FGF2, LIF, and IGF1 (FLI) supplementation during human in vitro maturation enhances markers of gamete competence.

STUDY QUESTION: Does a chemically defined maturation medium supplemented with FGF2, LIF, and IGF1 (FLI) improve in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) obtained from children, adolescents, and young adults undergoing ovarian tissue cryopreservation (OTC)? SUMMARY ANSWER: Although FLI supplementation did not increase the incidence of oocyte meiotic maturation during human IVM, it significantly improved quality outcomes, including increased cumulus cell expansion and mitogen-activated protein kinase (MAPK) expression as well as enhanced transzonal projection retraction. WHAT IS KNOWN ALREADY: During OTC, COCs, and denuded oocytes from small antral follicles are released into the processing media. Recovery and IVM of these COCs is emerging as a complementary technique to maximize the fertility preservation potential of the tissue. However, the success of IVM is low, especially in the pediatric population. Supplementation of IVM medium with FLI quadruples the efficiency of pig production through improved oocyte maturation, but whether a similar benefit occurs in humans has not been investigated. STUDY DESIGN, SIZE, DURATION: This study enrolled 75 participants between January 2018 and December 2021 undergoing clinical fertility preservation through the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago. Participants donated OTC media, accumulated during tissue processing, for research. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants who underwent OTC and include a pediatric population that encompassed children, adolescents, and young adults ≤22 years old. All participant COCs and denuded oocytes were recovered from media following ovarian tissue processing. IVM was then performed in either a standard medium (oocyte maturation medium) or one supplemented with FLI (FGF2; 40 ng/ml, LIF; 20 ng/ml, and IGF1; 20 ng/ml). IVM outcomes included meiotic progression, cumulus cell expansion, transzonal projection retraction, and detection of MAPK protein expression. MAIN RESULTS AND THE ROLE OF CHANCE: The median age of participants was 6.3 years, with 65% of them classified as prepubertal by Tanner staging. Approximately 60% of participants had been exposed to chemotherapy and/or radiation prior to OTC. On average 4.7 ± 1 COCs and/or denuded oocytes per participant were recovered from the OTC media. COCs (N = 41) and denuded oocytes (N = 29) were used for IVM (42 h) in a standard or FLI-supplemented maturation medium. The incidence of meiotic maturation was similar between cohorts (COCs: 25.0% vs 28.6% metaphase II arrested eggs in Control vs FLI; denuded oocytes: 0% vs 5.3% in Control vs FLI). However, cumulus cell expansion was 1.9-fold greater in COCs matured in FLI-containing medium relative to Controls and transzonal projection retraction was more pronounced (2.45 ± 0.50 vs 1.16 ± 0.78 projections in Control vs FLIat 16 h). Additionally, MAPK expression was significantly higher in cumulus cells obtained from COCs matured in FLI medium for 16-18 h (chemiluminescence corrected area 621,678 vs 2,019,575 a.u., P = 0.03). LIMITATIONS, REASONS FOR CAUTION: Our samples are from human participants who exhibited heterogeneity with respect to age, diagnosis, and previous treatment history. Future studies with larger sample sizes, including adult participants, are warranted to determine the mechanism by which FLI induces MAPK expression and activation. Moreover, studies that evaluate the developmental competence of eggs derived from FLI treatment, including assessment of embryos as outcome measures, will be required prior to clinical translation. WIDER IMPLICATIONS OF THE FINDINGS: FLI supplementation may have a conserved beneficial effect on IVM for children, adolescents, and young adults spanning the agricultural setting to clinical fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Department of Obstetrics and Gynecology startup funds (F.E.D.), Department of Surgery Faculty Practice Plan Grant and the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago (M.M.L. and E.E.R.). M.M.L. is a Gesualdo Foundation Research Scholar. Y.Y.'s research is supported by the internal research funds provided by Colorado Center of Reproductive Medicine. Y.Y., L.D.S., R.M.R., and R.S.P. have a patent pending for FLI. The remaining authors have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Associations of childhood allergies with parental reproductive and allergy history.

PURPOSE: There has been a noted parallel rise in both the use of Assisted Reproductive Technology (ART) to conceive and childhood allergies in the last few decades. The purpose of this study was to investigate the possible association between reproductive and allergy history in parents and allergies in their children. METHODS: This exploratory study used a cross-sectional study design and web-based survey to collect anonymous data on demographics, allergy, and health history from parents and about each of their children under 18 years of age. Children were stratified into two groups by allergy status (yes/no), and associations between each variable and the odds of allergies were tested using univariable and multivariable mixed logistic regression models. RESULTS: Of the 563 children in the study, 237 were reported to have allergies whereas 326 did not. Age, residential community, household income, mode of conception, paternal age at conception, biological parental allergy status, and history of asthma and eczema were significantly associated with allergies in univariable analysis. Multivariable analysis revealed household income ($50 k to $99 k vs ≥ $200 k adj OR = 2.72, 95% CI 1.11, 6.65), biological parental allergies (mother-adj OR 2.74, 95% CI 1.59, 4.72, father-adj OR 2.06, 95% CI 1.24, 3.41) and each additional year of age of children (adj OR 1.17, CI 1.10, 1.24) were significantly associated with odds of allergies in children. CONCLUSION: Although the exploratory nature of this convenience, snowballing sample limited the generalizability of the findings, initial observations warrant further investigation and validation in a larger more diverse population.

Low Molecular Weight Hyaluronan Induces an Inflammatory Response in Ovarian Stromal Cells and Impairs Gamete Development In Vitro.

The ovarian stroma, the microenvironment in which female gametes grow and mature, becomes inflamed and fibrotic with age. Hyaluronan is a major component of the ovarian extracellular matrix (ECM), and in other aging tissues, accumulation of low molecular weight (LMW) hyaluronan fragments can drive inflammation. Thus, we hypothesized that LMW hyaluronan fragments contribute to female reproductive aging by stimulating an inflammatory response in the ovarian stroma and impairing gamete quality. To test this hypothesis, isolated mouse ovarian stromal cells or secondary stage ovarian follicles were treated with physiologically relevant (10 or 100 µg/mL) concentrations of 200 kDa LMW hyaluronan. In ovarian stromal cells, acute LMW hyaluronan exposure, at both doses, resulted in the secretion of a predominantly type 2 (Th2) inflammatory cytokine profile as revealed by a cytokine antibody array of conditioned media. Additional qPCR analyses of ovarian stromal cells demonstrated a notable up-regulation of the eotaxin receptor Ccr3 and activation of genes involved in eosinophil recruitment through the IL5-CCR3 signaling pathway. These findings were consistent with an age-dependent increase in ovarian stromal expression of Ccl11, a major CCR3 ligand. When ovarian follicles were cultured in 10 or 100 µg/mL LMW hyaluronan for 12 days, gametes with compromised morphology and impaired meiotic competence were produced. In the 100 µg/mL condition, LMW hyaluronan induced premature meiotic resumption, ultimately leading to in vitro aging of the resulting eggs. Further, follicles cultured in this LMW hyaluronan concentration produced significantly less estradiol, suggesting compromised granulosa cell function. Taken together, these data demonstrate that bioactive LMW hyaluronan fragments may contribute to reproductive aging by driving an inflammatory stromal milieu, potentially through eosinophils, and by directly compromising gamete quality through impaired granulosa cell function.

Disruption of O-GlcNAc homeostasis during mammalian oocyte meiotic maturation impacts fertilization.

Meiotic maturation and fertilization are metabolically demanding processes, and thus the mammalian oocyte is highly susceptible to changes in nutrient availability. O-GlcNAcylation-the addition of a single sugar residue (O-linked ß-N-acetylglucosamine) on proteins-is a posttranslational modification that acts as a cellular nutrient sensor and likely modulates the function of oocyte proteins. O-GlcNAcylation is mediated by O-GlcNAc transferase (OGT), which adds O-GlcNAc onto proteins, and O-GlcNAcase (OGA), which removes it. Here we investigated O-GlcNAcylation dynamics in bovine and human oocytes during meiosis and determined the developmental sequelae of its perturbation. OGA, OGT, and multiple O-GlcNAcylated proteins were expressed in bovine cumulus oocyte complexes (COCs), and they were localized throughout the gamete but were also enriched at specific subcellular sites. O-GlcNAcylated proteins were concentrated at the nuclear envelope at prophase I, OGA at the cortex throughout meiosis, and OGT at the meiotic spindles. These expression patterns were evolutionarily conserved in human oocytes. To examine O-GlcNAc function, we disrupted O-GlcNAc cycling during meiotic maturation in bovine COCs using Thiamet-G (TMG), a highly selective OGA inhibitor. Although TMG resulted in a dramatic increase in O-GlcNAcylated substrates in both cumulus cells and the oocyte, there was no effect on cumulus expansion or meiotic progression. However, zygote development was significantly compromised following in vitro fertilization of COCs matured in TMG due to the effects on sperm penetration, sperm head decondensation, and pronuclear formation. Thus, proper O-GlcNAc homeostasis during meiotic maturation is important for fertilization and pronuclear stage development.

Histologic analysis and lipid profiling reveal reproductive age-associated changes in peri-ovarian adipose tissue.

BACKGROUND: Reproductive aging is a robust phenotype that occurs in all females and is characterized by a significant reduction in gamete quantity and quality, which can have negative consequences on both endocrine function and fertility. Age-associated differences in the oocyte, follicle, and ovary have been well-documented, but how the broader environment changes with age is less well understood. Fat is one of the largest organs in the body, and peri-gonadal adipose tissue surrounds the rodent ovary and comprises a local ovarian environment. The goal of this study was to characterize how peri-ovarian adipose tissue changes with advanced reproductive age. METHODS: We isolated peri-gonadal adipose tissue from two cohorts of CB6F1 mice: reproductively young (6-12 weeks) and reproductively old (14-17 months). A comparative histological analysis was performed to evaluate adipocyte architecture. We then extracted lipids from the tissue and performed multiple reaction monitoring (MRM)-profiling, a mass spectrometry-based method of metabolite profiling, to compare the lipid profiles of peri-gonadal adipose tissue in these age cohorts. RESULTS: We found that advanced reproductive age was associated with adipocyte hypertrophy and a corresponding decrease in the number of adipocytes per area. Of the 10 lipid classes examined, triacylglycerols (TAGs) had significantly different profiles between young and old cohorts, despite quantitative analysis revealing a decrease in the total amount of TAGs per weight of peri-gonadal adipose tissue with age. CONCLUSIONS: These findings pinpoint age-associated physiological changes in peri-gonadal adipose tissue with respect to adipocyte morphology and lipid profiles and lay the foundation for future studies to examine how these alterations may influence both adipocyte and ovarian function.

The oocyte microenvironment is altered in adolescents compared to oocyte donors.

Study question: Are the molecular signatures of cumulus cells (CCs) and follicular fluid (FF) of adolescents undergoing fertility preservation differ from that of reproductively adult oocyte donors? Summary answer: The microenvironment immediately surrounding the oocyte, including the CCs and FF, is altered in adolescents undergoing fertility preservation compared to oocyte donors. What is known already: Adolescents experience a period of subfecundity following menarche. Recent evidence suggests that this may be at least partially due to increased oocyte aneuploidy. Reproductive juvenescence in mammals is associated with suboptimal oocyte quality. Study design size duration: This was a prospective cohort study. Adolescents (10-19 years old, N=23) and oocyte donors (22-30 years old, N=31) undergoing ovarian stimulation and oocyte retrieval at the Northwestern Fertility and Reproductive Medicine Center between November 1, 2020 and May 1, 2023 were enrolled in this study. Participants/materials setting methods: Patient demographics, ovarian stimulation, and oocyte retrieval outcomes were collected for all participants. The transcriptome of CCs associated with mature oocytes was compared between adolescents (10-19 years old, n=19), and oocyte donors (22-30 years old, n=19) using bulk RNA-sequencing. FF cytokine profiles (10-19 years old, n=18 vs. 25-30 years old, n=16) were compared using cytokine arrays. Main results and the role of chance: RNA-seq analysis revealed 581 differentially expressed genes (DEGs) in cumulus cells of adolescents relative to oocyte donors, with 361 genes downregulated and 220 upregulated. Genes enriched in pathways involved in cell cycle and cell division (e.g., GO:1903047, p= 3.5 × 10-43; GO:0051983, p= 4.1 × 10-30; GO:0000281, p= 7.7 × 10-15; GO:0044839, p= 5.3 × 10-13) were significantly downregulated, while genes enriched in several pathways involved in cellular and vesicle organization (e.g., GO:0010256, p= 1.2 × 10-8; GO:0051129, p= 6.8 × 10-7; GO:0016050, p= 7.4 × 10-7; GO:0051640, p= 8.1 × 10-7) were upregulated in CCs of adolescents compared to oocyte donors. The levels of 9 cytokines were significantly increased in FF of adolescents compared to oocyte donors: IL-1 alpha (2-fold), IL-1 beta (1.7-fold), I-309 (2-fold), IL-15 (1.6-fold), TARC (1.9-fold), TPO (2.1-fold), IGFBP-4 (2-fold), IL-12-p40 (1.7-fold) and ENA-78 (1.4-fold). Interestingly, 7 of these cytokines have known pro-inflammatory roles. Importantly, neither the CC transcriptomes or FF cytokine profiles were different in adolescents with or without cancer. Large scale data: Original high-throughput sequencing data will be deposited in Gene Expression Omnibus (GEO) before publication, and the GEO accession number will be provided here. Limitations reasons for caution: This study aims to gain insights into the associated gamete quality by studying the immediate oocyte microenvironment. The direct study of oocytes is more challenging due to sample scarcity, as they are cryopreserved for future use, but will provide a more accurate assessment of oocyte reproductive potential. Wider implications of the findings: Understanding the underpinnings of altered immediate oocyte microenvironment of adolescent patients may provide insights into the reproductive potential of the associated gametes in the younger end of the age spectrum. This has implications for the fertility preservation cycles for very young patients. Study funding/competing interests: This project was supported by Friends of Prentice organization SP0061324 (M.M.L and E.B.), Gesualdo Family Foundation (Research Scholar: M.M.L.), and NIH/NICHD K12 HD050121 (E.B.). The authors have declared that no conflict of interest exists.

Exceptional longevity of mammalian ovarian and oocyte macromolecules throughout the reproductive lifespan.

The mechanisms contributing to age-related deterioration of the female reproductive system are complex, however aberrant protein homeostasis is a major contributor. We elucidated exceptionally stable proteins, structures, and macromolecules that persist in mammalian ovaries and gametes across the reproductive lifespan. Ovaries exhibit localized structural and cell-type specific enrichment of stable macromolecules in both the follicular and extrafollicular environments. Moreover, ovaries and oocytes both harbor a panel of exceptionally long-lived proteins, including cytoskeletal, mitochondrial, and oocyte-derived proteins. The exceptional persistence of these long-lived molecules suggest a critical role in lifelong maintenance and age-dependent deterioration of reproductive tissues.

Immature Follicular Origins and Disrupted Oocyte Growth Pathways Contribute to Decreased Gamete Quality During Reproductive Juvenescence in Mice.

Egg quality dictates fertility outcomes, and although there is a well-documented decline with advanced reproductive age, how it changes during puberty is less understood. Such knowledge is critical, since advances in Assisted Reproductive Technologies are enabling pre- and peri-pubertal patients to preserve fertility in the medical setting. Therefore, we investigated egg quality parameters in a mouse model of the pubertal transition or juvenescence (postnatal day; PND 11-40). Animal weight, vaginal opening, serum inhibin B levels, oocyte yield, oocyte diameter, and zona pellucida thickness increased with age. After PND 15, there was an age-associated ability of oocytes to resume meiosis and reach metaphase of meiosis II (MII) following in vitro maturation (IVM). However, eggs from the younger cohort (PND 16-20) had significantly more chromosome configuration abnormalities relative to the older cohorts and many were at telophase I instead of MII, indicative of a cell cycle delay. Oocytes from the youngest mouse cohorts originated from the smallest antral follicles with the fewest cumulus layers per oocyte, suggesting a more developmentally immature state. RNA Seq analysis of oocytes from mice at distinct ages revealed that the genes involved in cellular growth signaling pathways (PI3K, mTOR, and Hippo) were consistently repressed with meiotic competence, whereas genes involved in cellular communication were upregulated in oocytes with age. Taken together, these data demonstrate that gametes harvested during the pubertal transition have low meiotic maturation potential and derive from immature follicular origins.

Dental resins used in 3D printing technologies release ovo-toxic leachates.

We recently engineered the first female reproductive tract on a chip (EVATAR), to enable sex-based ex vivo research. To increase the scalability and accessibility of EVATAR, we turned to 3D printing (3DP) technologies, selecting two biocompatible 3DP resins, Dental SG (DSG) and Dental LT (DLT) to generate 3DP microphysiologic platforms. Due to the known sensitivity of reproductive cells to leachable compounds, we first screened for toxicity of these biomaterials using an in vitro mammalian oocyte maturation assay. Culture of mouse oocytes in 3DP plates using conventionally treated DSG resin resulted in rapid oocyte degeneration. Oxygen plasma treatment of the surface of printed DSG resin prevented this degeneration, and the majority of the resulting oocytes progressed through meiosis in vitro. However, 57.0% ± 37.2% of the cells cultured in the DSG resin plates exhibited abnormal chromosome morphology compared to 19.4% ± 17.3% of controls cultured in polystyrene. All tested DLT resin conditions, including plasma treatment, resulted in complete and rapid oocyte degeneration. To identify the ovo-toxic component of DLT, we analyzed DLT leachate using mass spectroscopy. We identified Tinuvin 292, a commercial light stabilizer, as a major component of the DLT leachate, which resulted in a dose-dependent disruption of meiotic progression and increase in chromosomal abnormalities with oocyte exposure, showing significant ovo-toxicity in mammals. Severe reproductive toxicity induced by in vitro exposure to these 3D-printed resins highlights potential risks of deploying insufficiently characterized materials for biomedical applications and underscores the need for more rigorous evaluation and designation of biocompatible materials.

Hyaluronan and Collagen Are Prominent Extracellular Matrix Components in Bovine and Porcine Ovaries.

The extracellular matrix (ECM) is a major component of the ovarian stroma. Collagen and hyaluronan (HA) are critical ovarian stromal ECM molecules that undergo age-dependent changes in the mouse and human. How these matrix components are regulated and organized in other mammalian species with reproductive characteristics similar to women such as cows and pigs, has not been systematically investigated. Therefore, we performed histological, molecular, and biochemical analyses to characterize collagen and HA in these animals. Bovine ovaries had more collagen than porcine ovaries when assessed biochemically, and this was associated with species-specific differences in collagen gene transcripts: Col3a1 was predominant in cow ovaries while Col1a1 was predominant in pig ovaries. We also observed more HA in the porcine vs. bovine ovary. HA was distributed across three molecular weight ranges (<100 kDa, 100-300 kDa, and >300 kDa) in ovarian tissue and follicular fluid, with tissue having more >300 kDa HA than the other two ranges. Transcripts for HA synthesis and degradation enzymes, Has3 and Hyal2, respectively, were predominant in cow ovaries, whereas Has2, Kiaa1199, and Tmem2 tended to be predominant in pig ovaries. Together, our findings have implications for the composition, organization, and regulation of the ovarian ECM in large mammalian species, including humans.

Characterization of Stress Responses in a Drosophila Model of Werner Syndrome.

As organisms age, their resistance to stress decreases while their risk of disease increases. This can be shown in patients with Werner syndrome (WS), which is a genetic disease characterized by accelerated aging along with increased risk of cancer and metabolic disease. WS is caused by mutations in WRN, a gene involved in DNA replication and repair. Recent research has shown that WRN mutations contribute to multiple hallmarks of aging including genomic instability, telomere attrition, and mitochondrial dysfunction. However, questions remain regarding the onset and effect of stress on early aging. We used a fly model of WS (WRNexoΔ) to investigate stress response during different life stages and found that stress sensitivity varies according to age and stressor. While larvae and young WRNexoΔ adults are not sensitive to exogenous oxidative stress, high antioxidant activity suggests high levels of endogenous oxidative stress. WRNexoΔ adults are sensitive to stress caused by elevated temperature and starvation suggesting abnormalities in energy storage and a possible link to metabolic dysfunction in WS patients. We also observed higher levels of sleep in aged WRNexoΔ adults suggesting an additional adaptive mechanism to protect against age-related stress. We suggest that stress response in WRNexoΔ is multifaceted and evokes a systemic physiological response to protect against cellular damage. These data further validate WRNexoΔ flies as a WS model with which to study mechanisms of early aging and provide a foundation for development of treatments for WS and similar diseases.

Ovulation and ovarian wound healing are impaired with advanced reproductive age.

Aging is associated with reduced tissue remodeling efficiency and increased fibrosis, characterized by excess collagen accumulation and altered matrix degradation. Ovulation, the process by which an egg is released from the ovary, is one of the most dynamic cycles of tissue wounding and repair. Because the ovary is one of the first organs to age, ovulation and ovarian wound healing is impaired with advanced reproductive age. To test this hypothesis, we induced superovulation in reproductively young and old mice and determined the numbers of eggs ovulated and corpora lutea (CLs), the progesterone producing glands formed post-ovulation. Reproductively old mice ovulated fewer eggs and had fewer CLs relative to young controls. Moreover, reproductively old mice exhibited a greater number of oocytes trapped within CLs and expanded cumulus oocyte complexes within unruptured antral follicles, indicative of failed ovulation. In addition, post-ovulatory tissue remodeling was compromised with age as evidenced by reduced CL vasculature, increased collagen, decreased hyaluronan, decreased cell proliferation and apoptosis, impaired wound healing capacity, and aberrant morphology of the ovarian surface epithelium (OSE). These findings demonstrate that ovulatory dysfunction is an additional mechanism underlying the age-related loss of fertility beyond the reduction of egg quantity and quality.

Ovarian stiffness increases with age in the mammalian ovary and depends on collagen and hyaluronan matrices.

Fibrosis is a hallmark of aging tissues which often leads to altered architecture and function. The ovary is the first organ to show overt signs of aging, including increased fibrosis in the ovarian stroma. How this fibrosis affects ovarian biomechanics and the underlying mechanisms are unknown. Using instrumental indentation, we demonstrated a quantitative increase in ovarian stiffness, as evidenced by an increase in Young's modulus, when comparing ovaries from reproductively young (6-12 weeks) and old (14-17 months) mice. This ovarian stiffness was dependent on collagen because ex vivo enzyme-mediated collagen depletion in ovaries from reproductively old mice restored their collagen content and biomechanical properties to those of young controls. In addition to collagen, we also investigated the role of hyaluronan (HA) in regulating ovarian stiffness. HA is an extracellular matrix glycosaminoglycan that maintains tissue homeostasis, and its loss can change the biomechanical properties of tissues. The total HA content in the ovarian stroma decreased with age, and this was associated with increased hyaluronidase (Hyal1) and decreased hyaluronan synthase (Has3) expression. These gene expression differences were not accompanied by changes in ovarian HA molecular mass distribution. Furthermore, ovaries from mice deficient in HAS3 were stiffer compared to age-matched WT mice. Our results demonstrate that the ovary becomes stiffer with age and that both collagen and HA matrices are contributing mechanisms regulating ovarian biomechanics. Importantly, the age-associated increase in collagen and decrease in HA are conserved in the human ovary and may impact follicle development and oocyte quality.

Evidence for premature aging in a Drosophila model of Werner syndrome.

Werner syndrome (WS) is an autosomal recessive progeroid disease characterized by patients' early onset of aging, increased risk of cancer and other age-related pathologies. WS is caused by mutations in WRN, a RecQ helicase that has essential roles responding to DNA damage and preventing genomic instability. While human WRN has both an exonuclease and helicase domain, Drosophila WRNexo has high genetic and functional homology to only the exonuclease domain of WRN. Like WRN-deficient human cells, Drosophila WRNexo null mutants (WRNexoΔ) are sensitive to replication stress, demonstrating mechanistic similarities between these two models. Compared to age-matched wild-type controls, WRNexoΔ flies exhibit increased physiological signs of aging, such as shorter lifespans, higher tumor incidence, muscle degeneration, reduced climbing ability, altered behavior, and reduced locomotor activity. Interestingly, these effects are more pronounced in females suggesting sex-specific differences in the role of WRNexo in aging. This and future mechanistic studies will contribute to our knowledge in linking faulty DNA repair mechanisms with the process of aging.