Increased ovarian volume is associated with hyperandrogenemia in women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a heterogeneous disorder characterized by chronic anovulation, hyperandrogenenism, and polycystic ovary morphology (PCOM). Although the role of hyperandrogenism in preantral follicle development remains unclear, recent studies report that androgen stimulates folliculogenesis, especially in small follicles and provides increased ovarian stromal volume. We performed this study to determine the relationship of hyperandrogenism and polycystic ovary morphology. We recruited 433 women with PCOS diagnosed by National Institute of Child Health and Human Disease (NICHD) criteria and 420 control women. In the early follicular phase, all subjects underwent venous blood sampling for glucose, insulin, testosterone, LH, FSH, and 75g 2-h oral glucose tolerance test after an overnight fast. Polycystic ovary morphology was diagnosed by 7MHz ultrasonography. In women with PCOS, PCOM was identified in 304 women (70.2%). Women with PCOS had significantly higher fasting insulin, 2-h post load glucose, 2-h post load insulin, ovarian volume and ovarian follicle number and lower metabolic clearance rate (MCR) compared with age-, BMI-matched controls (all Ps<0.01). PCOS women with PCOM had significantly higher total testosterone levels compared to PCOS women without PCOM (P<0.01). Total testosterone levels correlated significantly with ovarian volume (r=0.225, P<0.01), and logistic regression analysis showed association of ovarian volume with testosterone levels (β=0.163, P<0.05) in women with PCOS. Our results show that in Korean women with PCOS, high ovarian volume might be associated with hyperandrogenemia. ;다낭성 난소 증후군은 만성적인 무배란, 고안드로겐증, 그리고 다낭성 난소를 특징으로 하는 질병이다. 비록 고안드로겐증이 난포의 발달에 미치는 영향은 불분명하지만 최근 연구에 따르면 안드로겐이 작은 크기의 여포 발달을 자극하여 난소의 기질 부피를 증가시키는 것으로 보고되고 있다. 따라서 본 연구는 고안드로겐증이 다낭성 난소와 관련이 있는지를 알아보고자 하였다. National institute of Child Health and Human Disease (NICHD) 의 진단기준에 부합하는 433명의 다낭성 난소 증후군 환자와 420명의 대조군을 대상으로 하였으며 난포기 초기에 8시간 이상 금식한 후 혈액검사로 혈당, 인슐린, 테스토스테론, 황체 호르몬, 난포자극호르몬, 그리고 75g 포도당 부하 검사 후 혈당과 인슐린을 측정하였다. 다낭성 난소는 질 또는 직장 초음파로 측정하였다. 다낭성 난소 증후군 중 다낭성 난소는 304명 (70.2%)의 환자에서 발견되었다. 다낭성 난소 증후군 환자군은 대조군에 비해 통계적으로 유의하게 높은 공복 혈당과 인슐린, 테스토스테론, 75g 포도당 부하 혈당과 인슐린, 난소 부피와 난포 개수, 그리고 대사 청소율을 보였다 (P<0.01). 다낭성 난소 증후군 환자 중 다낭성 난소가 있는 군에서 다낭성 난소가 없는 군에 비해 통계적으로 높은 테스토스테론 수치를 보였다. 테스토스테론은 난소 부피와 높은 상관계수를 보였고 (r=0.225, P<0.01) 로지스틱 회기분석을 통해서도 테스토스테론과 난소부피와는 유의한 관계가 있었다 (β=0.136, P<0.01). 본 연구를 통해 한국 다낭성 난소 증후군 환자 중에서 난소 부피가 클수록 고안드로겐혈증과 관련이 있다는 것을 알 수 있었다.I. INTRODUCTION 1 II. MATERIALS AND METHODS 3 A. Subjects 3 B. Methods 3 C. Data analysis 5 III. RESULTS 6 IV. DISCUSSION 7 REFERENCES 15 KOREAN ABSTRACT 1

Inhibitory roles of TRP14 in RANKL-induced ostoeclast differentiation

Thioredoxin-related protein 14 (TRP14)는 14 kDa 정도 크기의 이황화물 환원 효소로써 이 단백질이 TNFα 자극에 의한 NF-κB와 mitogenactivated protein kinases (MAPKs)를 조절하는 것이 알려져 있다. 그리고파골 세포 (osteoclast)는 우리 몸에서 뼈를 분해함으로써 뼈의 재구성(remodeling) 과 항상성 (homeostasis)에 중요하게 작용하는 다핵세포(multinucleated cell)이다. Receptor activator of NF-κB ligand (RANKL)은 단핵/대식세포로부터 파골 세포로 분화하는데 필수적이고, 이러한 신호전달과정에서 NF-κB와 MAPK가 중요한 신호전달물질로 알려져 있다. TRP14이 파골 세포 분화에 영향을 미치는 지 알아보기 위해 TRP14의 양이 감소된 RAW264.7 stable cell line을 만들었으며 이 세포에서 대조군보다 파골 세포 분화가 촉진되는 것을 확인하였다. 또한 이 세포에서 RANKL 자극에 의해 IκBα와 JNK, p38의 인산화가 더 빠르고 강하게 되는 것을 알 수 있었다. 다음으로 파골 세포 분화에 필수적인 NFATc1의 발현에 TRP14이 영향을 주는 지에 대하여 알아 보았는데, TRP14의 발현이 감소된 세포에서 RANKL에 의한 NFATc1의 발현이 크게 증가하는 것을 볼 수 있었다. 성숙한 파골 세포에서 발견할 수 있는 actin ring의 형성도 TRP14의 발현 양이 감소된 세포에서 더 빠른 시간에 더 큰 actin ring 이 관찰되었다. 그리고 이렇게 RAW264.7 세포의 결과와 마찬가지로 TRP14의 발현이 억제된 BMM에서도 같은 결과를 볼 수 있었다. 즉 BMM에서도 TRP14 발현의 감소가 RANKL에 의한 파골 세포 분화, IκBα와 JNK의 인산화를 증가시켰다. 이러한 결과들은 TRP14이 RANKL에 의한 NF-κB 와 MAPK의 활성화를 저해함으로써 파골 세포 분화를 억제한다는 것을 시사한다.;Thioredoxin-related protein 14 (TRP14) is a novel 14-kDa disulfide reductase, which regulates NF-κB and mitogen-activated protein kinases (MAPKs) in TNFα signaling pathways. To demonstrate physiological relevance of TRP14 to the regulation of NF-κB and MAPK, I examined whether TRP14 can inhibit osteoclast differentiation. Osteoclast differentiation of RAW264.7 macrophages by receptor activator of NF-κB ligand (RANKL) was enhanced by depletion of TRP14, while it was inhibited by TRP14 overexpression, as revealed by tartrateresistant acid phosphatase staining and activity assay. When RAW264.7 cells was exposed to RANKL, depletion of TRP14 augmented phosphorylation and degradation of IκBα, and activation of c-Jun NH2-terminal kinase (JNK) and p38 as well as consequent induction of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) that is a pivotal determinant of osteoclastogenesis. In addition depletion of TRP14 strongly expanded actin ring formation that is a prerequisite for osteoclast bone resorption. Moreover, in bone marrow-derived macrophages, depletion of TRP14 augmented not only osteoclast differentiation, but also activation of NF-κB and JNK. These results suggest that TRP14 attenuates RANKL-induced osteoclast differentiation through inhibiting NF-κB and MAPK.1. INTRODUCTION 1 2. MATERIALS AND METHODS 5 2.1. Reagents and antibodies 5 2.2. Construction of plasmids that express small interfering RNA (siRNA) targeting TRP14 5 2.3. Cloning and mutagenesis of mouse TRP14 8 2.4. Establishment of RAW264.7 stable cells 8 2.5. Generation of TRP14 siRNA transgenic (Tg) mice 9 2.6. PCR for genotyping 11 2.7. Preparation of Bone marrow-derived macrophages (BMMs) 12 2.8. Osteoclast differentiation 12 2.9. Tartrate-resistant acid phosphatase (TRAP) staining 13 2.10. TRAP activity assay 13 2.11. Western blotting 14 2.12. Actin-ring staining 14 2.13.Statistical analysis 15 3. RESULTS 16 3.1. Establishment of RAW264.7 cell lines that stably express TRP14 siRNA or TRP14 16 3.2. TRP14 suppresses RANKL-induced osteoclast differentiation of RAW264.7 cells 23 3.3. TRP14 depletion enhances activation of NF-κB in RAW264.7 cells stimulated by RANKL 28 3.4. TRP14 depletion enhances activation of JNK and p38 in RAW264.7 cells stimulated by RANKL 31 3.5. Depletion of TRP14 augments the expression of TRAF6, c-Fos and NFATc1 in RAW264.7 cells stimulated by RANKL 34 3.6. Depletion of TRP14 promotes actin ring formation in RAW264.7 cells stimulated by RANKL 36 3.7. Generation of TRP14 siRNA transgenic mouse 38 3.8. TRP14 depletion augments RANKL-induced osteoclast differentiation of BMM cells in the presence of M-CSF 41 3.9. TRP14 depletion promotes activations of NF-kB and JNK in BMM cells stimulated by RANKL in the presence of M-CSF 44 4. DISCUSSION 46 5. REFERENCES 52 6. 국문 초록 5

다낭난소증후군의 질병 특징과 관련 유전자형의 관계

연구 배경: 다낭난소증후군은 유전적 인자와 환경적 인자가 복합적으로 작용하는 가임기 여성에서 흔한 내분비 질환이다. 다낭난소증후군은 다유전자 이상에 의한 것으로 여겨지는데 그동안 여러 후보 유전자들이 연구되어 왔으나 일치된 결과를 보이지 않았다. 최근 전장 유전체 연관 분석을 통해 다낭난소증후군의 단일염기다형성 마커들이 밝혀졌는데 관련 유전자들과 다낭난소증후군의 임상적 특징과의 관련성을 분석한 연구는 많지 않다. 본 연구의 목적은 다낭난소증후군의 질병의 특징과 전장 유전체 연관 분석을 통해 밝혀진 관련 유전자형과의 연관성을 알아보는 데에 있다. 연구 방법: 총 1810명의 가임기 여성을 대상으로 하였다. European Society of Human Reproduction and Endocrinology 의 진단기준에 따라 만성 무배란과 고안드로겐증, 다낭난소 중 두가지 이상이 있는 다낭난소증후군 여성 883명과 정상월경을 하는 건강한 대조군 927명을 연구에 포함시켰다. HumanOmni1-Quad v1 array 를 이용하여 다낭난소증후군과 관련된 13개의 단일염기다형성 관련 유전자형을 분석하였고 PLINK 를 통해 전장 유전체 연관 분석을 시행하였다. 연구 결과: 단일염기다형성 마커 중 LHCGR 과 관련 있는 rs10176989, TOX3 와 관련 있는 rs1107466, RAB5B 와 관련 있는 rs705704, 그리고 KHDRBS3 와 관련 있는 rs10505648 가 다낭난소증후군과 대조군 사이에서 유전자형 빈도에 차이가 있었다. FSHB 와 관련 있는 rs11031006 는 테스토스테론 농도 (P < 0.01), 월경 횟수 (P < 0.01), 그리고 황체형성호르몬 농도 (P = 0.01) 와 관련이 있었고 FSHR 와 관련 있는 rs2268361 는 테스토스테론 농도 (P = 0.03), 월경 횟수 (P = 0.03), 난포 개수 (P = 0.03), 그리고 75g 경구 당부하 검사 후의 인슐린 농도 (P = 0.03) 와 관련이 있었다. LHCGR 와 관련 있는 rs10176989 와 TOX3 와 관련 있는 rs11075466 는 테스토스테론 농도 및 월경 횟수와 관련이 있었으며 (각각 P < 0.01, P = 0.01) RAB5B 와 관련 있는 rs705704, KHDRBS3 와 관련 있는 rs10505648, 그리고 YAP1 와 관련 있는 rs1894116 은 월경 횟수와 (각각 P = 0.03, P < 0.01, P < 0.01) 난포 개수와 (각각 P < 0.01, P = 0.03, P = 0.03) 관련이 있었다. 결론: 다낭난소증후군의 전장 유전체 연관 분석을 통해 밝혀진 관련 유전자 중 FSHB, FSHR, LHCGR, TOX3, RAB5B, KHDRBS3, 그리고YAP1 은 다낭난소증후군의 특징적인 형질 – 테스토스테론 농도, 월경 횟수, 난포 개수, 황체형성호르몬 농도, 그리고 포도당 대사와 관련이 있었다. 다낭난소증후군의 유전적 다양성이 질병의 다양한 특징에 영향을 미칠 수 있음을 인지하고 이를 통해 다낭난소증후군의 병태생리를 이해하기 위해 더 많은 연구가 필요하겠다.;Polycystic ovary syndrome (PCOS) is a highly complex disorder with contributions from both genetic and environmental factors. Previous genome wide association studies (GWAS) in the Han Chinese, Korean, and European population identified multiple risk loci for PCOS, but a small number of studies reported the association of susceptibility genes with phenotypic characteristics of disease. The aim of this study was to investigate the association between the susceptibility genes of the PCOS from GWAS and clinical features of the disease. A total of 1,810 reproductive age women (18-39 years old) including 883 women with PCOS diagnosed by European Society for Human Reproduction and Embryology (ESHRE) criteria and 927 regular cycling healthy control women were recruited. The thirteen susceptibility single nucleotide polymorphisms were genotyped using HumanOmni1-Quad v1 array. Venous blood was drawn in early follicular phase to obtain baseline metabolic and hormonal parameter, and the 75g oral glucose tolerance test (OGTT) was performed. Hirsutism was assessed and pelvic ultrasound examinations were performed. The genotype frequencies in rs10176989 nearest to LHCGR, rs1107466 nearest to TOX3, rs705704 nearest to RAB5B, and rs10505648 nearest to KHDRBS3 were significantly different between the PCOS and the control women. The rs11031006 nearest to FSHB was associated with free testosterone level (P < 0.01), menstruation number per year (P < 0.01), and luteinizing hormone level (P = 0.01). The rs2268361 nearest to FSHR was associated with free testosterone level (P = 0.03), menstruation number per year (P = 0.03), ovarian follicular number (P = 0.03), and insulin level after 75g OGTT (P = 0.03). Insulin sensitivity index was marginally associated with FSHR genotype although it did not reach statistical significance (P = 0.06). The rs10176989 nearest to LHCGR and rs11075466 nearest to TOX3 were associated with free testosterone levels (P < 0.01 and P = 0.01, respectively) and menstruation number per year (P = 0.02, P = 0.01, respectively). The rs705704 nearest to RAB5B, rs10505648 nearest to KHDRBS3, and rs1894116 nearest to YAP1 were associated with menstruation number (P = 0.03, P < 0.01, and P < 0.01, respectively) and ovarian follicular number (P < 0.01, P = 0.03, and P = 0.03, respectively). The PCOS susceptibility genes based on GWAS - FSHB, FSHR, LHCGR, TOX3, RAB5B, KHDRBS3, YAP1, and THADA were differently associated with distinctive characteristics of PCOS. This result suggests that genetic heterogeneity may contribute to the complex phenotypes of PCOS.I. INTRODUCTION 1 II. SUBJECTS AND MEHTODS 3 A. Subjects 3 B. Methods 4 C. SNP determination 5 D. Statistical analysis 6 III. RESULTS 7 IV. DISCUSSION 10 REFERENCES 25 ABSTRACT (Korean) 3

Association of Vitamin D Deficiency with Diabetic Nephropathy

Background: Low 25-hydroxyvitamin D (25OHD) levels are associated with the incidence of type 2 diabetes mellitus (T2DM). However, the association between 25OHD and metabolic health status or diabetic complications is inconclusive. We evaluated this relationship between vitamin D status and metabolic parameters and complications of T2DM. Methods: This study included 1,392 patients with T2DM who visited Eulji and Ewha Diabetes Center between January 2011 and August 2016. Anthropometric parameters and laboratory tests including glycated hemoglobin (HbA1c), lipid profile, liver and kidney function, and urinary albumin-to-creatinine ratio (UACR) were evaluated. Diabetic macro- and microvascular complications were determined through a medical record review. Serum 25OHD concentrations were measured by chemiluminescent immunoassay. Results: The mean 25OHD level was 16.8 +/- 9.6 ng/mL. Vitamin D deficiency (<20 ng/mL) and severe deficiency (< 10 ng/mL) were observed in 990 (71.1%) and 351 (25.2%) participants, respectively. 25OHD level was positively correlated with age and high-density lipoprotein cholesterol (HDL-C) level and negatively correlated with HbA1c, triglyceride level, and UACR. HDL-C and UACR were significantly associated with 25OHD after adjusting for other variables. Vitamin D deficiency was independently related to nephropathy after adjusting for confounding variables. Conclusion: Vitamin D deficiency was common among Korean T2DM patients; it was independently associated with microalbuminuria and HDL level, and positively related to diabetic nephropathy

Urinary bisphenol A is associated with insulin resistance and obesity in reproductive-aged women

Background/ObjectivesThe prevalence of obesity has markedly increased and is closely related to insulin resistance. Although lifestyle and genetic predisposition are significant factors influencing the pathophysiology within the body, endocrine-disrupting chemicals (EDCs) are also important triggers of metabolic disturbance. We investigated the relationship between exposure to EDCs and insulin resistance and obesity in healthy, reproductive-aged women. Subjects/MethodsThis cross-sectional analysis included 296 healthy, reproductive-aged women between 30 and 49 years. Metabolically healthy was defined as an absence of the components of metabolic syndrome. Urinary levels of bisphenol A (BPA), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono-n-butyl phthalate (MnBP) were measured by high-performance liquid chromatography and tandem mass spectroscopy (LC-MS). Homoeostatic model analysis of insulin resistance (HOMA-IR) was utilized as an index of insulin resistance. ResultsUrinary BPA levels were positively correlated with BMI, waist circumference, fasting serum insulin and HOMA-IR. MEHHP, MEOHP and MnBP were not associated with any of the above parameters. In the multiple regression analysis, the BPA levels were significantly associated with BMI and waist circumference after adjusting for age, smoking and alcohol consumption status, triglycerides (TG), total cholesterol (TC) and high-density lipoprotein (HDL). Fasting insulin and HOMA-IR values were also significantly related to urinary BPA concentration after adjusting for confounding variables. Metabolically unhealthy women exhibited significantly higher levels of urinary BPA (P = 001) compared to metabolically healthy women. ConclusionsHigher urinary BPA levels are associated with obesity, insulin resistance and metabolic disruption in Korean reproductive-aged women. BPA could play an important role in the pathogenesis of metabolic abnormalities. Further studies are required to elucidate the relationship between EDCs and metabolic disturbances in various age and sex groups

해양과학기술 정보분석을 통한 해양산업 진흥 정책연구

한국해양과학기술