The immature human ovary shows loss of abnormal follicles and increasing follicle developmental competence through childhood and adolescence

Abstract

Study question: Do the ovarian follicles of children and adolescents differ in their morphology and in vitro growth potential from those of adults?

Summary answer: Pre-pubertal ovaries contained a high proportion of morphologically abnormal non-growing follicles, and follicles showed reduced capacity for in vitro growth.

What is known already: The pre-pubertal ovary is known to contain follicles at the early growing stages. How this changes over childhood and through puberty is unknown, and there are no previous data on the in vitro growth potential of follicles from pre-pubertal and pubertal girls.

Study design, size, duration: Ovarian biopsies from five pre-pubertal and seven pubertal girls and 19 adult women were analysed histologically, cultured in vitro for 6 days, with growing follicles then isolated and cultured for a further 6 days.

Participants/materials, setting, methods: Ovarian biopsies were obtained from girls undergoing ovarian tissue cryopreservation for fertility preservation, and compared with biopsies from adult women. Follicle stage and morphology were classified. After 6 days in culture, follicle growth initiation was assessed. The growth of isolated secondary follicles was assessed over a further 6 days, including analysis of oocyte growth.

Main results and the role of chance: Pre-pubertal ovaries contained a high proportion of abnormal non-growing follicles (19.4 versus 4.85% in pubertal ovaries; 4004 follicles analysed; P = 0.02) characterized by indistinct germinal vesicle membrane and absent nucleolus. Follicles with this abnormal morphology were not seen in the adult ovary. During 6 days culture, follicle growth initiation was observed at all ages; in pre-pubertal samples there was very little development to secondary stages, while pubertal samples showed similar growth activation to that seen in adult tissue (pubertal group: P = 0.02 versus pre-pubertal, ns versus adult). Isolated secondary follicles were cultured for a further 6 days. Those from pre-pubertal ovary showed limited growth (P < 0.05 versus both pubertal and adult follicles) and no change in oocyte diameter over that period. Follicles from pubertal ovaries showed increased growth; this was still reduced compared with follicles from adult women (P < 0.05) but oocyte growth was proportionate to follicle size.

Limitations, reasons for caution: These data derive from only a small number of ovarian biopsies, although large numbers of follicles were analysed. It is unclear whether the differences between groups are related to puberty, or just age.

Wider implications of the findings: These findings show that follicles from girls of all ages can be induced to grow in vitro, which has important implications for some patients who are at high risk of malignant contamination of their ovarian tissue. The reduced growth of isolated follicles indicates that there are true intrafollicular differences in addition to potential differences in their local environment, and that there are maturational processes occurring in the ovary through childhood and adolescence, which involve the loss of abnormal follicles, and increasing follicle developmental competence.

Study funding/competing interest(s): Funded by MRC grants G0901839 and G1100357. No competing interests.

Keywords: adolescence; childhood; follicle; ovary; puberty.

Figure 1

(A) Relationship between follicle number (per mm³ of tissue) and age. (B) Confocal image of ovarian cortex from a 3-year-old girl immunostained for acetylated tubulin (green) and f-actin (using phalloidin: red). (C) Distribution of follicle classes (as percentage of total) in ovarian tissue from pre-pubertal and pubertal girls, and adults. Blue: non-growing follicles; red: primary follicles; green: secondary follicles. A total of 2672, 1278 and 798 follicles are classified in the three age groups, respectively.

Figure 2

(A) Photomicrograph of ovarian tissue from pre-pubertal girl aged 8 years. Non-growing follicles with both abnormal (top inset) and normal (bottom inset) morphology are present. (B) Photomicrograph of ovarian tissue from pubertal girl aged 14 years showing only follicles of normal morphology (main image and inset). Scale bars 25 µm in inserts, 50 µm in main images. (C) Relationship between percentage of non-growing follicles with abnormal morphology and age. Red circles: pre-pubertal girls, blue circles, pubertal girls. (D) Diameter of normal (white bars) and abnormal (black bars) non-growing follicles in pre-pubertal and pubertal groups, and in adult ovary (which contains no abnormal non-growing follicles). Mean ± SEM, pre-pubertal group normal n = 2079, abnormal morphology n = 500; pubertal group normal n = 1357, abnormal morphology n = 68; adult n = 44, *P < 0.05 versus normal.

Figure 3

(A) Distribution of follicle stages (as percentage of total) in cultured ovarian tissue in pre-pubertal, pubertal and adult groups, before culture (Day 0) and after 6 days. Blue: non-growing follicles; red: primary follicles; green: secondary follicles. (B) Follicle diameter during culture of isolated secondary follicles. Follicles were isolated from tissue after 6 days of culture, and follicle diameter was determined 2, 4 and 6 days thereafter. Blue triangles, pre-pubertal group; red squares, pubertal group; green circles: adult group (mean ± sem, n = 15, 73 and 44 respectively). *P < 0.05 versus pre-pubertal group, ^†P < 0.05 versus adult group. (C) Oocyte diameter of isolated secondary follicles after culture for 6 days in tissue, then a further 6 days as isolated follicles, to Day 12. Blue columns: Day 6; yellow columns, Day 12. Mean ± SEM, n = 15 and 77 follicles in pre-pubertal and pubertal groups, respectively, n = 44 follicles in adult group.*P < 0.05 versus Day 6.

Figure 4

Photomicrographs of secondary follicles after 6 days of isolated follicle culture. (A) immunofluorescent image of an abnormal follicle from a pre-pubertal girl (age 8 years); acetylated tubulin (green) and f-actin (red) with DAPI nuclear counterstain (blue). (B, C and D) are secondary follicles from pubertal girls (ages 12.0 and 15.3 years) showing healthy morphology after culture; (E) and (F) are follicles from adult ovary (ages 31 and 34 years), showing early antrum formation. Note that in all cases some ovarian stroma remains attached to the individually dissected follicles.