Shear wave elastography to assess stiffness of the human ovary and other reproductive tissues across the reproductive lifespan in health and disease†.

The ovary is one of the first organs to show overt signs of aging in the human body, and ovarian aging is associated with a loss of gamete quality and quantity. The age-dependent decline in ovarian function contributes to infertility and an altered endocrine milieu, which has ramifications for overall health. The aging ovarian microenvironment becomes fibro-inflammatory and stiff with age, and this has implications for ovarian physiology and pathology, including follicle growth, gamete quality, ovulation dynamics, and ovarian cancer. Thus, developing a non-invasive tool to measure and monitor the stiffness of the human ovary would represent a major advance for female reproductive health and longevity. Shear wave elastography is a quantitative ultrasound imaging method for evaluation of soft tissue stiffness. Shear wave elastography has been used clinically in assessment of liver fibrosis and characterization of tendinopathies and various neoplasms in thyroid, breast, prostate, and lymph nodes as a non-invasive diagnostic and prognostic tool. In this study, we review the underlying principles of shear wave elastography and its current clinical uses outside the reproductive tract as well as its successful application of shear wave elastography to reproductive tissues, including the uterus and cervix. We also describe an emerging use of this technology in evaluation of human ovarian stiffness via transvaginal ultrasound. Establishing ovarian stiffness as a clinical biomarker of ovarian aging may have implications for predicting the ovarian reserve and outcomes of Assisted Reproductive Technologies as well as for the assessment of the efficacy of emerging therapeutics to extend reproductive longevity. This parameter may also have broad relevance in other conditions where ovarian stiffness and fibrosis may be implicated, such as polycystic ovarian syndrome, late off target effects of chemotherapy and radiation, premature ovarian insufficiency, conditions of differences of sexual development, and ovarian cancer. Summary sentence:  Shear Wave Elastography is a non-invasive technique to study human tissue stiffness, and here we review its clinical applications and implications for reproductive health and disease.

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.

Follicular fluid aids cell adhesion, spreading in an age independent manner and shows an age-dependent effect on DNA damage in fallopian tube epithelial cells.

Ovarian cancer (OC) is deadly, and likely arises from the fallopian tube epithelium (FTE). Despite the association of OC with ovulation, OC typically presents in post-menopausal women who are no longer ovulating. The goal of this study was to understand how ovulation and aging interact to impact OC progression from the FTE. Follicular fluid released during ovulation induces DNA damage in the FTE, however, the role of aging on FTE exposure to follicular fluid is unexplored. Follicular fluid samples were collected from 14 women and its effects on FTE cells was assessed. Follicular fluid caused DNA damage and lipid oxidation in an age-dependent manner, but instead induced cell proliferation in a dose-dependent manner, independent of age in FTE cells. Follicular fluid regardless of age disrupted FTE spheroid formation and stimulated attachment and growth on ultra-low attachment plates. Proteomics analysis of the adhesion proteins in the follicular fluid samples identified vitronectin, a glycoprotein responsible for FTE cell attachment and spreading.

Human follicular fluid elicits select dose- and age-dependent effects on mouse oocytes and cumulus-oocyte complexes in a heterologous in vitro maturation assay.

Follicular fluid (FF) is a primary microenvironment of the oocyte within an antral follicle. Although several studies have defined the composition of human FF in normal physiology and determined how it is altered in disease states, the direct impacts of human FF on the oocyte are not well understood. The difficulty of obtaining suitable numbers of human oocytes for research makes addressing such a question challenging. Therefore, we used a heterologous model in which we cultured mouse oocytes in human FF. To determine whether FF has dose-dependent effects on gamete quality, we performed in vitro maturation of denuded oocytes from reproductively young mice (6-12 weeks) in 10%, 50%, or 100% FF from participants of mid-reproductive age (32-36 years). FF impacted meiotic competence in a dose-dependent manner, with concentrations >10% inhibiting meiotic progression and resulting in spindle and chromosome alignment defects. We previously demonstrated that human FF acquires a fibro-inflammatory cytokine signature with age. Thus, to determine whether exposure to an aging FF microenvironment contributes to the age-dependent decrease in gamete quality, we matured denuded oocytes and cumulus-oocyte complexes (COCs) in FF from reproductively young (28-30 years) and old (40-42 years) participants. FF decreased meiotic progression of COCs, but not oocytes, from reproductively young and old (9-12 months) mice in an age-dependent manner. Moreover, FF had modest age-dependent impacts on mitochondrial aggregation in denuded oocytes and cumulus layer expansion dynamics in COCs, which may influence fertilization or early embryo development. Overall, these findings demonstrate that acute human FF exposure can impact select markers of mouse oocyte quality in both dose- and age-dependent manners.

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.

Oocyte quality is enhanced by hypoglycosylated FSH through increased cell-to-cell interaction during mouse follicle development.

Macroheterogeneity in follicle-stimulating hormone (FSH) ß-subunit N-glycosylation results in distinct FSH glycoforms. Hypoglycosylated FSH21 is the abundant and more bioactive form in pituitaries of females under 35 years of age, whereas fully glycosylated FSH24 is less bioactive and increases with age. To investigate whether the shift in FSH glycoform abundance contributes to the age-dependent decline in oocyte quality, the direct effects of FSH glycoforms on folliculogenesis and oocyte quality were determined using an encapsulated in vitro mouse follicle growth system. Long-term culture (10-12 days) with FSH21 (10 ng/ml) enhanced follicle growth, estradiol secretion and oocyte quality compared with FSH24 (10 ng/ml) treatment. FSH21 enhanced establishment of transzonal projections, gap junctions and cell-to-cell communication within 24 h in culture. Transient inhibition of FSH21-mediated bidirectional communication abrogated the positive effects of FSH21 on follicle growth, estradiol secretion and oocyte quality. Our data indicate that FSH21 promotes folliculogenesis and oocyte quality in vitro by increasing cell-to-cell communication early in folliculogenesis, and that the shift in in vivo abundance from FSH21 to FSH24 with reproductive aging may contribute to the age-dependent decline in oocyte quality.

Proteomic quantification of native and ECM-enriched mouse ovaries reveals an age-dependent fibro-inflammatory signature.

The ovarian microenvironment becomes fibrotic and stiff with age, in part due to increased collagen and decreased hyaluronan. However, the extracellular matrix (ECM) is a complex network of hundreds of proteins, glycoproteins, and glycans which are highly tissue specific and undergo pronounced changes with age. To obtain an unbiased and comprehensive profile of age-associated alterations to the murine ovarian proteome and ECM, we used a label-free quantitative proteomic methodology. We validated conditions to enrich for the ECM prior to proteomic analysis. Following analysis by data-independent acquisition (DIA) and quantitative data processing, we observed that both native and ECM-enriched ovaries clustered separately based on age, indicating distinct age-dependent proteomic signatures. We identified a total of 4,721 proteins from both native and ECM-enriched ovaries, of which 383 proteins were significantly altered with advanced age, including 58 ECM proteins. Several ECM proteins upregulated with age have been associated with fibrosis in other organs, but to date their roles in ovarian fibrosis are unknown. Pathways regulating DNA metabolism and translation were downregulated with age, whereas pathways involved in ECM remodeling and immune response were upregulated. Interestingly, immune-related pathways were upregulated with age even in ECM-enriched ovaries, suggesting a novel interplay between the ECM and the immune system. Moreover, we identified putative markers of unique immune cell populations present in the ovary with age. These findings provide evidence from a proteomic perspective that the aging ovary provides a fibroinflammatory milieu, and our study suggests target proteins which may drive these age-associated phenotypes for future investigation.

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.

CD38 regulates ovarian function and fecundity via NAD+ metabolism.

Mammalian female reproductive lifespan is typically significantly shorter than life expectancy and is associated with a decrease in ovarian NAD+ levels. However, the mechanisms underlying this loss of ovarian NAD+ are unclear. Here, we show that CD38, an NAD+ consuming enzyme, is expressed in the ovarian extrafollicular space, primarily in immune cells, and its levels increase with reproductive age. Reproductively young mice lacking CD38 exhibit larger primordial follicle pools, elevated ovarian NAD+ levels, and increased fecundity relative to wild type controls. This larger ovarian reserve results from a prolonged window of follicle formation during early development. However, the beneficial effect of CD38 loss on reproductive function is not maintained at advanced age. Our results demonstrate a novel role of CD38 in regulating ovarian NAD+ metabolism and establishing the ovarian reserve, a critical process that dictates a female's reproductive lifespan.

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.

Common mechanisms of physiological and pathological rupture events in biology: novel insights into mammalian ovulation and beyond.

Ovulation is a cyclical biological rupture event fundamental to fertilisation and endocrine function. During this process, the somatic support cells that surround the germ cell undergo a remodelling process that culminates in breakdown of the follicle wall and release of a mature egg. Ovulation is driven by known proteolytic and inflammatory pathways as well as structural alterations to the follicle vasculature and the fluid-filled antral cavity. Ovulation is one of several types of systematic remodelling that occur in the human body that can be described as rupture. Although ovulation is a physiological form of rupture, other types of rupture occur in the human body which can be pathological, physiological, or both. In this review, we use intracranial aneurysms and chorioamniotic membrane rupture as examples of rupture events that are pathological or both pathological and physiological, respectively, and compare these to the rupture process central to ovulation. Specifically, we compared existing transcriptomic profiles, immune cell functions, vascular modifications, and biomechanical forces to identify common processes that are conserved between rupture events. In our transcriptomic analysis, we found 12 differentially expressed genes in common among two different ovulation data sets and one intracranial aneurysm data set. We also found three genes that were differentially expressed in common for both ovulation data sets and one chorioamniotic membrane rupture data set. Combining analysis of all three data sets identified two genes (Angptl4 and Pfkfb4) that were upregulated across rupture systems. Some of the identified genes, such as Rgs2, Adam8, and Lox, have been characterised in multiple rupture contexts, including ovulation. Others, such as Glul, Baz1a, and Ddx3x, have not yet been characterised in the context of ovulation and warrant further investigation as potential novel regulators. We also identified overlapping functions of mast cells, macrophages, and T cells in the process of rupture. Each of these rupture systems share local vasoconstriction around the rupture site, smooth muscle contractions away from the site of rupture, and fluid shear forces that initially increase and then decrease to predispose one specific region to rupture. Experimental techniques developed to study these structural and biomechanical changes that underlie rupture, such as patient-derived microfluidic models and spatiotemporal transcriptomic analyses, have not yet been comprehensively translated to the study of ovulation. Review of the existing knowledge, transcriptomic data, and experimental techniques from studies of rupture in other biological systems yields a better understanding of the physiology of ovulation and identifies avenues for novel studies of ovulation with techniques and targets from the study of vascular biology and parturition.

Morphokinetic parameters of mouse oocyte meiotic maturation and cumulus expansion are not affected by reproductive age or ploidy status.

INTRODUCTION: Morphokinetic analysis using a closed time-lapse monitoring system (EmbryoScope + ™) provides quantitative metrics of meiotic progression and cumulus expansion. The goal of this study was to use a physiologic aging mouse model, in which egg aneuploidy levels increase, to determine whether there are age-dependent differences in morphokinetic parameters of oocyte maturation. METHODS: Denuded oocytes and intact cumulus-oocyte complexes (COCs) were isolated from reproductively young and old mice and in vitro matured in the EmbryoScope + ™. Morphokinetic parameters of meiotic progression and cumulus expansion were evaluated, compared between reproductively young and old mice, and correlated with egg ploidy status. RESULTS: Oocytes from reproductively old mice were smaller than young counterparts in terms of GV area (446.42 ± 4.15 vs. 416.79 ± 5.24 µm2, p < 0.0001) and oocyte area (4195.71 ± 33.10 vs. 4081.62 ± 41.04 µm2, p < 0.05). In addition, the aneuploidy incidence was higher in eggs with advanced reproductive age (24-27% vs. 8-9%, p < 0.05). There were no differences in the morphokinetic parameters of oocyte maturation between oocytes from reproductively young and old mice with respect to time to germinal vesicle breakdown (GVBD) (1.03 ± 0.03 vs. 1.01 ± 0.04 h), polar body extrusion (PBE) (8.56 ± 0.11 vs. 8.52 ± 0.15 h), duration of meiosis I (7.58 ± 0.10 vs. 7.48 ± 0.11 h), and kinetics of cumulus expansion (0.093 ± 0.002 vs. 0.089 ± 0.003 µm/min). All morphokinetic parameters of oocyte maturation were similar between euploid and aneuploid eggs irrespective of age. CONCLUSION: There is no association between age or ploidy and the morphokinetics of mouse oocyte in vitro maturation (IVM). Future studies are needed to evaluate whether there is an association between morphokinetic dynamics of mouse IVM and embryo developmental competence.

Ovarian volume partially explains associations of phthalate biomarkers with anti-Müllerian hormone and estradiol in midlife women.

BACKGROUND/OBJECTIVES: Women are ubiquitously exposed to endocrine disruptors, including phthalates. Ovarian follicles undergoing folliculogenesis (indirectly measured by ovarian volume) produce anti-Müllerian hormone (AMH) and estradiol (E2). We evaluated associations of phthalates with ovarian volume to assess whether this explained prior positive associations of phthalates with AMH and E2. METHODS: Women ages 45-54 years (n = 614) had transvaginal ultrasounds of right/left ovaries to calculate mean ovarian volume. Women provided up-to-four urine and blood samples for quantifying AMH (first serum sample), E2 (all serum samples), and nine phthalate metabolites (from pooled urine, representing six parent phthalates). Multivariable linear or logistic regression models (for individual phthalate biomarkers), as well as weighted quantile sum (WQS) regression (for mixture analyses) evaluated associations of phthalate biomarkers with ovarian volume. Using cross-sectional mediation analysis, we assessed whether associations of phthalates with ovarian volume partially explained those of phthalates with AMH or E2. RESULTS: Most women were non-Hispanic White (68%) and pre-menopausal (67%) with higher urinary phthalate metabolite concentrations than U.S. women. In single-pollutant models, 10% increases in mono(3-carboxypropyl) phthalate (MCPP) and monobenzyl phthalate (MBzP) were associated with 0.44% (95% CI: -0.02%, 0.91%) and 0.62% (95% CI: 0.02%, 1.23%) larger ovarian volumes, respectively. As a cumulative mixture, 10% increases in the phthalate mixture were associated with 2.89% larger ovarian volume (95%CI: 0.27, 5.59) with MCPP (35%) and MBzP (41%) identified as major contributors. Higher ovarian volume due to a 10% increase in MBzP (indirect effect OR: 1.004; 95% CI: 1.00, 1.01) explained 16% of the positive association between MBzP and higher AMH, whereas higher ovarian volume due to a 10% increase in MCPP (indirect effect %Δ: 0.11; 95% CI: -0.01, 0.22) explained 23% of the positive association between MCPP and E2. CONCLUSION: In this cross-sectional study, phthalates were associated with increased ovarian volume, with implications for midlife hormone production.

An ex vivo ovulation system enables the discovery of novel ovulatory pathways and nonhormonal contraceptive candidates†.

Ovulation is an integral part of women's menstrual cycle and fertility. Understanding the mechanisms of ovulation has broad implications for the treatment of anovulatory diseases and the development of novel contraceptives. Now, few studies have developed effective models that both faithfully recapitulate the hallmarks of ovulation and possess scalability. We established a three-dimensional encapsulated in vitro follicle growth (eIVFG) system that recapitulates folliculogenesis and produces follicles that undergo ovulation in a controlled manner. Here, we determined whether ex vivo ovulation preserves molecular signatures of ovulation and demonstrated its use in discovering novel ovulatory pathways and nonhormonal contraceptive candidates through a high-throughput ovulation screening. Mature murine follicles from eIVFG were induced to ovulate ex vivo using human chorionic gonadotropin and collected at 0, 1, 4, and 8 hours post-induction. Phenotypic analyses confirmed key ovulatory events, including cumulus expansion, oocyte maturation, follicle rupture, and luteinization. Single-follicle RNA-sequencing analysis revealed the preservation of ovulatory genes and dynamic transcriptomic profiles and signaling. Soft clustering identified distinct gene expression patterns and new pathways that may critically regulate ovulation. We further used this ex vivo ovulation system to screen 21 compounds targeting established and newly identified ovulatory pathways. We discovered that proprotein convertases activate gelatinases to sustain follicle rupture and do not regulate luteinization and progesterone secretion. Together, our ex vivo ovulation system preserves molecular signatures of ovulation, presenting a new powerful tool for studying ovulation and anovulatory diseases as well as for establishing a high-throughput ovulation screening to identify novel nonhormonal contraceptives for women.

Anatomic nomenclature and 3-dimensional regional model of the human ovary: call for a new paradigm.

The ovaries are the female gonads that are crucial for reproduction, steroid production, and overall health. Historically, the ovary was broadly divided into regions defined as the cortex, medulla, and hilum. This current nomenclature lacks specificity and fails to consider the significant anatomic variations in the ovary. Recent technological advances in imaging modalities and high-resolution omic analyses have brought about the need for revision of the existing definitions, which will facilitate the integration of generated data and enable the characterization of organ subanatomy and function at the cellular level. The creation of these high-resolution multimodal maps of the ovary will enhance collaboration and communication among disciplines and between clinicians and researchers. Beginning in March 2021, the Pediatric and Adolescent Gynecology Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development invited subject-matter experts to participate in a series of workshops and meetings to standardize ovarian nomenclature and define the organ's features. The goal was to develop a spatially defined and semantically consistent terminology of the ovary to support collaborative, team science-based endeavors aimed at generating reference atlases of the human ovary. The group recommended a standardized, 3-dimensional description of the ovary and an ontological approach to the subanatomy of the ovary and definition of follicles. This new greater precision in nomenclature and mapping will better reflect the ovary's heterogeneous composition and function, support the standardization of tissue collection, facilitate functional analyses, and enable clinical and research collaborations. The conceptualization process and outcomes of the effort, which spanned the better part of 2021 and early 2022, are introduced in this article. The institute and the workshop participants encourage researchers and clinicians to adopt the new systems in their everyday work to advance the overarching goal of improving human reproductive health.

Recapitulating folliculogenesis and oogenesis outside the body: encapsulated in vitro follicle growth†.

Folliculogenesis is a tightly coordinated process essential for generating a fertilization-competent gamete while also producing gonadal hormones that sustain endocrine function. In vitro follicle growth systems have been critical to our understanding of key events in folliculogenesis, such as gonadotropin-independent and dependent growth, steroid hormone production, and oocyte growth and maturation (cytoplasmic and meiotic). Although there are several successful follicle culture strategies, the following protocol details an encapsulated in vitro follicle growth (eIVFG) system for use with mouse ovarian follicles. Encapsulated IVFG is performed with alginate hydrogels, which are biologically inert, maintains cell-to-cell interactions between granulosa cells and the oocyte, and preserves follicle architecture as found in the ovary. The system supports follicle growth, development, and differentiation from the early primary follicle to the antral follicle stage. Moreover, post-folliculogenesis events including meiotic maturation, ovulation, and luteinization are also supported. Importantly, the culture of secondary follicles has successfully resulted in viable pups after blastocyst transfer. This alginate-based eIVFG system is versatile and has broad applications as a tool for interrogating the fundamental biology of the ovarian follicle in a controlled manner, a screening platform for toxicity and bioactivity, and a potential fertility preservation method for endangered species as well as humans.