjamini-Hochberg adjustment). RESULT(S): Reproductive hormone levels did not differ between the two groups. Mean BMI, serum leptin concentration, and bioelectrical impedance analysis indices of adiposity were higher in OW than in NWW. Histological examination of the endometrial samples confirmed normal-appearing endometrium in both OW and NWW. A total of 2,930 proteins were detected across all samples, with an average number of proteins per sample of 2,059 ± 482 in NWW and 2,437 ± 187 in OW. A total of 17 proteins were differentially expressed in OW vs. NWW; 2 were more abundant, whereas 15 were underexpressed in OW, including the progesterone receptor. CONCLUSION(S): In this well-phenotyped population of healthy women, obesity was associated with significant endometrial proliferative phase proteomic differences affecting the hormonal and immunologic pathways. These could contribute to an increased risk of menstrual bleeding abnormalities and create an altered environment for future luteinization.

Placental Transcriptome Adaptations to Maternal Nutrient Restriction in Sheep.

Placental development is modified in response to maternal nutrient restriction (NR), resulting in a spectrum of fetal growth rates. Pregnant sheep carrying singleton fetuses and fed either 100% (n = 8) or 50% (NR; n = 28) of their National Research Council (NRC) recommended intake from days 35-135 of pregnancy were used to elucidate placentome transcriptome alterations at both day 70 and day 135. NR fetuses were further designated into upper (NR NonSGA; n = 7) and lower quartiles (NR SGA; n = 7) based on day 135 fetal weight. At day 70 of pregnancy, there were 22 genes dysregulated between NR SGA and 100% NRC placentomes, 27 genes between NR NonSGA and 100% NRC placentomes, and 22 genes between NR SGA and NR NonSGA placentomes. These genes mediated molecular functions such as MHC class II protein binding, signaling receptor binding, and cytokine activity. Gene set enrichment analysis (GSEA) revealed significant overrepresentation of genes for natural-killer-cell-mediated cytotoxicity in NR SGA compared to 100% NRC placentomes, and alterations in nutrient utilization pathways between NR SGA and NR NonSGA placentomes at day 70. Results identify novel factors associated with impaired function in SGA placentomes and potential for placentomes from NR NonSGA pregnancies to adapt to nutritional hardship.

Transcriptome Analyses of Myometrium from Fibroid Patients Reveals Phenotypic Differences Compared to Non-Diseased Myometrium.

Uterine fibroid tissues are often compared to their matched myometrium in an effort to understand their pathophysiology, but it is not clear whether the myometria of uterine fibroid patients represent truly non-disease control tissues. We analyzed the transcriptomes of myometrial samples from non-fibroid patients (M) and compared them with fibroid (F) and matched myometrial (MF) samples to determine whether there is a phenotypic difference between fibroid and non-fibroid myometria. Multidimensional scaling plots revealed that M samples clustered separately from both MF and F samples. A total of 1169 differentially expressed genes (DEGs) (false discovery rate < 0.05) were observed in the MF comparison with M. Overrepresented Gene Ontology terms showed a high concordance of upregulated gene sets in MF compared to M, particularly extracellular matrix and structure organization. Gene set enrichment analyses showed that the leading-edge genes from the TGFß signaling and inflammatory response gene sets were significantly enriched in MF. Overall comparison of the three tissues by three-dimensional principal component analyses showed that M, MF, and F samples clustered separately from each other and that a total of 732 DEGs from F vs. M were not found in the F vs. MF, which are likely understudied in the pathogenesis of uterine fibroids and could be key genes for future investigation. These results suggest that the transcriptome of fibroid-associated myometrium is different from that of non-diseased myometrium and that fibroid studies should consider using both matched myometrium and non-diseased myometrium as controls.

Identification of Loci and Pathways Associated with Heifer Conception Rate in U.S. Holsteins.

Heifer conception rate (HCR) is defined as the percentage of inseminated heifers that become pregnant at each service. The genome-wide association analyses in this study focused on identifying the loci associated with Holstein heifer (n = 2013) conception rate at first service (HCR1) and the number of times bred (TBRD) to achieve a pregnancy. There were 348 unique loci associated (p < 5 × 10-8) with HCR1 and 615 unique loci associated (p < 5 × 10-8) with TBRD. The two phenotypes shared 302 loci, and 56 loci were validated in independent cattle populations. There were 52 transcription factor binding sites (TFBS) and 552 positional candidate genes identified in the HCR1- and TBRD-associated loci. The positional candidate genes and the TFBS associated with HCR1 and TBRD were used in the ingenuity pathway analysis (IPA). In the IPA, 11 pathways, 207 master regulators and 11 upstream regulators were associated (p < 1.23 × 10-5) with HCR1 and TBRD. The validated loci associated with both HCR1 and TBRD make good candidates for genomic selection and further investigations to elucidate the mechanisms associated with subfertility and infertility.

Protein Synthesis by Day 16 Bovine Conceptuses during the Time of Maternal Recognition of Pregnancy.

Interferon Tau (IFNT), the conceptus-derived pregnancy recognition signal in cattle, significantly modifies the transcriptome of the endometrium. However, the endometrium also responds to IFNT-independent conceptus-derived products. The aim of this study was to determine what proteins are produced by the bovine conceptus that may facilitate the pregnancy recognition process in cattle. We analysed by mass spectrometry the proteins present in conceptus-conditioned media (CCM) after 6 h culture of Day 16 bovine conceptuses (n = 8) in SILAC media (arginine- and lysine-depleted media supplemented with heavy isotopes) and the protein content of extracellular vesicles (EVs) isolated from uterine luminal fluid (ULF) of Day 16 pregnant (n = 7) and cyclic (n = 6) cross-bred heifers on day 16. In total, 11,122 proteins were identified in the CCM. Of these, 5.95% (662) had peptides with heavy labelled amino acids, i.e., de novo synthesised by the conceptuses. None of these proteins were detected in the EVs isolated from ULF. Pregnancy-associated glycoprotein 11, Trophoblast Kunitz domain protein 1 and DExD-Box Helicase 39A were de novo produced and present in the CCM from all conceptuses and in previously published CCM data following 6 and 24 h. A total of 463 proteins were present in the CCM from all the conceptuses in the present study, and after 6 and 24 h culture in a previous study, while expression of their transcripts was not detected in endometrium indicating that they are likely conceptus-derived. Of the proteins present in the EVs, 67 were uniquely identified in ULF from pregnant heifers; 35 of these had been previously reported in CCM from Day 16 conceptuses. This study has narrowed a set of conceptus-derived proteins that may be involved in EV-mediated IFNT-independent embryo-maternal communication during pregnancy recognition in cattle.

Extracellular vesicles: Novel regulators of conceptus-uterine interactions?

This review focuses on extracellular vesicles (EV) in the uterus and their potential biological roles as mediators of conceptus-uterine interactions essential for implantation and pregnancy establishment. Growing evidence supports the idea that EV are produced by both the endometrium and conceptus during pregnancy. Exosomes and microvesicles, collectively termed EV, mediate cell-cell communication in other tissues and organs. EV have distinct cargo, including lipids, proteins, RNAs, and DNA, that vary depending on the cell of origin and regulate processes including angiogenesis, adhesion, proliferation, cell survival, inflammation, and immune response in recipient cells. Molecular crosstalk between the endometrial epithelium and the blastocyst/conceptus, particularly the trophectoderm, regulates early pregnancy events and is a prerequisite for successful implantation. Trafficking of EV between the conceptus and endometrium may represent a key form of communication important for pregnancy establishment. Increased understanding of EV in the uterine environment and their physiological roles in endometrial-conceptus interactions is expected to provide opportunities to improve pregnancy success.

Characterization and regulation of extracellular vesicles in the lumen of the ovine uterus†.

Secretions of the endometrium are vital for peri-implantation growth and development of the sheep conceptus. Extracellular vesicles (EVs) are present in the uterine lumen, emanate from both the endometrial epithelia of the uterus and trophectoderm of the conceptus, and hypothesized to mediate communication between those cell types during pregnancy establishment in sheep. Size-exclusion chromatography and nanoparticle tracking analysis determined that total EV number in the uterine lumen increased from days 10 to 14 of the cycle but was lower on days 12 and 14 of pregnancy in sheep. Intrauterine infusions of interferon tau (IFNT) did not affect total EV number in the uterine lumen. Quantitative mass spectrometric analyses defined proteins and lipids in EVs isolated from the uterine lumen of day 14 cyclic and pregnant sheep. In vitro analyses found that EVs decreased ovine trophectoderm cell proliferation and increased IFNT production without effects on gene expression as determined by RNA-seq. Collective results support the idea EVs impact conceptus growth during pregnancy establishment via effects on trophectoderm cell growth.

Prostaglandin-endoperoxide synthase 2 is not required for preimplantation ovine conceptus development in sheep.

Conceptus development and elongation is required for successful pregnancy establishment in ruminants and is coincident with the production of interferon τ (IFNT) and prostaglandins (PGs). In both the conceptus trophectoderm and endometrium, PGs are primarily synthesized through a prostaglandin-endoperoxide synthase 2 (PTGS2) pathway and modify endometrial gene expression and thus histotroph composition in the uterine lumen to promote conceptus growth and survival. Chemical inhibition of PG production by both the endometrium and the conceptus prevented elongation in sheep. However, the contributions of conceptus-derived PGs to preimplantation conceptus development remain unclear. In this study, CRISPR-Cas9 genome editing was used to inactivate PTGS2 in ovine embryos to determine the role of PTGS2-derived PGs in conceptus development and elongation. PTGS2 edited conceptuses produced fewer PGs, but secreted similar amounts of IFNT to their Cas9 control counterparts and elongated normally. Expression of PTGS1 was lower in PTGS2 edited conceptuses, but PPARG expression and IFNT secretion were unaffected. Content of PGs in the uterine lumen was similar as was gene expression in the endometrium of ewes who received either Cas9 control or PTGS2 edited conceptuses. These results support the idea that intrinsic PTGS2-derived PGs are not required for preimplantation embryo or conceptus survival and development in sheep.

Genomic Analysis of Spontaneous Abortion in Holstein Heifers and Primiparous Cows.

BACKGROUND: The objectives of this study were to identify loci, positional candidate genes, gene-sets, and pathways associated with spontaneous abortion (SA) in cattle and compare these results with previous human SA studies to determine if cattle are a good SA model for humans. Pregnancy was determined at gestation day 35 for Holstein heifers and cows. Genotypes from 43,984 SNPs of 499 pregnant heifers and 498 pregnant cows that calved at full term (FT) were compared to 62 heifers and 28 cows experiencing SA. A genome-wide association analysis, gene-set enrichment analysis-single nucleotide polymorphism, and ingenuity pathway analysis were used to identify regions, pathways, and master regulators associated with SA in heifers, cows, and a combined population. RESULTS: Twenty-three loci and 21 positional candidate genes were associated (p < 1 × 10-5) with SA and one of these (KIR3DS1) has been associated with SA in humans. Eight gene-sets (NES > 3.0) were enriched in SA and one was previously reported as enriched in human SA. Four master regulators (p < 0.01) were associated with SA within two populations. CONCLUSIONS: One locus associated with SA was validated and 39 positional candidate and leading-edge genes and 2 gene-sets were enriched in SA in cattle and in humans.

Identification of loci associated with conception rate in primiparous Holstein cows.

BACKGROUND: Subfertility is a major issue facing the dairy industry as the average US Holstein cow conception rate (CCR) is approximately 35%. The genetics underlying the physiological processes responsible for CCR, the proportion of cows able to conceive and maintain a pregnancy at each breeding, are not well characterized. The objectives of this study were to identify loci, positional candidate genes, and transcription factor binding sites (TFBS) associated with CCR and determine if there was a genetic correlation between CCR and milk production in primiparous Holstein cows. Cows were bred via artificial insemination (AI) at either observed estrus or timed AI and pregnancy status was determined at day 35 post-insemination. Additive, dominant, and recessive efficient mixed model association expedited (EMMAX) models were used in two genome-wide association analyses (GWAA). One GWAA focused on CCR at first service (CCR1) comparing cows that conceived and maintained pregnancy to day 35 after the first AI (n = 494) to those that were open after the first AI (n = 538). The second GWAA investigated loci associated with the number of times bred (TBRD) required for conception in cows that either conceived after the first AI (n = 494) or repeated services (n = 472). RESULTS: The CCR1 GWAA identified 123, 198, and 76 loci associated (P < 5 × 10- 08) in additive, dominant, and recessive models, respectively. The TBRD GWAA identified 66, 95, and 33 loci associated (P < 5 × 10- 08) in additive, dominant, and recessive models, respectively. Four of the top five loci were shared in CCR1 and TBRD for each GWAA model. Many of the associated loci harbored positional candidate genes and TFBS with putative functional relevance to fertility. Thirty-six of the loci were validated in previous GWAA studies across multiple breeds. None of the CCR1 or TBRD associated loci were associated with milk production, nor was their significance with phenotypic and genetic correlations to 305-day milk production. CONCLUSIONS: The identification and validation of loci, positional candidate genes, and TFBS associated with CCR1 and TBRD can be utilized to improve, and further characterize the processes involved in cattle fertility.

Validation of 46 loci associated with female fertility traits in cattle.

BACKGROUND: Subfertility is one challenge facing the dairy industry as the average Holstein heifer conception rate (HCR), the proportion of heifers that conceive and maintain a pregnancy per breeding, is estimated at 55-60%. Of the loci associated with HCR, few have been validated in an independent cattle population, limiting their usefulness for selection or furthering our understanding of the mechanisms involved in successful pregnancy. Therefore, the objectives here were to identify loci associated with HCR: 1) to the first artificial insemination (AI) service (HCR1), 2) to repeated AI services required for a heifer to conceive (TBRD) and 3) to validate loci previously associated with fertility. Breeding and health records from 3359 Holstein heifers were obtained after heifers were bred by AI at observed estrus, with pregnancy determined at day 35 via palpation. Heifer DNA was genotyped using the Illumina BovineHD BeadChip, and genome-wide association analyses (GWAA) were performed with additive, dominant and recessive models using the Efficient Mixed Model Association eXpedited (EMMAX) method with a relationship matrix for two phenotypes. The HCR1 GWAA compared heifers that were pregnant after the first AI service (n = 497) to heifers that were open following the first AI service (n = 405), which included those that never conceived. The TBRD GWAA compared only those heifers which did conceive, across variable numbers of AI service (n = 712). Comparison of loci previously associated with fertility, HCR1 or TBRD were considered the same locus for validation when in linkage disequilibrium (D' > 0.7). RESULTS: The HCR1 GWAA identified 116, 187 and 28 loci associated (P < 5 × 10- 8) in additive, dominant and recessive models, respectively. The TBRD GWAA identified 235, 362, and 69 QTL associated (P < 5 × 10- 8) with additive, dominant and recessive models, respectively. Loci previously associated with fertility were in linkage disequilibrium with 22 loci shared with HCR1 and TBRD, 5 HCR1 and 19 TBRD loci. CONCLUSIONS: Loci associated with HCR1 and TBRD that have been identified and validated can be used to improve HCR through genomic selection, and to better understand possible mechanisms associated with subfertility.

Integrative analysis of the forkhead box A2 (FOXA2) cistrome for the human endometrium.

The pioneer forkhead box (FOX)A2 transcription factor is specifically expressed in the glands of the uterus, which are central to endometrial function and fertility. In mice, FOXA2 is a critical regulator of uterine gland development in the neonate and gland function in the adult. An integrative approach was used here to define the FOXA2 cistrome in the human endometrium. Genome-wide mapping of FOXA2 binding intervals by chromatin immunoprecipitation sequencing was performed using proliferative (P)- and midsecretory (MS)-phase endometrium and integrated with the transcriptome determined by RNA sequencing. Distinctive FOXA2 binding intervals, enriched for different transcription factor binding site motifs, were detected in the P and MS endometrium. Pathway analysis revealed different biologic processes regulated by genes with FOXA2 binding intervals in the P and MS endometrium. Thus, FOXA2 is postulated to regulate gene expression in concert with other transcription factors and impact uterine gland development and function in a cycle phase-dependent manner. Analyses also identified potential FOXA2-regulated genes that influence uterine receptivity, blastocyst implantation, and stromal cell decidualization, which are key events in pregnancy establishment.-Kelleher, A. M., Behura, S. K., Burns, G. W., Young, S. L., DeMayo, F. J., Spencer, T. E. Integrative analysis of the forkhead box A2 (FOXA2) cistrome for the human endometrium.

Bovine Genome Database: Tools for Mining the Bos taurus Genome.

The Bovine Genome Database (BGD; http://bovinegenome.org ) is a web-accessible resource that supports bovine genomics research by providing genome annotation and data mining tools. BovineMine is a tool within BGD that integrates BGD data, including the genome, genes, precomputed gene expression levels and variant consequences, with external data sources that include quantitative trait loci (QTL), orthologues, Gene Ontology, gene interactions, and pathways. BovineMine enables researchers without programming skills to create custom integrated datasets for use in downstream analyses. This chapter describes how to enhance a bovine genomics project using the Bovine Genome Database, with data mining examples demonstrating BovineMine.

Hymenoptera Genome Database: Using HymenopteraMine to Enhance Genomic Studies of Hymenopteran Insects.

The Hymenoptera Genome Database (HGD; http://hymenopteragenome.org ) is a genome informatics resource for insects of the order Hymenoptera, which includes bees, ants and wasps. HGD provides genome browsers with manual annotation tools (JBrowse/Apollo), BLAST, bulk data download, and a data mining warehouse (HymenopteraMine). This chapter focuses on the use of HymenopteraMine to create annotation data sets that can be exported for use in downstream analyses. HymenopteraMine leverages the InterMine platform to combine genome assemblies and official gene sets with data from OrthoDB, RefSeq, FlyBase, Gene Ontology, UniProt, InterPro, KEGG, Reactome, dbSNP, PubMed, and BioGrid, as well as precomputed gene expression information based on publicly available RNAseq. Built-in template queries provide starting points for data exploration, while the QueryBuilder tool supports construction of complex custom queries. The List Analysis and Genomic Regions search tools execute queries based on uploaded lists of identifiers and genome coordinates, respectively. HymenopteraMine facilitates cross-species data mining based on orthology and supports meta-analyses by tracking identifiers across gene sets and genome assemblies.