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Jepsen M.R.,University of Aarhus | Kloverpris S.,University of Aarhus | Botkjaer J.A.,Copenhagen University | Wissing M.L.,Holbaek Fertility Clinic | And 2 more authors.
Human Reproduction | Year: 2016

STUDY QUESTION Is the proteolytic activity of pregnancy-associated plasma protein-A (PAPP-A) regulated by the stanniocalcins (STC1 and STC2) during human follicle maturation? SUMMARY ANSWER The STCs and PAPP-A show similar expression by immunohistochemistry in developing follicles, and regulation of PAPP-A proteolytic activity is suggested by the identification of inhibited protein complexes between PAPP-A and STC1 or STC2 in human follicular fluid (FF). WHAT IS KNOWN ALREADY The insulin-like growth factor (IGF)-regulating proteinase PAPP-A is secreted by the granulosa cells of estrogen-dominant follicles and is involved in follicle growth. STC1 and STC2 have recently been identified as novel PAPP-A inhibitors, and their expression in non-human mammalian ovaries has previously been observed. STUDY DESIGN, SIZE, DURATION The proteolytic activity of PAPP-A in human follicular fluid was assessed, and the interaction between PAPP-A and the STCs in human ovarian tissues and follicular fluid was analyzed using immunoassays. From 21 women, matched pairs of follicular fluid were obtained from one follicle just prior to final maturation of follicles with human chorionic gonadotrophin (hCG), and from another follicle in connection with oocyte aspiration after hCG treatment. Ovarian tissues were obtained from women having one ovary removed for fertility preservation by cryopreservation prior to gonadotoxic treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS The concentration and activity of PAPP-A were determined in all samples of follicular fluid. Furthermore, to investigate PAPP-A regulation during follicle development, immunohistochemical staining of PAPP-A, STC1, and STC2 was performed on pre-antral and antral human follicles. To attempt the demonstration of native complexes between PAPP-A and the STCs, immunoprecipitation from a pool of human follicular fluid was performed. MAIN RESULTS AND THE ROLE OF CHANCE The concentration of PAPP-A antigen in follicular fluid increased upon stimulation of ovulation with hCG (P < 0.02), but at the same time, PAPP-A activity was decreased. PAPP-A, STC1, and STC2 were localized together in primordial, late primary, and antral follicles, indicating that complex formation is possible in ovarian tissue. Covalent PAPP-A:STC2 and non-covalent PAPP-A:STC1 complexes were immunoprecipitated from follicular fluid, documenting for the first time native inhibited complexes between PAPP-A and the STCs. LIMITATIONS, REASONS FOR CAUTION We have demonstrated the presence of native complexes between PAPP-A and the STCs in the human ovary, indicating STC-mediated PAPP-A proteolytic inhibition. Further investigation is required to extend this principle to other tissues. WIDER IMPLICATIONS OF THE FINDINGS Our data suggest that the STCs contribute to PAPP-A regulation during folliculogenesis and support a general model in which STC1 and STC2 are regulators of mammalian IGF activity through inhibition of PAPP-A. We suggest that future functional studies take both PAPP-A and the STCs into consideration. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.


Botkjaer J.A.,Copenhagen University | Jeppesen J.V.,Copenhagen University | Wissing M.L.,Holbaek Fertility Clinic | Kloverpris So.,University of Aarhus | And 4 more authors.
Fertility and Sterility | Year: 2015

Objective To evaluate follicular fluid (FF) levels of pregnancy-associated plasma protein A (PAPP-A) in relation to levels of intrafollicular hormones. Furthermore, immunostaining of human follicles of varying diameters was studied for PAPP-A, antimüllerian hormone (AMH), and aromatase, and the biological activity of PAPP-A in FF was evaluated. Design Laboratory investigation. Setting University hospital. Patient(s) A total of 43 women with a total of 80 samples were obtained from three different size-groups of antral follicles collected before and after the LH surge. Intervention(s) ELISA measurement of steroids, PAPP-A, and AMH, immunohistochemistry of PAPP-A, AMH, and aromatase on follicles of different diameter, and proteolytic activity of PAPP-A toward insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4). Main Outcome Measure(s) Association between FF levels of PAPP-A and measured ovarian hormones, PAPP-A activity in FF, localization of PAPP-A, AMH, and aromatase in antral follicles. Result(s) A highly significant association between FF levels of PAPP-A and all measured hormones were obtained with positive associations toward E2 and P, whereas AMH, T, and A showed strong negative associations. PAPP-A proteolytic activity toward IGFBP-4 was detected in human FF. PAPP-A immunostaining shifted from being primarily present in theca cells of small antral follicles to being expressed in granulosa cells (GCs) of preovulatory follicles. In contrast, AMH expression became reduced with increasing follicular diameter. Aromatase expression was highly specifically localized to GCs of preovulatory follicles. Conclusion(s) The results suggest that PAPP-A is specifically involved in the regulation of steroidogenesis in human antral follicles. Local regulation of IGF-II activity may represent a mechanism by which PAPP-A exerts this function and highlights the importance of IGF signaling during follicular development. © 2015 American Society for Reproductive Medicine.


Wissing M.L.,Holbaek Fertility Clinic | Sonne S.B.,Copenhagen University | Westergaard D.,Technical University of Denmark | Nguyen Kd.,Technical University of Denmark | And 3 more authors.
Journal of ovarian research | Year: 2014

BACKGROUND: Corona radiata cells (CRCs) refer to the fraction of cumulus cells just adjacent to the oocyte. The CRCs are closely connected to the oocyte throughout maturation and their gene expression profiles might reflect oocyte quality. Polycystic ovary syndrome (PCOS) is a common cause of infertility. It is controversial whether PCOS associate with diminished oocyte quality. The purpose of this study was to compare individual human CRC samples between PCOS patients and controls.METHODS: All patients were stimulated by the long gonadotropin-releasing hormone (GnRH) agonist protocol. The CRC samples originated from individual oocytes developing into embryos selected for transfer. CRCs were isolated in a two-step denudation procedure, separating outer cumulus cells from the inner CRCs. Extracted RNA was amplified and transcriptome profiling was performed with Human Agilent® arrays.RESULTS: The transcriptomes of CRCs showed no individual genes with significant differential expression between PCOS and controls, but gene set enrichment analysis identified several cell cycle- and DNA replication pathways overexpressed in PCOS CRCs (FDR < 0.05). Five of the genes contributing to the up-regulated cell cycle pathways in the PCOS CRCs were selected for qRT-PCR validation in ten PCOS and ten control CRC samples. qRT-PCR confirmed significant up-regulation in PCOS CRCs of cell cycle progression genes HIST1H4C (FC = 2.7), UBE2C (FC = 2.6) and cell cycle related transcription factor E2F4 (FC = 2.5).CONCLUSION: The overexpression of cell cycle-related genes and cell cycle pathways in PCOS CRCs could indicate a disturbed or delayed final maturation and differentiation of the CRCs in response to the human chorionic gonadotropin (hCG) surge. However, this had no effect on the in vitro development of the corresponding embryos. Future studies are needed to clarify whether the up-regulated cell cycle pathways in PCOS CRCs have any clinical implications.


Wissing M.L.,Holbaek Fertility Clinic | Bjerge M.R.,Holbaek Fertility Clinic | Olesen A.I.G.,Holbaek Fertility Clinic | Hoest T.,Holbaek Fertility Clinic | Mikkelsen A.L.,Holbaek Fertility Clinic
Reproductive BioMedicine Online | Year: 2014

This study investigated whether polycystic ovary syndrome (PCOS) affected early embryo development assessed by time-lapse analysis of embryo kinetics from fertilization to the blastocyst stage. This was a prospective cohort study of two pronuclei (2PN) embryos from 25 hyperandrogenic PCOS patients (110 2PN embryos), 26 normoandrogenic PCOS patients (140 2PN embryos) and 20 healthy, regularly cycling women (controls, 97 2PN embryos). Patients underwent the same baseline evaluation and the same ovarian stimulation from April 2010 to February 2013. Oocytes were fertilized by intracytoplasmic sperm injection and incubated in an EmbryoScope with pictures taken every 20 min in seven focal planes. Time to 2PN breakdown, first cleavage and cleavage to 3, 4, 5, 6, 7 and 8 cells, morula and blastocyst (t2, t3, t4, t 5, t6, t7, t8, tM, t B) were annotated. Differences in embryo kinetics between groups were assessed by mixed modelling. Compared with controls, embryos from hyperandrogenic PCOS patients were significantly delayed at 2PN breakdown, t2, t3, t4 and t7 but not at t 5, t6, t8, tM or tB. Embryos from hyperandrogenic PCOS women had developed slower from fertilization to the 8-cell stage compared with embryos from controls. We investigated whether polycystic ovary syndrome (PCOS) affected early embryo development assessed by time-lapse analysis of embryo kinetics from fertilization to the blastocyst stage. This was a prospective cohort study of two pronuclei (2PN) embryos from 25 hyperandrogenic PCOS patients (110 2PN embryos), 26 normoandrogenic PCOS patients (140 2PN embryos) and 20 healthy, regularly cycling women (controls, 97 2PN embryos). Patients underwent the same baseline evaluation and the same ovarian stimulation from April 2010 to February 2013. Oocytes were fertilized by intracytoplasmic sperm injection and incubated in an EmbryoScope with pictures taken every 20 min in seven focal planes. Time to 2PN breakdown, first cleavage, cleavage to 3, 4, 5, 6, 7 and 8 cells, morula and blastocyst (t2, t3, t4, t5, t6, t7, t8, tM, tB) were annotated. Differences in embryo kinetics between groups were assessed by mixed modelling. With mixed modelling, the 'patient factor' is adjusted for, as each patient contributed several embryos for analysis. Compared with embryos from controls, embryos from hyperandrogenic PCOS patients were significantly delayed at 2PN breakdown (P = 0.0128), t2 (P = 0.0061), t3 (P = 0.0171), t4 (P = 0.0017), t7 (P = 0.04) and but not at t5, t 6, t8, tM or tB. We concluded that embryos from hyperandrogenic PCOS women developed slower from fertilization to the 7-cell stage compared with embryos from controls. The clinical impact of the embryo delay was unknown, since this study was not powered to detect differences in implantation and pregnancy rates between groups. © 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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