Longitudinal assessment of circulating insulin-like peptide 3 levels in healthy peripubertal girls  Casper P. Hagen, Ph.D., Mikkel G. Mieritz, M.D., John E. Nielsen, M.Sc., Ravinder Anand-Ivell, Ph.D., Richard Ivell, Ph.D., Anders Juul, D.M.Sc.  Fertility and Sterility  Volume 103, Issue 3, Pages 780-786.e1 (March 2015) DOI: 10.1016/j.fertnstert.2014.11.014 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Serum levels of INSL3 in 10 healthy peripubertal girls (A–J) according to age (A) and time to pubertal onset (B). The detection limit of INSL3 (0.01 ng/mL) is marked with a black, dashed line. When all samples were included in a cross-sectional model, serum levels of INSL3 were lower in samples from prepubertal girls (B1) vs. girls in late puberty (B4+B5): 0.01 (<0.01–0.13) vs. 0.05 (0.01–0.25) ng/mL, P=.031 (C). Median, 10th and 90th percentile (black line and grey box). The postpubertal increase of INSL3 was confirmed by a component variant model taking longitudinal values into account (D). Geometric mean (fat black line), ±1.96 SD (thin black lines) corresponding to the 95% confidence interval. Insulin-like peptide 3 levels at first examination after pubertal onset vs. >2 years after pubertal onset: 0.03 (0.02–0.06) vs. 0.15 (0.07–0.33) ng/mL, P=.004. Fertility and Sterility 2015 103, 780-786.e1DOI: (10.1016/j.fertnstert.2014.11.014) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Two representative examples (girl A: left; and girl B: right) of individual variation of INSL3 levels, LH, and theca cell products (T and A) (upper panel) as well as FSH and granulosa cell products (E2, inhibin B, and AMH) (lower panel) according to pubertal onset (marked with vertical grey line). The detection limit of INSL3 (0.01 ng/mL) is marked with a black, dashed line. Fluctuations of INSL3 were not always parallel with gonadotropins or ovarian hormones. Insulin-like peptide 3 reflected products from large follicles (T, A, E2, inhibin B) better than products from small follicles (AMH). Fertility and Sterility 2015 103, 780-786.e1DOI: (10.1016/j.fertnstert.2014.11.014) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Positive immunohistochemical intracytoplasmatic staining of INSL3 (A) and CYP11A1 (B) in theca cells of a large follicle (>4 mm). Control with omission of INSL3 antibody (D). The majority of granulosa cells of preantral follicles (<1 mm) did not stain for INSL3 (C). A, B, and D are serial sections from a 15-year-old girl. C is from a 14-year-old girl. Fertility and Sterility 2015 103, 780-786.e1DOI: (10.1016/j.fertnstert.2014.11.014) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Insulin-like peptide 3 correlated with T (P=.002) (A), A (P=.003) (B), E2 (P=.004) (C), inhibin B (P=.006) (D), LH (P=.003), and FSH (P=.029) (red) (E) but not with AMH (P=.549) (F). All samples (n = 84) from the 10 girls were included. Fertility and Sterility 2015 103, 780-786.e1DOI: (10.1016/j.fertnstert.2014.11.014) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions