A blueprint of the topology and mechanics of the human ovary for next-generation bioengineering and diagnosis

Although the first dissection of the human ovary dates back to the 17th century, the biophysical characteristics of the ovarian cell microenvironment are still poorly understood. However, this information is vital to deciphering cellular processes such as proliferation, morphology and differentiation, as well as pathologies like tumor progression, as demonstrated in other biological tissues. Here, we provide the first readout of human ovarian fiber morphology, interstitial and perifollicular fiber orientation, pore geometry, topography and surface roughness, and elastic and viscoelastic properties. By determining differences between healthy prepubertal, reproductive-age, and menopausal ovarian tissue, we unravel and elucidate a unique biophysical phenotype of reproductive-age tissue, bridging biophysics and female fertility. While these data enable to design of more biomimetic scaffolds for the tissue-engineered ovary, our analysis pipeline is applicable for the characterization of other organs in physiological or pathological states to reveal their biophysical markers or design their bioinspired analogs.

P–699 Multi-scale study of the architecture, topography and mechanics of the human ovary from prepuberty to menopause: a blueprint for next-generation bioengineering and diagnosis

Study question Does the ovarian ECM have a precise and unique biophysical phenotype, specific to each age, from prepuberty to menopause? Summary answer Differences between healthy prepubertal, reproductive-age, and menopausal ovarian tissue, unravel and elucidate a unique biophysical phenotype of reproductive-age tissue, bridging biophysics and female fertility. What is known already Ovarian engineering has recently emerged to respond to patient needs and offer reliable models for basic research. It has relied on synthetic and natural biomaterials and microfluidics. However, these techniques were designed based on knowledge acquired from 2D cell culture and animal models. Our lack of information on the human ovary hampers our ability to mimic the main features of this organ, for clinical applications. The complex composition and hierarchical structure of its ECM complicates the design of truly biomimetic constructs, notably: fiber morphology, interstitial and perifollicular fiber orientation, porosity, topography, and viscoelasticity, which all play a role in mechanotransduction. Study design, size, duration Ovarian biopsies were taken from prepubertal (mean age [±SD]=7±3 years, n = 21), reproductive age (mean age [±SD]=27±5, n = 26 ) and menopausal (mean age [±SD]=61±6 years, n = 29) patients after obtaining their informed consent. All participating adult subjects were undergoing laparoscopic surgery for benign gynecological diseases not affecting the ovaries. Prepubertal tissue was derived from young cancer patients scheduled for ovarian cortex cryopreservation as a fertility preservation strategy, before being subjected to acute gonadotoxic cancer treatments. Participants/materials, setting, methods All samples were cryopreserved by slow freezing and kept frozen until the day of their analysis. Tissues provided from the same patients (n = 5 per age group) were investigated by scanning electron microscopy (SEM) (fiber, pore and topography analyses) and atomic force microscopy (AFM). A larger number of paraffin-fixed biopsies (prepubertal, n = 16, reproductive-age, n = 21, and menopausal, n = 24) obtained from the biobank of St-Luc’s Hospital were used to conduct computed fiber orientation analysis. Main results and the role of chance Our results revealed a unique ECM architecture at reproductive age, where fibers of intermediate diameter are assembled into thickest bundles compared to prepubertal and menopausal tissues(p < 0.0001). Indeed, during prepuberty the bundles assemble into a tight network with high number of small pores while reproductive-age ovary gain more porosity(p < 0.0001). However, at menopause tissue pore number and area change significantly(p < 0.001). These pore geometry and distribution changes contribute to diffusion and access of key molecules to/from cells, which can be translated into changes in permeability and molecule selectivity with age. Fiber directionality around follicle borders at preantral stages revealed that before and after puberty, secondary follicles appear to modify their microenvironment arrangement locally compared to follicles at earlier stages of development (p < 0.01), by reorienting the majority of collagen fibers below 50°.This could indicate that follicles at this stage require higher fiber contact and adhesion signaling to complete their development and maturation towards ovulation. AFM evidenced a relatively rigid ovarian tissue at prepuberty, softening significantly at reproductive age, then stiffening considerably upon menopause. These differences(p < 0.01) are not only structure-dependent, but also related to biochemical differences in ECM composition, as previously demonstrated in our follow-up of variations in elastic matrisome components from prepuberty to menopause. Limitations, reasons for caution The samples represent single time points from each age group which could present limitations, since following ovary dynamics from prepuberty to menopause in the same patient is not feasible. Wider implications of the findings: Our study provides the first conclusive proof of a link between ECM biophysics and fertility by comparing different stages of ovarian transformation related to a woman’s reproductive life, which will oriente new strategies for infertility prognoses based on ECM biophysics and may become a blueprint for designing functional engineered ovaries. Trial registration number Not applicable

IMPLICATION OF SIRTUINS AND KISSPEPTIN IN OVARIAN AGING

Цель работы - исследование экспрессии сируинов-1 и -6 и кисспептина в ткани яичников человека, взятых в разные периоды онтогенеза - от формирования пула фолликулов в антенатальном периоде до угасания функции яичников в постменопаузе. Выявлено, что сиртуины экспрессируются в ткани яичника человека во всех возрастных группах. Максимальный уровень экспрессии SIRT1 в ткани яичника наблюдали в периоды внутриутробного развития и перименопаузы, SIRT6 - в репродуктивный период и перименопаузу. Показано участие SIRT1 в пренатальном отборе ооцитов у плодов человека, так как именно в этой группе выявлена повышенная экспрессия данного маркера. Уровень экспрессии маркера кисспептина растет по мере формирования яичников и включения репродуктивной функции, пик экспрессии наблюдается в период перед наступлением климакса. Проведенное исследование по выявлению экспрессии этих белков в яичнике человека расширяет представление о регуляции фолликулярного резерва яичников и репродуктивном старении. The aim of the work was to study the expression of sirtuins 1 and 6 and kisspeptin in human ovarian tissue taken in different periods of ontogenesis: from the formation of a pool of follicles in the antenatal period to the extinction of ovarian function in postmenopausal women. It was revealed that sirtuins are expressed in human ovary tissue in all age groups. The maximum expression level of SIRT1 in ovarian tissue was observed during intrauterine development and during the perimenopause, SIRT6 during the reproductive period and perimenopause. The participation of SIRT1 in prenatal selection of oocytes in human fetuses was shown, since it was in this group that increased expression of this marker was revealed. The expression level of the kisspeptin marker increases with the formation of the ovaries and the inclusion of reproductive function; the peak of expression is observed in the period before the onset of menopause. The study conducted to identify the expression of these proteins in the human ovary expands the understanding of the regulation of the ovarian follicular reserve and reproductive aging.

Vaspin, a novel adipokine in woman granulosa cells physiology and PCOS pathogenesis?

Vaspin is a novel adipokine mainly expressed in visceral adipose tissue and closely related to obesity and insulin-resistance. Currently, data about its ovarian expression are limited to animal models and its role in human reproduction is largely unexplored. Our study’s aims were thento characterise vaspin expression in the human ovary and to study in vitro its effects on granulosa cells physiology. Secondly, we assessed vaspin and its receptor GRP78 variations in granulosa cells and follicular fluid of a cohort of 112 infertile women undergoing an in vitro fertilisation procedure and allocated to 3 groups, each including normal-weight and obese subjects: 34 PCOS patients, 33 women with isolated polycystic ovary morphology (ECHO group) and 45 controls. Vaspin and GRP78 expression in the ovary was assessed by immunohistochemistry, RT-qPCR and Western blot. Granulosa cells and follicular fluid were analysed by RT-qPCR and ELISA, respectively. In vitro, granulosa cells metabolism was studied after stimulation with recombinant human vaspin, with and without a small interfering RNA directed against GRP78. Vaspin was highly expressed in the human ovary and concentration-dependently enhanced granulosa cells steroidogenesis, proliferation and viability through GRP78 (p<0.0001). Vaspin levels in both granulosa cells and follicular fluid were significantly higher in obese women (p<0.0001) and in the normal-weight ECHO group (p<0.001), which also had the highest expression rates of GRP78 (p<0.05).Although further investigation is needed, vaspin appears as a novel modulator of human granulosa cells physiology and possibly plays a role in PCOS pathogenesis, notably protecting from insulin-resistance induced complications.

Fertilization and development of oocytes with separated and conjoined zona pellucida recovered from polyovular follicles: description of two cases and a literature review

Summary Recovery of more than one oocyte from a single follicle during laparoscopic egg collection has been reported sporadically and accepted as confirmation of the presence of polyovular or binovular follicles in the human ovary at reproductive age. Most of these reports include conjoined oocytes that share common or fused zona pellucida, and are generally accepted as evidence for true polyovularity due to its certain characteristics. In this study, we report one case of a conjoined oocyte and another case of the recovery of two separate oocytes in a cumulus cell complex and details of their early embryonic development. To our knowledge, this report of the recovery of two separate oocytes without zonal contact is the first in the literature. We reviewed the relevant literature to evaluate information regarding the origin, incidence and significance of polyovularity in reproductive health.

Mapping the mechanical microenvironment in the ovary

Follicle development in the human ovary must be tightly regulated to ensure cyclical release of oocytes (ovulation), and disruption of this process is a common cause of infertility. Recent ex vivo studies suggest that follicle growth may be mechanically regulated, however the actual mechanical properties of the follicle microenvironment have remained unknown. Here we map and quantify the mechanical microenvironment in mouse ovaries using colloidal probe atomic force microscope (AFM) indentation, finding an overall mean Young’s Modulus 3.3 ± 2.5 kPa. Spatially, stiffness is low at the ovarian edge and centre, which are dominated by extra-follicular ECM, and highest in an intermediate zone dominated by large follicles. This suggests that large follicles should be considered as mechanically dominant structures in the ovary, in contrast to previous expectations. Our results provide a new, physiologically accurate framework for investigating how mechanics impacts follicle development and will underpin future tissue engineering of the ovary.