Granular assessment of paediatric and adolescent ovaries following low levels of alkylating treatment reveals significant morphologic differences in early stage follicles.

STUDY QUESTION: Are subtle changes in the paediatric adolescent/young adult ovarian follicles and microenvironment following low-dose acute alkylating chemotherapy detectable by granular assessment tools? SUMMARY ANSWER: Low-dose alkylating agents impact the ovaries of pre- and post-pubertal participants with significant alterations to the follicle and oocyte area, number of granulosa cells (GCs) per follicle, and number of immature vessels, when compared to ovaries from untreated participants. WHAT IS KNOWN ALREADY: The metric known as cyclophosphamide equivalent dose (CED) was developed to counsel patients on the risk of acute and long-term adverse outcomes following exposure to alkylating agents. Likewise, CED is used to evaluate the risk of developing premature ovarian insufficiency and can support decisions to undergo fertility preservation. It is well known that high levels of alkylating agents significantly impact the ovarian reserve and extrafollicular microenvironment. However, there is limited data on the impact of low levels of CED on the human ovary. STUDY DESIGN, SIZE, DURATION: This is a retrospective study of 75 participants aged from 0.53 to 22.82 years, all of whom had undergone ovarian tissue cryopreservation (OTC) at a children's hospital. We assessed the short-term effects of low-dose alkylating agents (CED ≤8 g/m2) on the paediatric and adolescent ovary using immunohistochemical assessments and a comprehensive morphological assessment of ovarian follicles. The median time from first alkylating treatment to tissue collection was 1.35 months. The pre-pubertal cohort consisted of 50 participants; 4 had non-alkylating treatments (NA), 23 participants had alkylating treatments (A), and 23 were untreated (U). The post-pubertal cohort consisted of 25 participants: 5 with NA, 10 with A, and 10 who were U. PARTICIPANTS/MATERIALS, SETTING, METHODS: Biopsies for routine pathology were obtained during a unilateral oophorectomy performed for OTC. Formalin-fixed paraffin-embedded ovarian tissue was analyzed retrospectively. The extrafollicular environment was analyzed for collagen density, stromal fibre architecture, vascularity and vessel maturity, apoptosis, proliferation, and DNA damage. Digital scans of hematoxylin and eosin-stained pathology slides were used to classify and morphologically assess follicles in adjacent sections. MAIN RESULTS AND THE ROLE OF CHANCE: We first determined the effect of acute low-dose alkylating agents on the extrafollicular environment. Collagen was significantly more abundant in pre-pubertal ovarian tissue compared to post-pubertal (20.97 ± 3.30 vs. 7.11 ±1.82%, P ≤ 0.0008), but there was no significant difference in collagen abundance within either group based on exposure. Interestingly, collagen fibres in the cortex of post-pubertal ovaries exposed to non-alkylating agents were significantly narrower and less straight compared to untreated ovaries (7.85 ± 0.05 vs. 8.85 ± 0.13 µm, P = 0.01 and 92.24 ± 0.123 vs. 93.27 ± 0.252, P = 0.01). Consistent with previous work in reproductively aged individuals, there were significantly more immature vessels in pre-pubertal ovaries exposed to alkylating agents compared to untreated (3.29 ± 0.94 vs. 0.104 ± 0.07 vessel count/tissue area mm2, P = 0.037). Next, follicles within these cohorts were assessed using a granular and comprehensive morphological follicle analysis pipeline. Ovaries from pre-pubertal participants that were exposed to non-alkylating and alkylating therapy had significantly smaller primordial follicles (PMF) (1321 ± 14.52 (NA), 1379 ± 8.42 (A), 1533 ± 10.07 μm2 (U), P ≤ 0.0001) and transitional follicles (TF) (1356 ± 14.04 (NA), 1384 ± 10.05 (A), 1639 ± 14.04 μm2 (U), P ≤ 0.0001) with significantly fewer GCs per PMF (9.0 ± 0.15 (NA), 8.0 ± 0.08 (A), 10.0 ± 0.10 (U), P ≤ 0.0001) and TF compared to untreated ovaries (12.0 ± 0.16 (NA), 11.0 ± 0.09 (A), 13.0 ± 0.16 (U), P ≤ 0.0001, 0.0002). Ovaries from post-pubertal participants exposed to non-alkylating therapies had significantly smaller TFs (1437 ± 72.58 (NA), 1709 ± 131.8 (A), 2727 ± 308.9 μm2 (U), P = 0.021) and smaller primary follicles (1479 ± 71.26 (NA), 1709 ± 131.8 (A), 2981 ± 375.7 μm2 (U), P ≤ 0.0001). PMF (10.0 ± 0.26 (NA), 10.0 ± 0.28 (A), 8.0 ± 0.19 (U), P ≤ 0.0001, 0.0005) and TFs (12.0 ± 0.23 (NA), 12.0 ± 0.25 (A), 11.0 ± 0.20 (U), P ≤ 0.0001, 0.0013) had significantly more GCs per follicle, while primary follicles had significantly fewer GCs (16.0 ± 0.92 (NA), 17.0 ± 0.99 (A), 26.0 ± 2.85 (U), P ≤ 0.0001, 0.0003) compared to untreated ovaries. Then, we examined oocyte areas and found that oocytes from PMF (1032 ± 12.35 (NA), 1039 ± 9.29 (A), 1157 ± 10.81 μm2 (U), P ≤ 0.0001) and TFs (1030 ± 12.76 (NA), 1000 ± 12.53 (A), 1235 ± 18.53 μm2 (U), P ≤ 0.0001) of pre-pubertal ovaries exposed to non-alkylating and alkylating treatments were significantly smaller in area compared to untreated ovaries. In contrast, in post-pubertal ovaries, PMF (1051 ± 23.81 (NA), 1203 ± 18.74 (A), 980.0 ± 17.27 μm2 (U), P = 0.038, <0.0001) and TF (1257 ± 20.01 (NA), 1094 ± 19.43 (A), 963.2 ± 18.51 μm2 (U), P ≤ 0.0001, 0.0004) oocytes were significantly larger in exposed ovaries compared to untreated ovaries. In addition, there were significantly more anti-gamma H2A histone family member X (γH2AX)-expressing oocytes in ovaries from post-pubertal participants exposed to alkylating therapies compared to untreated participants (61.37 ± 9.13 (A) vs. 24.08 ± 8.37% (U), P = 0.0329). Finally, we found that PMFs containing γH2AX-expressing oocytes were significantly larger in both pre- and post-pubertal ovaries exposed to alkylating therapies compared to those without γH2AX-expressing oocytes (985.6 ± 14.04 (A) vs. 932.3 ± 10.32 μm2 (U), P = 0.0096 and 1062 ± 25.28 (A) vs. 872.1 ± 28.26 μm2 (U), P ≤ 0.0001). LIMITATIONS, REASONS FOR CAUTION: This study analyzed 3-4 mm ovarian biopsies, which may not be representative of the whole ovary, and provide a snapshot in time and not a functional assessment. Additionally, no participants in the study have yet opted for ovarian tissue transplantation, so the ability of this tissue to restore fertility and hormones after transplant cannot be correlated with these histological assessments. Due to tissue size and sections available for research, immunohistological assessments were limited. WIDER IMPLICATIONS OF THE FINDINGS: This is one of the few studies to assess the short-term consequences of alkylating agents on paediatric and adolescent human ovarian tissue. We did not find any significant differences in the follicle density between exposed and untreated cohorts. However, upon a more detailed analysis, significant differences in follicle area, oocyte area, and number of GCs per follicle were found between exposed and untreated cohorts and the observations differed based on pubertal status. Additionally, these findings improve our understanding of the short-term effects of non-alkylating and low levels of alkylating agents on human paediatric and adolescent ovarian tissue. Future research defining the functional impact of these differences is required to determine if they can inform clinicians how and when to counsel potential OTC patients. This work describes the importance of analyzing more granular parameters in addition to follicle counts and provides an example of how such analyses can uncover otherwise hidden differences. FUNDING: This work was funded by T32HD094699 (H.B.M.), U01HD110336 (M.M.L.), the Gesualdo Foundation Research Scholar funds (M.M.L.), the Center for Reproductive Science Marcia L. Storch Scholarship (S.P.), and the Office of Undergraduate Research (S.P.). DISCLOSURES: The authors have no conflict of interests to disclose. TRIAL REGISTRATION NUMBER: N/A.