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. 2018 Jan 12:8:376.
doi: 10.3389/fendo.2017.00376. eCollection 2017.

Hallmarks of Human Small Antral Follicle Development: Implications for Regulation of Ovarian Steroidogenesis and Selection of the Dominant Follicle

Affiliations

Hallmarks of Human Small Antral Follicle Development: Implications for Regulation of Ovarian Steroidogenesis and Selection of the Dominant Follicle

Stine G Kristensen et al. Front Endocrinol (Lausanne). .

Abstract

Regulation of human ovarian steroidogenesis differs from other species and precise knowledge on how human small antral follicles (hSAF) develop and acquire competence for continued growth and steroid output is still incomplete. The present study has characterized almost 1,000 normal hSAF collected in connection with cryopreservation of ovarian tissue for fertility preservation. The antral follicles (ranging from 3 to 13 mm) were generally aspirated from one ovary surgically removed during the natural cycle, and the follicular fluid (FF) and the granulosa cells (GC) were isolated and snap-frozen. In FF, the following hormones were measured: inhibin-B, inhibin-A, AMH, follistatin, PAPP-A, estradiol, progesterone, testosterone, and androstenedione. In GC, mRNA gene expressions using q-PCR were measured for the following genes: FSHR, AMH, CYP19, and AR. All samples in which one of the abovementioned parameters was measured were included, but typically multiple parameters were measured. Highly significant differences in concentration and follicular content in relation to follicular diameter were found for all measured hormones despite massive variability in-between follicles for any given diameter. The results demonstrate that profound changes take place in the hormonal microenvironment around follicular diameters of 8-11 mm corresponding to when follicular selection occurs. At this point, inhibin-B and inhibin-A showed distinct peaks concomitant with a significant reduction in both AMH protein and mRNA expression. Concentrations of inhibins, androgens, FSHR, and AR were intimately associated, and it is suggested that inhibin-B in combination with PAPP-A and thereby IGF2 activity exerts important paracrine signaling at follicular selection. At the same time upregulation of estradiol synthesis and CYP19 mRNA expression increased steroid output profoundly. Furthermore, the highly significant association between FSHR and AR mRNA gene expression enforces important functions of androgens in follicular development. Collectively, these data reintroduce the understanding of the follicular phase as two parted in which regulation of steroidogenesis differs. The profound changes taking place around follicular selection highlight important paracrine actions of TGF-β family members and IGFs for securing dominance of the selected follicle.

Keywords: follicle-stimulating hormone; follicular fluid; granulosa cells; human ovarian steroidogenesis; human small antral follicles; inhibin-B; luteinizing hormone; paracrine signaling.

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Figures

Figure 1
Figure 1
Number and diameter of follicles collected per woman. (A) Bars depict the frequency of small antral follicles in relation to the specific diameters. (B) Bars depict the number of small antral follicles collected per woman.
Figure 2
Figure 2
Intrafollicular content and concentrations of peptide and steroid hormones. Graphs show the follicular fluid concentrations of estradiol, progesterone, androstenedione, testosterone, inhibin B, inhibin A, AMH, PAPP-A, and follistatin, and follicular content in nanograms (bottom panel) of inhibin B, inhibin A, and AMH according to follicular diameter. Values are presented as mean ± SEM. Statistically significant differences were observed in intrafollicular hormone levels of estradiol [analysis of variance (ANOVA); N = 264; P < 0.001], androstenedione (ANOVA; N = 276; P < 0.03), progesterone (ANOVA; N = 267; P < 0.003), PAPPA (ANOVA; N = 96; P < 0.03), inhibin-B (ANOVA; N = 627; P < 0.002), inhibin-A (ANOVA; N = 258; P < 0.001), AMH (ANOVA; N = 655; P < 0.001), but not testosterone (ANOVA; N = 566; P > 0.10) and follistatin (ANOVA; N = 148; P > 0.10). Regarding follicular content (bottom panel), the intrafollicular contents of inhibin-B (ANOVA; N = 611; P < 0.001), inhibin-A (ANOVA; N = 258; P < 0.001), and AMH (ANOVA; N = 654; P < 0.001) were observed to be significantly different in relation to follicular diameter. Specific numbers for the analyzed follicle groups divided according to follicular diameter are shown in Tables S1 and S2 in Supplementary Material.
Figure 3
Figure 3
Granulosa cell expression of selected genes in human small antral follicles. Graphs show relative expression levels of FSHR, CYP19a1, AR, and AMH in relation to follicular diameter. Values are presented as mean ± SEM. Statistically significant differences were observed in the mRNA expression levels of FSHR [analysis of variance (ANOVA); N = 315; P < 0.002] and AR (ANOVA; N = 316; P < 0.03), but not for CYP19a1 (ANOVA; N = 252; P > 0.10) and AMH (ANOVA; N = 277; P > 0.10). Specific numbers for the analyzed follicle groups divided according to follicular diameter are shown in Table S3 in Supplementary Material.
Figure 4
Figure 4
Hallmarks of human small antral follicle development: implications for regulation of ovarian steroidogenesis and selection of the dominant follicle.

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