103 The Tunica Albuginea Increases in Thickness and Density Correlatively with the Age of the Patient in Human Ovaries

Abstract

A decline in fertility occurs with age. Because the extracellular matrix has a vital role in maintaining the integrity and determining the tissue function of numerous organs, the reasons for this fertility reduction may be associated with changes occurring in the ovarian matrix. To demonstrate this hypothesis, by using Picrosirius Red (PSR), a connective tissue dye specific for fibres of collagen I and III, we examined the ovarian tunica albuginea (TA), a part of the cortex composed by dense connective tissue that protects the ovaries and whose rupture is critical for the occurrence of ovulation events. According to an approval from the Ethics Committee of Okayama University (approval number 1818), ovaries were obtained from patients with gender identity disorder at the Okayama University Hospital. Sliced ovaries were fixed in 4% paraformaldehyde and included in paraffin. Sections of 5 μm thickness were stained with PSR. For each individual, 2 non-overlapping brightfield images were taken of each section with a fluorescence microscope and the attached viewer software (Keyence BZ-X710, Keyence Corp., Osaka, Japan). ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to quantify the distance of the dense connective tissue by selecting 5 random points manually per image and, to quantify the density of the tissue; we detected PSR staining above a determined threshold kept constant for all the images analysed. Statistical analyses of data from 3 replicates per individual were performed by Pearson’s correlation, and a P-value < 0.05 was considered significant. We examined samples from 18 individuals from 21 to 46 years old (mean age ± SD: 30.1 ± 7.5 years). When we observed the PSR histological sections, we could not detect any correlation between dosage and length of the androgenic treatment received by the patients and the thickness and density of the TA (P = 0.76 and P = 0.14, respectively). However, when we contrasted the age of the donor with the previous parameters, we detected a significant positive correlation (r = 0.52, P < 0.05) between the thickness of the TA and the age of the subject, indicating that this layer becomes thicker with increasing age. Furthermore, when we assessed its density, the results indicated a significant positive correlation (r = 0.55, P < 0.05) between age and TA density, meaning that this layer also becomes more compact in older individuals. The thickness of the TA seems to increase due to fibrotic growth underneath this layer, which causes an increase of the total thickness of the dense connective tissue. These areas of fibrosis have no follicles and usually present a clear limit with the rest of the cortical tissue area. In conclusion, we demonstrated that the ovarian cortical area comprising dense connective tissue grew thicker with increasing age without any contribution of the dosage or length of the androgenic treatment. The implications of these findings regarding ovulation, follicular growth, and the potential effects of related hormones such as LH and FSH on the ovaries must be investigated further.