ORIGINAL ARTICLES: REPRODUCTIVE ENDOCRINOLOGY
Longitudinal assessment of circulating insulin-like peptide 3 levels in healthy peripubertal girls Casper P. Hagen, Ph.D.,a Mikkel G. Mieritz, M.D.,a John E. Nielsen, M.Sc.,a Ravinder Anand-Ivell, Ph.D.,b Richard Ivell, Ph.D.,b and Anders Juul, D.M.Sc.a a Department of Growth and Reproduction, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; and b School of Biosciences, The University of Nottingham, Nottingham, United Kingdom
Objective: To elucidate the natural course of circulating insulin-like peptide 3 (INSL3) levels according to puberty as well as its relation to other reproductive hormones. Design: Population-based cohort study. Setting: Not applicable. Patient(s): Healthy peripubertal girls (n ¼ 10) examined every 6 months; total number of examinations was 84; median (range) per girl: 9 (4–10), including staging of pubertal breast development and blood samples. Intervention(s): None. Main Outcome Measure(s): Serum levels of INSL3, inhibin B, E2, antim€ ullerian hormone, LH, and FSH (validated immunoassays), and T and androstenedione (liquid chromatography–tandem mass spectrometry). Result(s): Serum levels of INSL3 varied considerably between girls (range, 0.01–0.27 ng/mL) and within each girl as puberty progressed; intraindividual variation, median (range) 102% (65%–143%). Insulin-like peptide 3 increased in late puberty (B1 to B4þB5); geometric mean 0.03 ng/mL to 0.15 ng/mL. Insulin-like peptide 3 levels reflected markers of large follicles (T, androstendione, inhibin B, and E2) better than markers of small follicles (antim€ ullerian hormone), and INSL3 staining was localized in theca interna cells of antral follicles. Conclusion(s): Insulin-like peptide 3 increased in late puberty, albeit inter- and intraindividual variations were substantial. Immunohistochemistry and intraindividual variation, as well as relations to other ovarian hormones, reveal that INSL3 in girls is a unique and specific marker of theca cells surrounding antral follicles. The potential clinical use of INSL3 for Use your smartphone evaluation of ovarian function in girls remains to be elucidated. (Fertil SterilÒ 2015;103:780–6. to scan this QR code Ó2015 by American Society for Reproductive Medicine.) and connect to the Key Words: INSL3, puberty, theca, girl, follicle Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/hagenc-insl3-healthy-peripubertal-girls/
I
n pediatric endocrinology, ovarian function is assessed clinically (breast development, menarche, menstrual bleeding regularity) and biochemically, including serum levels of gonadotropins (pituitary function) as ullerian well as E2, inhibin B, and antim€
hormone (AMH) (granulosa cell function of follicles in different stages). Although sensitive and specific liquid chromatography–tandem mass spectrometry (LC-MS/MS) technology has enabled valid assessment of circulating androgens even in peripubertal girls,
Received September 16, 2014; revised October 26, 2014; accepted November 10, 2014; published online December 13, 2014. C.P.H. has nothing to disclose. M.G.M. has nothing to disclose. J.E.N. has nothing to disclose. R.A.-I. has nothing to disclose. R.I. has nothing to disclose. A.J. has nothing to disclose. This study was funded by the Danish Agency for Science, Technology and Innovation (09-067180), Capital Region of Denmark (Dec. 2011), and The Research Fund of Rigshospitalet (9615.06.1.15). Reprint requests: Casper P. Hagen, M.D., Ph.D., University of Copenhagen, Department of Growth and Reproduction, GR, Rigshospitalet, Section 5064, Blegdamsvej 9, DK-2100 Copenhagen, Denmark (E-mail: [email protected]). Fertility and Sterility® Vol. 103, No. 3, March 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.11.014 780
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
T and androstenedione (A) are also produced by the adrenals and therefore not specific for ovarian function. Undetectable serum levels of insulin-like peptide 3 (INSL3) in postmenopausal women (1) as well as tissue RNA microarrays (2) indicate that INSL3 in females is exclusively produced by the ovaries. Insulin-like peptide 3 is expressed by theca interna cells surrounding medium and large growing follicles, as well as in corpora lutea (3, 4). Theca cells are responsible for converting cholesterol to androgens—the substrate for aromatization to estrogens performed by neighboring granulosa cells (5). VOL. 103 NO. 3 / MARCH 2015
Fertility and Sterility® Paracrine INSL3 signaling through the receptor—relaxin/ insulin-like family peptide receptor 2 (RXFP2)—seems to be responsible for maintaining androgen biosynethesis in theca interna cells (6). In adult women, interindividual variation of INSL3 is substantial, circulating levels of INSL3 decrease with age, and intraindividual fluctuations during the menstrual cycle seem to reflect waves of FSH-induced antral follicles recruited from preantral follicles growing independently of FSH stimulation (3, 7). As a marker of theca cell activity, serum levels of INSL3 are elevated in patients with polycystic ovary syndrome (PCOS) with disrupted follicle maturation resulting in an abundant number of INSL3producing follicles (3). Circulating levels of INSL3 have not previously been described in girls. By thorough longitudinal evaluation of INSL3 in peripubertal healthy girls, we aimed to elucidate the natural course of INSL3 according to puberty, as well as its relation to other reproductive hormones.
MATERIALS AND METHODS Subjects
This is a secondary analysis of the longitudinal part of the Copenhagen Puberty Study (8–11), which includes a total of 208 children. Blood sampling and clinical examination, including pubertal staging, were carried out every 6 months between 2006 and 2011. A nested cohort of 10 girls was selected from the 208 participants according to three criteria: [1] female gender, [2] Caucasian origin, and [3] pubertal onset during follow-up. The nested cohort did not differ from the main cohort concerning anthropometrics, circulating hormone levels, or age at breast development (data not shown). The girls had no history of gynecologic, endocrine, or cerebral illness. The total number of examinations was 84; median (range) per girl: 9 (4–10). Selection of the 10 girls was blinded to all circulating hormone values. Other parts of the data (FSH, AMH, and T) have previously been published (9, 12).
Clinical Examination Breast stage (B1–5) was classified according to Tanner's classification (13). Pubertal onset was defined as advancement from B1 to RB2 at the following examination. Menarche was recorded in five girls. The median number (range) of postmenarcheal samples was 2 (1–3), corresponding to a gynecologic age of 0.9 (0.5–1.4) years. Drawing of blood samples was not standardized according to menstrual cycles.
Hormone Assays Blood was drawn from an antecubital vein in the morning between 8:00 and 10:00 AM. Immediately after blood sampling, the samples were centrifuged and aliquoted into cryotubes, which were stored at 20 C until hormone analyses were performed. Samples were blinded for the technician for pubertal staging. Serum concentrations of INSL3 were measured using a well-established time-resolved VOL. 103 NO. 3 / MARCH 2015
fluorescent immunoassay (14), recently modified for higher sensitivity (3). The detection limit was 0.01 ng/mL, with intra- and interassay coefficients of variation (CVs) across the range of measurement of
Longitudinal assessment of circulating insulin-like peptide 3 levels in healthy peripubertal girls Casper P. Hagen, Ph.D.,a Mikkel G. Mieritz, M.D.,a John E. Nielsen, M.Sc.,a Ravinder Anand-Ivell, Ph.D.,b Richard Ivell, Ph.D.,b and Anders Juul, D.M.Sc.a a Department of Growth and Reproduction, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; and b School of Biosciences, The University of Nottingham, Nottingham, United Kingdom
Objective: To elucidate the natural course of circulating insulin-like peptide 3 (INSL3) levels according to puberty as well as its relation to other reproductive hormones. Design: Population-based cohort study. Setting: Not applicable. Patient(s): Healthy peripubertal girls (n ¼ 10) examined every 6 months; total number of examinations was 84; median (range) per girl: 9 (4–10), including staging of pubertal breast development and blood samples. Intervention(s): None. Main Outcome Measure(s): Serum levels of INSL3, inhibin B, E2, antim€ ullerian hormone, LH, and FSH (validated immunoassays), and T and androstenedione (liquid chromatography–tandem mass spectrometry). Result(s): Serum levels of INSL3 varied considerably between girls (range, 0.01–0.27 ng/mL) and within each girl as puberty progressed; intraindividual variation, median (range) 102% (65%–143%). Insulin-like peptide 3 increased in late puberty (B1 to B4þB5); geometric mean 0.03 ng/mL to 0.15 ng/mL. Insulin-like peptide 3 levels reflected markers of large follicles (T, androstendione, inhibin B, and E2) better than markers of small follicles (antim€ ullerian hormone), and INSL3 staining was localized in theca interna cells of antral follicles. Conclusion(s): Insulin-like peptide 3 increased in late puberty, albeit inter- and intraindividual variations were substantial. Immunohistochemistry and intraindividual variation, as well as relations to other ovarian hormones, reveal that INSL3 in girls is a unique and specific marker of theca cells surrounding antral follicles. The potential clinical use of INSL3 for Use your smartphone evaluation of ovarian function in girls remains to be elucidated. (Fertil SterilÒ 2015;103:780–6. to scan this QR code Ó2015 by American Society for Reproductive Medicine.) and connect to the Key Words: INSL3, puberty, theca, girl, follicle Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/hagenc-insl3-healthy-peripubertal-girls/
I
n pediatric endocrinology, ovarian function is assessed clinically (breast development, menarche, menstrual bleeding regularity) and biochemically, including serum levels of gonadotropins (pituitary function) as ullerian well as E2, inhibin B, and antim€
hormone (AMH) (granulosa cell function of follicles in different stages). Although sensitive and specific liquid chromatography–tandem mass spectrometry (LC-MS/MS) technology has enabled valid assessment of circulating androgens even in peripubertal girls,
Received September 16, 2014; revised October 26, 2014; accepted November 10, 2014; published online December 13, 2014. C.P.H. has nothing to disclose. M.G.M. has nothing to disclose. J.E.N. has nothing to disclose. R.A.-I. has nothing to disclose. R.I. has nothing to disclose. A.J. has nothing to disclose. This study was funded by the Danish Agency for Science, Technology and Innovation (09-067180), Capital Region of Denmark (Dec. 2011), and The Research Fund of Rigshospitalet (9615.06.1.15). Reprint requests: Casper P. Hagen, M.D., Ph.D., University of Copenhagen, Department of Growth and Reproduction, GR, Rigshospitalet, Section 5064, Blegdamsvej 9, DK-2100 Copenhagen, Denmark (E-mail: [email protected]). Fertility and Sterility® Vol. 103, No. 3, March 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.11.014 780
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
T and androstenedione (A) are also produced by the adrenals and therefore not specific for ovarian function. Undetectable serum levels of insulin-like peptide 3 (INSL3) in postmenopausal women (1) as well as tissue RNA microarrays (2) indicate that INSL3 in females is exclusively produced by the ovaries. Insulin-like peptide 3 is expressed by theca interna cells surrounding medium and large growing follicles, as well as in corpora lutea (3, 4). Theca cells are responsible for converting cholesterol to androgens—the substrate for aromatization to estrogens performed by neighboring granulosa cells (5). VOL. 103 NO. 3 / MARCH 2015
Fertility and Sterility® Paracrine INSL3 signaling through the receptor—relaxin/ insulin-like family peptide receptor 2 (RXFP2)—seems to be responsible for maintaining androgen biosynethesis in theca interna cells (6). In adult women, interindividual variation of INSL3 is substantial, circulating levels of INSL3 decrease with age, and intraindividual fluctuations during the menstrual cycle seem to reflect waves of FSH-induced antral follicles recruited from preantral follicles growing independently of FSH stimulation (3, 7). As a marker of theca cell activity, serum levels of INSL3 are elevated in patients with polycystic ovary syndrome (PCOS) with disrupted follicle maturation resulting in an abundant number of INSL3producing follicles (3). Circulating levels of INSL3 have not previously been described in girls. By thorough longitudinal evaluation of INSL3 in peripubertal healthy girls, we aimed to elucidate the natural course of INSL3 according to puberty, as well as its relation to other reproductive hormones.
MATERIALS AND METHODS Subjects
This is a secondary analysis of the longitudinal part of the Copenhagen Puberty Study (8–11), which includes a total of 208 children. Blood sampling and clinical examination, including pubertal staging, were carried out every 6 months between 2006 and 2011. A nested cohort of 10 girls was selected from the 208 participants according to three criteria: [1] female gender, [2] Caucasian origin, and [3] pubertal onset during follow-up. The nested cohort did not differ from the main cohort concerning anthropometrics, circulating hormone levels, or age at breast development (data not shown). The girls had no history of gynecologic, endocrine, or cerebral illness. The total number of examinations was 84; median (range) per girl: 9 (4–10). Selection of the 10 girls was blinded to all circulating hormone values. Other parts of the data (FSH, AMH, and T) have previously been published (9, 12).
Clinical Examination Breast stage (B1–5) was classified according to Tanner's classification (13). Pubertal onset was defined as advancement from B1 to RB2 at the following examination. Menarche was recorded in five girls. The median number (range) of postmenarcheal samples was 2 (1–3), corresponding to a gynecologic age of 0.9 (0.5–1.4) years. Drawing of blood samples was not standardized according to menstrual cycles.
Hormone Assays Blood was drawn from an antecubital vein in the morning between 8:00 and 10:00 AM. Immediately after blood sampling, the samples were centrifuged and aliquoted into cryotubes, which were stored at 20 C until hormone analyses were performed. Samples were blinded for the technician for pubertal staging. Serum concentrations of INSL3 were measured using a well-established time-resolved VOL. 103 NO. 3 / MARCH 2015
fluorescent immunoassay (14), recently modified for higher sensitivity (3). The detection limit was 0.01 ng/mL, with intra- and interassay coefficients of variation (CVs) across the range of measurement of
