1249 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Letters to the Editor remission after a few cycles of chemotherapy (Shapira et al., 2018). Thus, restriction of pre-harvesting chemotherapy may have serious medical consequences and cannot be recommended based on the data presented in this study. The other conclusions reached by the authors are similarly prob- lematic. The authors state that they observed no increased activa- tion of primordial follicles in ovarian tissue exposed to chemother- apy based on morphometric assessment of follicle populations and FOXO3A immunostaining. However, more than half of the samples used for histological counting were from frozen/thawed tissue and not fresh embedded tissue. The process of freezing/thawing alters follicle morphology (Demirci et al., 2002, Rimon et al., 2005), pre- venting accurate assessment of follicle stage and atresia. Studies show that after freezing/thawing ∼30% of detected follicles are not viable (Gandolfi et al., 2006; Campos et al., 2011). Additionally, analysis was conducted on a pooled data base of tissue from patients exposed to high, low and no alkylating agent chemotherapy. Given the difer- ent outcomes of each of these treatment groups on follicle popula- tions, a combined assessment is invalid. Furthermore, immunostaining for FOXO3A was performed on ovaries removed between 14 and 35 days after treatment (from only three treated patients), a time frame long after any change in FOXO3A expression would be evi- dent. As a result of these methodological errors, the authors cannot draw any conclusions from this data regarding follicle activation after chemotherapy. In summary, we feel that the authors’ conclusions, in particular the recommendation to perform OTC before initiation of any chemother- apy, are not supported by these results and may have critical medical consequences. Confict of interest None. References Campos JR, Rosa-e-Silva JC, Carvalho BR, Vireque AA, Silva-de-Sa MF, Rosa-e-Silva AC. Cryopreservation time does not decrease follicular viability in ovarian tissue frozen for fertility preservation. Clinics (Sao Paulo, Brazil) 2011;66:2093–2097. Demirci B, Salle B, Frappart L, Franck M, Guerin JF, Lornage J. Morphological alterations and DNA fragmentation in oocytes from primordial and primary follicles after freezing-thawing of ovarian cortex in sheep. Fertil Steril 2002;77:595–600. Gandolfi F, Pafoni A, Papasso Brambilla E, Bonetti S, Brevini TA, Ragni G. Efciency of equilibrium cooling and vitrification pro- cedures for the cryopreservation of ovarian tissue: comparative analysis between human and animal models. Fertil Steril 2006;85: 1150–1156. Green DM, Nolan VG, Srivastava DK, Leisenring W, Neglia JP, Sklar CA, Hudson MM, Diller L, Stovall M, Robison LL. Quantifying alkylat- ing agent exposure: evaluation of the cyclophosphamide equivalent dose—a report from the Childhood Cancer Survivor Study. J Clin Oncol 2011;29:9547–9547. Lee SJ, Schover LR, Partridge AH, Patrizio P, Wallace WH, Hagerty K, Beck LN, Brennan LV, Oktay K. American Society of Clini- cal O. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. J Clin Oncol 2006;24: 2917–2931. Meirow D, Biederman H, Anderson RA, Wallace WH. Toxicity of chemotherapy and radiation on female reproduction. Clin Obstet Gynecol 2010;53:727–739. Meirow D, Ra’anani H, Shapira M, Brenghausen M, Derech Chaim S, Aviel-Ronen S, Amariglio N, Schif E, Orvieto R, Dor J. Transplanta- tions of frozen-thawed ovarian tissue demonstrate high reproductive performance and the need to revise restrictive criteria. Fertil Steril 2016;106:467–474. Pampanini V, Wagner M, Asadi-Azarbaijani B, Oskam IC, Sheikhi M, Sjodin MOD, Lindberg J, Hovatta O, Sahlin L, Bjorvang RD et al. Impact of first-line cancer treatment on the follicle quality in cryo- preserved ovarian samples from girls and young women. Hum Reprod 2019;34:1674–1685. Poirot C, Fortin A, Lacorte JM, Akakpo JP, Genestie C, Vernant JP, Brice P, Morice P, Leblanc T, Gabarre J et al. Impact of cancer chemotherapy before ovarian cortex cryopreservation on ovarian tissue transplantation. Hum Reprod 2019;34:1083–1094. Rimon E, Cohen T, Dantes A, Hirsh L, Amit A, Lessing JB, Freimann S, Amsterdam A, Azem F. Apoptosis in cryopreserved human ovarian tissue obtained from cancer patients: a tool for evaluating cryop- reservation utility. Int J Oncol 2005;27:345–353. Shapira M, Raanani H, Barshack I, Amariglio N, Derech-Haim S, Marciano MN, Schif E, Orvieto R, Meirow D. First delivery in a leukemia survivor after transplantation of cryopreserved ovarian tis- sue, evaluated for leukemia cells contamination. Fertil Steril 2018;109: 48–53. von Tresckow B, Moskowitz CH. Treatment of relapsed and refractory Hodgkin lymphoma. Semin Hematol 2016;53:180–185. M. Shapira, D. Meirow*, H. Raanani, and H. Roness Morris Kahn Fertility Preservation Center, Sheba Medical Center, Tel Hashomer, 52621, Israel *Correspondence address. E-mail: dror.meirow@sheba.health.gov.il doi:10.1093/humrep/deaa036 Advance Access Publication on May 13, 2020 Reply: Impact of first-line cancer treatment on follicle quality in cryopreserved ovarian samples Sir, On behalf of all co-authors, we thank M. Shapira and colleagues for their valuable criticism that has enabled us to raise public awareness about the impact of inclusion criteria and timing of fertility preservation in relation to therapy exposures in pediatric patients. It is of utmost importance for us that the correct message is conveyed from our study (Pampanini et al., 2019). Shapira et al. wrote: ‘the authors define all first-line chemotherapy received by their treatment group as low-risk in terms of gonado- Downloaded from https://academic.oup.com/humrep/article-abstract/35/5/1249/5818928 by guest on 16 July 2020