Abstract
Purpose: To investigate the effect of androgen (A2) at different concentrations on human endometrial epithelial cancer cell line (HHUA) using iTRAQ and liquid chromatography–mass spectrometry (LCMS/MS) analysis.
 Methods: Human endometrial cells were cultured in Dulbecco Modified Eagle Medium. Total protein was isolated from human endometrial cells and HHUA cells were treated with A2 in three different concentrations (10-9 , 10-8 and 10-7 M). The proteins were digested using filter-aided sample preparation (FASP) method, and labeled using iTRAQ. The proteins were then subjected to LC-MS/MS. The resultant peptides were identified using Expasy tool, and the up-stream and down-stream proteins were confirmed. Moreover, the up-regulated proteins: isoform 2 of nuclear ubiquitous casein and cyclindependent kinase substrate 1 (NUCKS1), isoform 2 of endothelial differentiation-related factor 1 (EDF1), and acid phosphatase 1 (ACP1) were independently confirmed using western blotting and immunohistochemistry techniques.
 Results: Transforming growth factor (TGF), p38 mitogen-activated protein kinase (p38-MAPK), cyclindependent kinase (CDK), and tumor protein p53 (TP53) were the major regulators of the upstream process, and were upregulated RM-linked endometrial epithelial cells. Androgen-associated proteins NUCKS1, EDF1, and ACP1 were significantly responsible for miscarriage-related changes in endometrial tissues (p < 0.05). The results showed that a high level of androgen significantly (p < 0.05) distorts the expression levels of proteins associated with endometrium development, resulting in impaired endometrial accessibility which led to miscarriage.
 Conclusion: Androgen-associated proteins are responsible for changes in endometrial tissues which, in turn, lead to recurrent miscarriage.
Highlights
Recurrent miscarriage (RM) is defined as pregnancy loss in more than three clinical pregnancies starting from conception time to 25 weeks [1]
The results revealed that NUCKS1 was down-regulated by A2 at doses of 10-7 and 10–8 M A2 (Figure 2 A), while endothelial differentiation-related factor 1 (EDF1) (Figure 2 B) and acid phosphatase 1 (ACP1) (Figure 2 C) were up-regulated by A2 at doses of 10–7 and 10–8 M, relative to the dose of 10–9 M
It has been reported that Polycystic ovarian syndrome (PCOS) is a major factor responsible for high incidence of RM, and that the underlying causes of RM are genetic factors, as well as endocrine and autoimmune diseases [12]
Summary
Recurrent miscarriage (RM) is defined as pregnancy loss in more than three clinical pregnancies starting from conception time to 25 weeks [1]. The causes of RM are not fully understood, but it is associated with multiple risk factors in many cases. In majority of RM cases, the underlying causes are not clear. It has been suggested that miscarriage is associated with mental disorders and anxiety, thereby affecting the quality of life [3]. Polycystic ovarian syndrome (PCOS) accounts for more than 80 % of RM in women. It is a hormonal disorder which occurs in 8 – 12 % of women population in their reproductive period, and it is mostly associated with anovulation with high levels of androgen. Women with high levels of androgen tend to have a higher risk of miscarriage and implantation failure than normal females [4]
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