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.

Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980–2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.

Objectives: To compare different actigraphy scoring settings with polysomnography (PSG) for 1 night of total sleep time (TST), sleep efficiency (SE), wake after sleep onset (WASO), and sleep onset latency (SOL) in healthy pregnant women between 6 and 7 months of gestation. Design: Secondary analysis using data from a case-control study. Setting: A large university-affiliated hospital in the Midwestern United States. Participants: A total of 78 pregnant women were recruited, among which 38 healthy women with uncomplicated pregnancies were included for this analysis. Measurements: Participants had an overnight PSG assessment at a sleep center while simultaneously wearing an actigraph (Minimitter; Philips Respironics, Andover, MA). Sleep parameters from both devices included TST, SE, WASO, and SOL. Four scoring settings were used to obtain these parameters from actigraphy. Bland-Altman tests were used to evaluate the agreement between sleep variables scored independently from actigraphy and PSG. Results: The default scoring setting (10-by-40) yielded significantly different results from the PSG (P < .01). The 10 immobile/mobile minutes for sleep onset/end with an activity threshold of 10 (10-by-10) produced estimations of TST, SE, and WASO closest to those produced by PSG. When this setting was used, the mean differences between PSG- and actigraphy-assessed TST, SE, and WASO were −1.9 minutes, −0.4%, and 7.4 minutes. When the 10 and 15 immobile/mobile minutes for sleep onset/end were used, the difference between PSG- and actigraphy-assessed SOL was approximately 4 to 5 minutes. Conclusions Findings from this study do not support the use of default actigraph settings in pregnant women. In contrast, the 10-by-10 scoring setting provided the greatest agreement and least bias in comparison with PSG for sleep measurements. The 10-by-10 scoring setting is recommended to be used in studies consisting of pregnant women.

Female reproductive aging is characterized by a rise in follicle-stimulating hormone (FSH) levels during peri-menopause. N-linked glycans are co-translationally attached to the Asn7 and Asn24 residues on the FSHβ subunit. Differences in the number of N-glycans on the FSHβ subunit result in distinct glycoforms: hypo-glycosylated (FSH21/18, glycans absent on either Asn24 or Asn7, respectively) or fully-glycosylated (FSH24, glycans present on both Asn7 and Asn24). The relative abundance of FSH glycoforms changes with advanced reproductive age, shifting from predominantly FSH21/18 in younger women to FSH24 in older women. Previous in vitro studies in granulosa cell lines and in vivo studies using Fshb-null mice showed these glycoforms elicit differential bioactivities. However, the direct effects of FSH glycoforms on the mouse ovarian follicle have not yet been determined. In this study, we isolated secondary follicles from pre-pubertal mice and treated them with 20- or 100 ng/mL purified recombinant FSH glycoforms for 1 h or 18–20 h. Analysis of phosphorylated PKA substrates showed that glycoforms were bioactive in follicles following 1-h treatment, although differential bioactivity was only observed with the 100 ng/mL dose. Treatment of follicles with 100 ng/mL of each glycoform also induced distinct expression patterns of FSH-responsive genes as assessed by qPCR, consistent with differential function. Our results, therefore, indicate that FSH glycoforms are bioactive in isolated murine follicles.

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