Regulation of Pregnancy-Associated Plasma Protein-A and Insulin-like Growth Factor activity in human ovaries and its importance for a successful pregnancy

Causes of infertility are highly diverse, often complex, and not fully elucidated. Various biological systems are known to affect fecundability. A wider knowledge of these systems is needed to help clarify complications behind infertility and this may in the end lead to better treatment options and guidance at the fertility clinics. The insulin-like growth factor (IGF) system has been shown to affect ovarian steroidogenesis and pregnancy outcome. IGF signaling is tightly governed with multiple layers of regulatory proteins. One key regulator is pregnancy-associated plasma protein-A (PAPP-A), which cleaves IGF binding protein (IGFBP)-2, -4, and -5 and thereby upregulates IGF bioavailability. This thesis aims to examine how PAPP-A and IGF activity is regulated within human ovarian follicles. Aberrant levels and genetic variations in PAPP-A have been shown to be associated with adverse pregnancy outcomes. This thesis further aims to investigate the effect of some of these genetic variations on PAPP-A level and the intrafollicular environment in human ovarian follicles. In addition, this thesis includes studies of the underlying mechanisms of genetic variations in PAPP-A. The first paper in this thesis identified significant correlations between intrafollicular PAPP-A levels and central-ovarian-produced hormones. PAPP-A antigen was found in the theca cell layer of human small antral follicles 4-6 mm in diameter. In follicles exceeding 8-9 mm in diameter, PAPP-A antigen expression shifted to the GC layer with highest intensity in GCs from preovulatory follicles. Additionally, we determined that PAPP-A present in human follicle fluid was able to cleave IGFBP-4 in vitro. The second paper evaluated the expression profile of key IGF-genes in stage-specific human follicles and GCs, including different sizes of preantral, small antral, and preovulatory follicles. This study demonstrated an increased expression of genes that augment IGF signaling with increasing follicle size. This suggested that the upregulation of IGF-genes is important for the high rate of GC proliferation and steroid synthesis observed in the final stages of follicular development. The third paper examined single nucleotide polymorphisms (SNPs) in the PAPP-A gene known to be associated with preeclampsia and recurrent pregnancy loss. A significant effect of the missense SNP rs7020782, also called the 1224 SNP (serine<tyrosine), was found. Significantly lower PAPP-A concentrations in FF was found in small antral follicles in women carrying the minor allele (serine variant) compared to women with the major allele. In addition, significantly reduced cleavage levels of IGFBP-4 in vitro was found when incubated with FF from women with the minor allele, suggesting a reduced IGF activity. These women also presented a non-significant altered hormone profile, which was suggested to be influenced by an altered regulation of bioactive IGF. The fourth paper demonstrates a significant effect of the rs7020782 SNP in PAPP-A on the proteolytic specificity of PAPP-A comparing recombinant PAPP-A proteins. A reduced cleavage rate of IGFBP-4 in vitro was reported for the serine variant of rs7020782 compared to the tyrosine variant. In addition, a non-significant reduced cleavage rate of the IGFBP-2 and IGFBP-5 was displayed for the serine variant. PAPP-A cell-surface adhesion to human embryonic kidney 293T (HEK293T) cells was not observed to be affected by the rs7020782 SNP. Additionally, complex formation between PAPP-A and its inhibitors, STC2 or proMBP, and the inhibitory effect of STC1 on PAPP-A mediated IGFBP-4 cleavage, were not influenced by the rs7020782 SNP. This study suggests that the serine variant of rs7020782 reduces PAPP-A substrate binding and thereby cleavage activity, leading to reduced IGF bioavailability. The rs7020782 SNP has previously been associated with recurrent pregnancy loss and this study suggests that this SNP may have potential to be used as a prognostic biomarker.