New methods to improve the safety assessment of cryopreserved ovarian tissue for fertility preservation in breast cancer patients Beatriz Rodríguez-Iglesias,

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New methods to improve the safety assessment of cryopreserved ovarian tissue for fertility preservation in breast cancer patients Beatriz Rodríguez-Iglesias, Ph.D., Edurne Novella-Maestre, Ph.D., Sonia Herraiz, Ph.D., César Díaz-García, M.D., Nuria Pellicer, B.Sc., Antonio Pellicer, M.D. Fertility and Sterility Volume 104, Issue 6, Pages 1493-1502.e2 (December 2015) DOI: 10.1016/j.fertnstert.2015.08.009 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Genomic expression of molecular markers selected for the definitive diagnostic tool. (A) Ovarian cortex from healthy patients (group 1) showed the same expression levels as the normalizing tissue control tissue for all the analyzed molecules. (B) In group 2, GCDFP15 was statistically significantly overexpressed in 6 of 8 of the metastatic ovarian cortex tissues and in all the nonovarian breast cancer metastatic tissues. Expression of both MGB1 and SBEM was increased in 7 of 8 ovarian cortex tissues and in 2 of 5 and 3 of 5 of nonovarian metastasis tissues, respectively. (C) Although breast cancer primary tumor samples showed a heterogeneous pattern for the expression of the selected markers, in all cases the expression of at least two markers was enhanced. Fertility and Sterility 2015 104, 1493-1502.e2DOI: (10.1016/j.fertnstert.2015.08.009) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 GCDFP15 immunostaining was found to be negative in (A) group 1 samples and (B) breast cancer (BC) diagnosed metastasis in the eye. Positive signal was detected in (C) BC metastatic ovarian cortex and (D) liver. The MGB1 signal was negative in (E) ovarian cortex from healthy patients, (F) slightly positive in the BC metastasis located in uterine ligament, and increased in BC metastatic (G) ovarian cortex and (H) liver. The SBEM expression was negative in (I) the ovarian cortex of healthy patients and (J) BC metastatic uterine ligament; it was highly positive in (K) ovarian cortex metastatic samples and (L) BC metastasis in liver. Scale bar = 100 μm; original magnification, ×20. Fertility and Sterility 2015 104, 1493-1502.e2DOI: (10.1016/j.fertnstert.2015.08.009) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Cell invasion assay. When compared with the negative control reference sample, increased values were detected in the MDA-MB-231 (***P<.001) and MDA-MB-468 (***P<.001) cell lines. When the ovarian cortex and ovarian medulla diagnosed as negative metastasis with the diagnostic tool were compared with the negative control, no differences were detected. Fertility and Sterility 2015 104, 1493-1502.e2DOI: (10.1016/j.fertnstert.2015.08.009) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Captions from the IVIS Spectrum Preclinical In Vivo Imaging System showing the fluorescent cells injected into the adrenal gland. Fluorescence images at weeks 1 and 24 in each group: (A) animals monitored from weeks 1–24 after cell injection; (B) positive control mice. Captions show increased fluorescent signal and cell migration on day 104 (D104). Fertility and Sterility 2015 104, 1493-1502.e2DOI: (10.1016/j.fertnstert.2015.08.009) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Scheme of experimental design and sample distribution. After xenotransplantation, the follow-up evaluation was performed with the IVIS Spectrum Preclinical In Vivo Imaging System. Fertility and Sterility 2015 104, 1493-1502.e2DOI: (10.1016/j.fertnstert.2015.08.009) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 High-magnification images obtained from the adrenal gland of a mice injected with breast cancer metastatic cell line (positive control) showing positive signal for (A) GDFP15, (B) MGB1, and (C) SBEM. Adrenal gland of a mice microinjected with cells obtained from the ovarian cortex of a healthy patient showing negative immunostaining for (D) GDFP15, (E) MGB1, and (F) SBEM. Fertility and Sterility 2015 104, 1493-1502.e2DOI: (10.1016/j.fertnstert.2015.08.009) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions