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.

Phthalates are detected in the follicular fluid of adolescents and oocyte donors with associated changes in the cumulus cell transcriptome.

Purpose: To investigate follicular fluid (FF) phthalate levels in adolescents undergoing fertility preservation compared to oocyte donors and explore its association with ovarian reserve and cumulus cell gene expression. Methods: 20 Adolescents (16.7 ± 0.6 years old) and 24 oocyte donors (26.2 ± 0.4 years old) undergoing fertility preservation were included in the study. Patient demographics, ovarian stimulation and oocyte retrieval outcomes were analyzed for each group. FF levels of 9 phthalate metabolites were assessed individually and as molar sums representative of common compounds (all phthalates: ΣPhthalates; DEHP: ΣDEHP), exposure sources (plastics: ΣPlastic; personal care products: ΣPCP), and modes of action (anti-androgenic: ΣAA) and compared between the two groups. Results: Follicular fluid ΣPlastic and ΣPCP levels were significantly higher in adolescents compared to oocyte donors (p<0.05). Follicular fluid ΣDEHP, ΣPlastic, ΣPCP, ΣAA, and ΣPhthalates levels were positively associated with antral follicle count (AFC) (p<0.05) in oocyte donors when adjusted for age, BMI, and race/ethnicity. RNA-seq analysis revealed 248 differentially expressed genes (DEGs) in cumulus cells of adolescents within the top quartile (n=4) of FF ΣPhthalates levels compared to the adolescents within the bottom half (n=9). Genes enriched in pathways involved in cell motility and development were significantly downregulated. Conclusion: Adolescents undergoing fertility preservation cycles demonstrate higher levels of phthalate metabolites in their follicular fluid compared to oocyte donors. Phthalate metabolite levels in FF are associated with higher AFC levels in oocyte donors. Higher phthalate levels in FF are associated with alterations in the cumulus cells transcriptome in adolescents.

Cumulus expansion is impaired with advanced reproductive age due to loss of matrix integrity and reduced hyaluronan.

Reproductive aging is associated with ovulatory defects. Age-related ovarian fibrosis partially contributes to this phenotype as short-term treatment with anti-fibrotic compounds improves ovulation in reproductively old mice. However, age-dependent changes that are intrinsic to the follicle may also be relevant. In this study, we used a mouse model to demonstrate that reproductive aging is associated with impaired cumulus expansion which is accompanied by altered morphokinetic behavior of cumulus cells as assessed by time-lapse microscopy. The extracellular matrix integrity of expanded cumulus-oocyte complexes is compromised with advanced age as evidenced by increased penetration of fluorescent nanoparticles in a particle exclusion assay and larger open spaces on scanning electron microscopy. Reduced hyaluronan (HA) levels, decreased expression of genes encoding HA-associated proteins (e.g., Ptx3 and Tnfaip6), and increased expression of inflammatory genes and matrix metalloproteinases underlie this loss of matrix integrity. Importantly, HA levels are decreased with age in follicular fluid of women, indicative of conserved reproductive aging mechanisms. These findings provide novel mechanistic insights into how defects in cumulus expansion contribute to age-related infertility and may serve as a target to extend reproductive longevity.

Photodynamic augmentation of oncolytic virus therapy for central nervous system malignancies.

Oncolytic viruses (OVs) have emerged as a clinical therapeutic modality potentially effective for cancers that evade conventional therapies, including central nervous system malignancies. Rationally designed combinatorial strategies can augment the efficacy of OVs by boosting tumor-selective cytotoxicity and modulating the tumor microenvironment (TME). Photodynamic therapy (PDT) of cancer not only mediates direct neoplastic cell death but also primes the TME to sensitize the tumor to secondary therapies, allowing for the combination of two potentially synergistic therapies with broader targets. Here, we created G47Δ-KR, clinical oncolytic herpes simplex virus G47Δ that expresses photosensitizer protein KillerRed (KR). Optical properties and cytotoxic effects of G47Δ-KR infection followed by amber LED illumination (peak wavelength: 585-595 nm) were examined in human glioblastoma (GBM) and malignant meningioma (MM) models in vitro. G47Δ-KR infection of tumor cells mediated KR expression that was activated by LED and produced reactive oxygen species, leading to cell death that was more robust than G47Δ-KR without light. In vivo, we tested photodynamic-oncolytic virus (PD-OV) therapy employing intratumoral injection of G47Δ-KR followed by laser light tumor irradiation (wavelength: 585 nm) in GBM and MM xenografts. PD-OV therapy was feasible in these models and resulted in potent anti-tumor effects that were superior to G47Δ-KR alone (without laser light) or laser light alone. RNA sequencing analysis of post-treatment tumor samples revealed PD-OV therapy-induced increases in TME infiltration of variable immune cell types. This study thus demonstrated the proof-of-concept that G47Δ-KR enables PD-OV therapy for neuro-oncological malignancies and warrants further research to advance potential clinical translation.

Do live birth rate and obstetric outcomes vary between immediate and delayed embryo transfers following freeze-all cycles?

RESEARCH QUESTION: Do live birth rates (LBR), obstetric and perinatal outcomes vary between women who underwent frozen embryo transfer (ET) in the immediately subsequent menstrual cycle, and with those who underwent delayed frozen ET. DESIGN: Retrospective cohort study (n = 198) consisting of 119 women who underwent immediate transfer within 30 days of oocyte retrieval (OR) and 79 women who underwent delayed transfer which was performed after >30 days following OR. Either flexible antagonist or flexible progestin-primed ovarian stimulation protocols were started after a baseline ultrasonography on the second or third day of menstrual cycle. Only freeze all cycles were included in the study and all transfers were with hormonal endometrial preparation. Main outcome measures were LBR, birth weight, gestational day at birth and pregnancy complications. RESULTS: Peak estradiol level on trigger day (2746 vs 2081 pg/ml) and number of metaphase-two oocytes (13 vs 10) were significantly higher in the immediate transfer group. Clinical pregnancy rate per ET was similar between the groups (50.4% vs 44.3%). However, miscarriage rate per positive pregnancy was significantly higher (12.3% vs 31.1%) while LBR per ET was significantly lower (42.9% vs 26.6%) in the delayed transfer group. Median gestational age at delivery were 267.5 and 268 days in the immediate and delayed transfer groups. Median birthweight was significantly higher in the delayed transfer group (3520 vs 3195 g). Adjusted analyses also suggest similar LBR with immediate and delayed transfer. CONCLUSION(S): Frozen ET in the immediate menstrual cycle and delayed ET, after a freeze all strategy did not show significant difference in terms of LBR after adjustment. Obstetric and perinatal outcomes of frozen ET in the immediate menstrual cycle appear reassuring.