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Padmanabhan, V.

Publications and source records attributed to Padmanabhan, V..

15 recordsLinked to original sources
bioRxiv2026

Microporous Immune-Isolating Capsule with Improved Diffusion for Restored Dynamic Bidirectional Hormone Signaling in a Murine Model of Premature Ovarian Insufficiency

Pediatric cancer survivors treated with gonadotoxic chemotherapy or radiation face lifelong premature ovarian insufficiency (POI), leading to elevated risk of cardiovascular disease, osteoporosis, and metabolic dysfunction. Pharmacological hormone replacement therapy (HRT) cannot replicate the pulsatile, bidirectional signaling of the hypothalamic-pituitary-gonadal (HPG) axis, leaving a critical therapeutic gap. Immune-isolating hydrogel capsules offer a promising strategy for the implantation of donor ovarian tissue without immunosuppression yet they require optimization for human applications. Here, we engineer a microporous immune-isolating capsule by incorporating thermosensitive gelatin microgels as sacrificial porogens. Microfluidic fabrication yielded monodisperse microgels that dissolved at 37{degrees}C generating disconnected micropores within a non-degradable poly(ethylene glycol) (PEG) matrix. Critically, the diffusion of FSH-scale analogs (40 kDa) increased by almost two-fold through the microporous capsules relative to nanoporous controls, while antibody-scale molecules (150 kDa) were blocked, demonstrating size-discriminating permeability. In ovariectomized mice implanted with encapsulated ovarian xenografts for 20 weeks, microporous capsules restored dynamic HPG-axis signaling evidenced by elevated levels of estradiol and progesterone, FSH suppression, and fluctuating hormone levels that resembled physiological patterns. Microporosity also improved graft viability, increasing stromal cellularity and reducing follicular apoptosis. These findings support microporous immune-isolating capsules as a platform for physiologically authentic therapy for POI.

bioengineering↗
bioRxiv2026

Sex-specific multigenerational epigenetic responses to real-world chemical mixture exposure in an outbred sheep model

Establishing whether real-world environmental chemical (EC) exposure can induce heritable epigenetic modifications in large, outbred mammals is key to determining long-term developmental impacts of the human exposome. Using an established biosolids-treated pasture (BS) sheep model, we investigated whether gestational exposure to low-level mixtures of EC induced heritable changes in DNA methylation across three generations of sheep. Reduced-representation bisulfite sequencing of liver, blood, and sperm, combined with a structured, lineage-controlled breeding design, revealed widespread but lineage- and sex-specific differentially methylated loci (DML) in F1 offspring, with detectable alterations evident in F2 and F3 descendants. Although most DML were unique to individual sire lineages, or to a single generation, subsets of loci showed repeated involvement across generations and were associated with altered gene expression in F3 descendants. Sperm from F1 males exhibited reduced methylation at numerous loci and, together with seminal plasma, revealed differential expression of several microRNAs. These effects, however, showed limited persistence in F2 males, indicative of intergenerational rather than fully transgenerational persistence. Collectively, these findings demonstrate that complex, low-level chemical exposures can elicit recurrent, sexually dimorphic epigenetic responses in outbred species, but underscore the challenge of disentangling exposure-induced inheritance from genetically regulated methylation variation. Significance StatementEnvironmental chemical (EC) exposures are ubiquitous, yet their capacity to induce heritable epigenetic changes in large, genetically diverse mammals is poorly understood. Using a real-world exposome-based sheep model, we demonstrate that low-level gestational EC exposure leads to sexually-dimorphic and lineage-dependent alterations in DNA methylation that can extend to unexposed descendants. Although genetic ancestry exerts a dominant influence over these responses, repeated alterations at specific loci suggests that environmentally induced epimutations can reoccur across generations in certain genomic contexts.

developmental biology↗
bioRxiv2025

MAP hydrogel promotes revascularization of human ovarian graft and integration with the hypothalamic-pituitary axis in a mouse model of primary ovarian insufficiency

Ovarian tissue cryopreservation and autotransplantation (OTCT) is a crucial fertility preservation strategy for patients facing gonadotoxic cancer treatments, but its clinical success is hampered by ischemic injury and follicle loss following transplantation. This study aimed to enhance OTCT outcomes by employing microporous annealed particle (MAP) hydrogels to promote human ovarian graft revascularization. Unlike non-encapsulated tissue grafts, which exhibited early but transient and disorganized host vascular infiltration followed by regression, tissue grafts encapsulated in MAP hydrogels (OvaMAPs) demonstrated delayed yet organized and stable, long-term revascularization. OvaMAPs had significantly greater mouse CD31+ tissue area and vessel length after 3 and 6 weeks post-transplantation in ovariectomized immunodeficient mice compared to non-encapsulated grafts. By 20 weeks, both groups restored physiological estradiol levels (with OvaMAPs reaching 158 pg/mL) and suppressed follicle-stimulating hormone, confirming integration of the grafts with the hosts hypothalamic-pituitary axes. Notably, OvaMAPs achieved comparable endocrine function restoration with reduced estradiol variability, indicating more consistent graft function. In conclusion, MAP hydrogel encapsulation promoted long-term graft revascularization and vascular stability after OTCT, ultimately supporting consistent endocrine integration with host physiology.

bioengineering↗
bioRxiv2025

Transcriptomic and proteomic dynamics of ovarian follicle group culture resemble in vivo folliculogenesis

The prohibitively low yield of fertilizable oocytes obtained from cultured ovarian follicles limits clinical translation of in vitro follicle maturation for fertility preservation. This is in part due to an incomplete understanding of the process of follicle development. Previous work has demonstrated that group culture of primary murine follicles had a synergistic effect on growth and maturation in contrast to single follicles, but mechanisms remained unknown. Here, we cultured primary follicles in groups of 5 (5X) or 10 (10x) for twelve days, separated the somatic cells from oocytes, and analyzed the temporal transcriptional signatures every two days. In total, 13,461 genes in somatic cells and 10,091 genes in oocytes were computationally sorted into ten temporally distinct gene expression patterns. The somatic cell temporal gene expression patterns showed strong concordance with the granulosa and theca cell markers reported in a recent single-cell whole ovary RNA sequencing study. Importantly, canonical markers of steroidogenesis in cultured follicles followed expected trajectories of decreasing Amh expression and increasing Inhba, Inhbb, Cyp11a1, Cyp17a1, Cyp19a1, Lhcgr, and Fshr over the culture period. Furthermore, when comparing the 10X and 5X culture groups, we identified 306 and 14 differentially expressed genes in somatic cells and oocytes, respectively. Shotgun proteomics data was aligned with the somatic cell transcriptomic data and identified four L-R pairs that were differentially expressed between the two conditions. These comprehensive datasets uncovered temporal dynamics of in vitro folliculogenesis in a compartment-specific manner, serving as a valuable resource for optimizing future follicle culture systems for fertility preservation.

cell biology↗
bioRxiv2025

Functional Integration of Different-Sex Gonad Transplants into the Adult Mouse Hypothalamic Pituitary Gonadal Axis

Gender-affirming hormone therapy (GAHT) relies on exogenous hormones to produce hormonal milieus that achieve and/or maintain embodiment goals. Another potential route to these endpoints is transplantation of novel steroidogenic tissue. To develop a pre-clinical model, we asked whether different-sex gonad transplants can be functionally integrated into the adult mouse hypothalamic-pituitary-gonadal (HPG) axis. Adult male and female mice were gonadectomized and implanted with gonads from genetically matched but different-sex pups. Controls received gonads from same-sex pups. Temporal changes to gonadotropin and steroid hormone levels reveal the decoupling of the HPG following gonadectomy and gonad-dependent levels after transplanting donor gonads. After six weeks, histological structures in transplanted gonads were consistent with expected steroidogenesis and gametogenesis. Interestingly, pituitary, ARC and AVPV mRNA showed gonad- and sex-dependent expression patterns. Future work with this technique could lead to translation to gender affirming care and explorations of gonad-dependent sex differences in biomedical and basic research.

cell biology↗
bioRxiv2025

Single-oocyte transcriptional profile of early-stage human oocytes reveals differentially expressed genes in the primordial and transitioning stages.

The critical initial step in human oocyte maturation - the transition of ovarian follicles from dormancy to activation - remains poorly understood. Here we performed RNA sequencing on single oocytes isolated from early-stage follicles from nine healthy reproductive-age donors. Data for 133 high-quality oocytes formed two connected clusters, C1 and C2, with 5,449 significantly differentially expressed genes. Using recently reported gene lists for early-stage follicles we found that C1 oocytes likely came from earlier, dormant primordial follicles, while C2 oocytes match later-stage primordial or transitioning follicles. We sought to validate two DE genes, UHRF1 for C1 and CCN2 for C2, by their RNA-FISH in situ pattern in morphologically classified follicles, but did not observe statistically significant differences between follicle stages. This apparent discrepancy between follicles stage determined by its morphology and oocytes transcriptional state, if replicated in additional studies, may indicate a lack of closely coupled synchrony between follicle morphology and oocytes functional state.

cell biology↗
bioRxiv2025

Restoration of ovarian endocrine function with encapsulated immune isolated human ovarian xenograft in ovariectomized mice

Anti-cancer treatments cause premature depletion of the non-renewable ovarian reserve of follicles, the source of key steroid hormones, leading to premature ovarian insufficiency (POI) in 50% of pediatric cancer survivors. Patients with POI, especially at the onset of pubertal development, experience significant endocrine complications, including delayed growth, elevated risks of obesity and diabetes, and accelerated cardiovascular, musculoskeletal and neurological disorders as adults. The only approved pharmacological treatment for POI is an off-label prescribed hormone replacement therapy, which does not replace physiologically functioning ovaries. To restore production of ovarian hormones and protect against immune-mediated injury, we developed a hydrogel-based capsule for implantation of donor ovarian tissue. We evaluated the restoration of ovarian endocrine function in ovariectomized immunodeficient (NOD scid gamma, NSG) mice implanted with encapsulated xenografts over 20 weeks through daily vaginal cytology, hormone measurements and histological analysis of explanted human xenografts. The encapsulated xenografts integrated into the murine hypothalamus-pituitary-gonad (HPG) axis responding to circulating murine gonadotropins and restoring ovarian endocrine function. As controls, we implanted non encapsulated human ovarian xenografts comparable in size. Without the need for exogeneous stimulation, the estrous cyclicity resumed in both groups of mice 12 weeks post implantation and all mice regularly cycled experiencing between 3 to 8 estrous cycles in 20 weeks. The levels of estradiol gradually increased reaching on average 50pg/mL 20 weeks post implantation. Morphological analysis of the encapsulated grafts revealed presence of large antral follicles, [~]3mm in diameter, consistent with regular cyclicity and measurable levels of circulating hormones. This work demonstrates that endocrine function of encapsulated human ovarian tissue was not affected by the encapsulation and integrated with the host physiology similarly to the non-encapsulated controls.

bioengineering↗
bioRxiv2024

Functional Changes to Achilles Tendon and Enthesis in a Mouse Model of an Adolescent Masculine Gender-Affirming Hormone Treatment

Many transgender youth seek gender affirming care, such as puberty suppression, to prolong decision-making and to align their physical sex characteristics with their gender identity. During peripubertal growth, connective tissues such as tendon rapidly adapt to applied mechanical loads (e.g., exercise) yet if and how tendon adaptation is influenced by sex and gender affirming hormone therapy during growth remains unknown. The goal of this study was to understand the how pubertal suppression influences the structural and functional properties of the Achilles tendon using an established mouse model of transmasculine gender affirming hormone therapy. C57BL/6N female-born mice were assigned to experimental groups to mimic gender-affirming hormone therapy in human adolescents, and treatment was initiated prior to the onset of puberty (at postnatal day 26, P26). Experimental groups included controls and mice serially treated with gonadotropin release hormone analogue (GnRHa), delayed Testosterone (T), or GnRHa followed by T. We found that puberty suppression using GnRHa, with and without T, improved the overall tendon load capacity in female-born mice. Treatment with T resulted in an increase in the maximum load that tendon can withstand before failure. Additionally, we found that GnRHa, but not T, treatment resulted in a significant increase in cell density at the Achilles enthesis. NEW & NOTEWORTHYThese findings demonstrate that puberty suppression or testosterone does not negatively influence tendon structural or functional properties in a mouse model of transmasculine gender affirming care. In all treatment groups, the ability of the tendon to withstand load was significantly increased. Puberty suppression with GnRHa significantly increased enthesis cell density, suggesting an extended growth phase. These findings elucidate the effects of gender affirming care on the structural and functional properties of the tendon and enthesis.

physiology↗
bioRxiv2024

Placental and Immune Cell DNA Methylation Reference Panel for Bulk Tissue Cell Composition

To distinguish DNA methylation (DNAm) from cell proportion changes in whole placental tissue research, we developed a robust cell type-specific DNAm reference to estimate cell composition. We collated newly collected and existing cell type DNAm profiles quantified via Illumina EPIC or 450k microarrays. To estimate cell composition, we deconvoluted whole placental samples (n=36) with robust partial correlation based on the top 50 hyper- and hypomethylated sites per cell type. To test deconvolution performance, we evaluated RMSE in predicting principal component one of DNAm variation in 204 external placental samples. We analyzed DNAm profiles (n=368,435 sites) from 12 cell types: cytotrophoblasts (n=18), endothelial cells (n=19), Hofbauer cells (n=26), stromal cells (n=21), syncytiotrophoblasts (n=4), six lymphocyte types (n=36), and nucleated red blood cells (n=11). Median cell composition was consistent with placental biology: 60.4% syncytiotrophoblast, 17.1% stromal, 8.8% endothelial, 4.5% cytotrophoblast, 3.9% Hofbauer, 1.7% nucleated red blood cells, and 1.2% neutrophils. Our expanded reference outperformed an existing reference in predicting DNAm variation (15.4% variance explained, IQR=21.61) with cell composition estimates (RMSE:10.51 vs. 11.43, p-value<0.001). This cell type reference can robustly estimate cell composition from whole placental DNAm data to detect important cell types, reveal biological mechanisms, and improve casual inference.

genomics↗
bioRxiv2023

Bone quality following peripubertal growth in a mouse model of transmasculine gender-affirming hormone therapy

During peri-puberty, bone growth and the attainment peak bone mass is driven predominantly by sex steroids. This is important when treating transgender and gender diverse youth, who have become increasingly present at pediatric clinics. Analogues of gonadotropin-releasing hormone (GnRH) are commonly prescribed to transgender and gender diverse youth prior to starting gender-affirming hormone therapy (GAHT). However, the impact of GnRH agonists on long bones with the addition of GAHT is relatively unknown. To explore this, we developed a trans-masculine model by introducing either GnRHa or vehicle treatment to female-born mice at a pre-pubertal age. This treatment was followed by male GAHT (testosterone, T) or control treatment three weeks later. Six weeks after T therapy, bone quality was compared between four treatment groups: Control (vehicle only), GnRHa-only, GnRHa + T, and T-only. Bone length/size, bone shape, mechanical properties, and trabecular morphology were modulated by GAHT. Independent of GnRHa administration, mice treated with T had shorter femurs, larger trabecular volume and increased trabecular number, higher trabecular bone mineral density, and wider superstructures on the surface of bone (e.g., third trochanters) when compared to control or GnRHa-only mice. In conclusion, prolonged treatment of GnRHa with subsequent GAHT treatment directly affect the composition, parameters, and morphology of the developing long bone. These findings provide insight to help guide clinical approaches to care for transgender and gender diverse youth.

physiology↗
bioRxiv2023

A Template for Translational Bioinformatics: Facilitating Multimodal Data Analyses in Preclinical Models of Neurological Injury

BackgroundPediatric neurological injury and disease is a critical public health issue due to increasing rates of survival from primary injuries (e.g., cardiac arrest, traumatic brain injury) and a lack of monitoring technologies and therapeutics for the treatment of secondary neurological injury. Translational, preclinical research facilitates the development of solutions to address this growing issue but is hindered by a lack of available data frameworks and standards for the management, processing, and analysis of multimodal data sets. MethodsHere, we present a generalizable data framework that was implemented for large animal research at the Childrens Hospital of Philadelphia to address this technological gap. The presented framework culminates in an interactive dashboard for exploratory analysis and filtered data set download. ResultsCompared with existing clinical and preclinical data management solutions, the presented framework accommodates heterogeneous data types (single measure, repeated measures, time series, and imaging), integrates data sets across various experimental models, and facilitates dynamic visualization of integrated data sets. We present a use case of this framework for predictive model development for intra-arrest prediction of cardiopulmonary resuscitation outcome. ConclusionsThe described preclinical data framework may serve as a template to aid in data management efforts in other translational research labs that generate heterogeneous data sets and require a dynamic platform that can easily evolve alongside their research.

bioinformatics↗
bioRxiv2022

Developmental exposure to a real-life environmental chemical mixture alters testicular transcription factor expression in neonatal and pre-pubertal rams, with morphological changes persisting into adulthood

Environmental chemical (EC) exposure may be impacting male reproductive health. The translationally relevant biosolids treated pasture (BTP) sheep model was used to investigate gestational low-level EC mixture exposure on the testis of F1 male offspring. Adult rams from ewes exposed to BTP 1 month before and throughout pregnancy had more seminiferous tubules with degeneration and depletion of elongating spermatids, indicating "recovery" from previously reported testicular dysgenesis syndrome-like phenotype in neonatal and pre-pubertal BTP lambs. Expression of transcription factors CREB1 (neonatal) and BCL11A and FOXP2 (pre-pubertal) were significantly higher in the BTP exposed testes, with no changes seen in the adults. Increased CREB1, which is crucial for testes development and regulation of steroidogenic enzymes, could be an adaptive response to gestational EC exposure to facilitate the phenotypic recovery. Overall, this demonstrates that testicular effects from gestational exposure to low-level mixtures of ECs can last into adulthood, potentially impacting fertility and fecundity.

pharmacology and toxicology↗
bioRxiv2021

Placental gene expression-based cell type deconvolution: Cell proportions drive preeclampsia gene expression differences

The placenta mediates adverse pregnancy outcomes, including preeclampsia, which is characterized by gestational hypertension and proteinuria. Placental cell type heterogeneity in preeclampsia is not well-understood and limits mechanistic interpretation of bulk gene expression measures. We generated single-cell RNA-sequencing samples for integration with existing data to create the largest deconvolution reference of 19 fetal and 8 maternal cell types from placental villous tissue at term (n=15,532 cells). We deconvoluted eight published microarray case-control studies of preeclampsia (n=330). Deconvolution revealed excess extravillous trophoblasts and fewer mesenchymal cells. Adjustment for cellular composition reduced preeclampsia-associated differentially expressed genes (FDR<0.05) from 1,224 to 0, whereas pathway alterations exhibiting a metabolic adaptation to hypoxia were robust to cell type adjustment. Cellular composition explained 35.1% of the association between preeclampsia and FLT1 overexpression. Our findings indicate substantial placental cellular heterogeneity in preeclampsia that predicts previously observed bulk gene expression differences. Our deconvolution reference lays the groundwork for cellular heterogeneity-aware investigation into placental dysfunction and adverse birth outcomes.

developmental biology↗
bioRxiv2019

Developmental programming: Transcriptional regulation of visceral and subcutaneous adipose by prenatal bisphenol-A in female sheep

BackgroundBisphenol-A (BPA) exposure is widespread and early life exposure is associated with metabolic syndrome. While visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) are implicated in the development of metabolic syndrome, the adipose depot-specific effects of prenatal BPA treatment are poorly understood. ObjectiveTo determine the impact of prenatal BPA exposure on the transcriptome of VAT and SAT adipose depots. MethodsRNA sequencing was performed on SAT and VAT from 21-month old control and prenatal BPA-treated female sheep. Differences in transcriptional profiling of SAT and VAT in controls and the effect of prenatal BPA treatment on individual genes and gene pathways were determined. ResultsThere were 179 differentially expressed genes (padjusted<0.05, log2-fold change >2.5) between SAT and VAT. Development and immune response pathways were upregulated in SAT, while metabolic pathways were upregulated in VAT. In SAT, BPA-treatment resulted in differential expression of 108 genes (78% upregulated with BPA) and altered pathways (immune response downregulated, RNA processing upregulated). In contrast in VAT, BPA-treatment differentially expressed 4 genes and upregulated chromatin and RNA processing pathways. ConclusionPrenatal BPA-treatment induces adult depot-specific alterations in RNA expression in inflammation, RNA processing, and chromatin, reflecting the diverse roles of SAT and VAT in regulating lipid storage and insulin sensitivity. These adipose tissue transcriptional dysregulations may contribute to the metabolic disorders observed in prenatal BPA-treated female sheep.

pharmacology and toxicology↗