Effect of large dietary doses of ß-carotene on plasma retinoid AND ß-carotene levels and ON progesterone production in the granulosa cells of Japanese quail

An experiment was conducted to study the effect of large-dose (-carotene supplementation on blood retinoid and (-carotene levels as well as on the progesterone secretion of the granulosa cells in Japanese quail. Laying quails were assigned to three dietary groups. The control group (Group C) received the basal diet (laying feed containing 9000 IU vitamin A/kg). In the treated groups (Groups BC1 and BC2) the basal diet was supplemented with 102and 103mg/kg (-carotene (BC), respectively. At the end of the two-week feeding period, 10 birds from each group were euthanised. Blood samples were analysed for retinol, retinyl palmitate and (-carotene concentrations. Granulosa cells were isolated from ovarian follicles (F1and F2), and PMSG-inducedin vitroprogesterone (P4) secretion was measured. Similar retinol concentrations were found in both (-carotene supplemented groups, indicating saturation of the retinol-transporting system. (-carotene supplementation was accompanied by hypercarotenaemia, but did not increase the retinyl palmitate levels in the blood. PMSG-induced P4production of the granulosa cells decreased significantly in Groups BC1 and BC2 in a dose-dependent manner.

Download Full-text

Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation

Journal of Endocrinology ◽

10.1677/joe.1.05550 ◽

2004 ◽

Vol 183 (1) ◽

pp. 51-60 ◽

Cited By ~ 28

Author(s):

E C Chin ◽

D R E Abayasekara

Keyword(s):

Protein Kinase ◽

Adenylate Cyclase ◽

Granulosa Cells ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Nucleotide Exchange ◽

Independent Manner ◽

Human Granulosa Cells ◽

Progesterone Secretion ◽

Dose Dependent

The corpus luteum formed after luteinization of follicular cells secretes progesterone under the control of luteinizing hormone (LH). Binding of LH to its G-protein-coupled receptor leads to the activation of the adenylate cyclase/ cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) signalling pathway. The identification of a new class of cAMP-binding proteins termed ‘guanine nucleotide exchange factors’ (cAMP-GEFs) provides a means by which changes in cAMP could yield actions that are independent of PKA. Hence, in this study, we have explored the hypothesis that steroidogenesis in luteinizing cells is mediated in both a cAMP/PKA-dependent and cAMP-dependent, but PKA-independent, manner. Human granulosa cells were isolated from follicular aspirates of women undergoing assisted conception. Luteinizing human granulosa cells were cultured for up to 3 days in the presence of human (h)LH and the adenylate cyclase activator forskolin in the added presence or absence of increasing doses of the PKA inhibitors H89 (N-[2-(4-bromocinnamylamino)ethyl] 5-isoquinoline) and PKI (myristoylated protein kinase A inhibitor amide 14–22) or the cAMP antagonist, Rp-cAMP. Agonist-stimulated progesterone secretion was inhibited in a dose-dependent manner by the PKA inhibitors and the cAMP antagonist, with decreasing sensitivity as luteinization progressed. Pretreatment of granulosa cells for 4 h with human (h)LH reduced the effectiveness of H89 in inhibiting progester-one secretion. Under basal conditions, cAMP-GEFI expression increased progressively throughout culture, and this could be further enhanced when cells were incubated with increasing doses of LH and forskolin. Furthermore, incubation of cells in the presence of increasing concentrations of the novel cAMP-GEF-specific cAMP analogue, 8 CPT-2 ME-cAMP (8-(4-chloro-phenylthio)-2′-0-methyladenosine-3′,5′-cyclic monophosphate), increased progesterone secretion in a dose-dependent manner. The results show that increases in cAMP generated by LH and forskolin, in addition to activating PKA, also induce increases in cAMP-GEFI protein expression in luteinizing human granulosa cells. In addition, activation of cAMP-GEFI results in increased progesterone secretion. Hence, increases in cAMP lead to the activation of PKA-dependent, as well as PKA-independent but cAMP-dependent (via cAMP-GEFI), signalling mechanisms. Since cAMP-GEFs have the capacity to activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB) signalling pathways, these may provide the potential mechanisms by which cAMP-dependent but PKA-independent progesterone synthesis is regulated.

Download Full-text

Roles of vitamin D and its receptor in the proliferation and apoptosis of luteinised granulosa cells in the goat

Reproduction Fertility and Development ◽

10.1071/rd18442 ◽

2020 ◽

Vol 32 (3) ◽

pp. 335 ◽

Cited By ~ 1

Author(s):

Xiaolei Yao ◽

Zhibo Wang ◽

M. A. El-Samahy ◽

Caifang Ren ◽

Zifei Liu ◽

...

Keyword(s):

Cell Cycle ◽

Vitamin D ◽

Granulosa Cells ◽

Expression Patterns ◽

Follicular Development ◽

Proliferation Rate ◽

Control Group ◽

Dependent Manner ◽

Proliferation And Apoptosis ◽

Dose Dependent

The objective of this study was to investigate the dose-dependent effect of 1α,25-(OH)2VD3 (Vit D3) on invitro proliferation of goat luteinised granulosa cells (LGCs) and to determine the underlying mechanisms of its action by overexpressing and silencing vitamin D receptor (VDR) in LGCs. Results showed that VDR was prominently localised in GCs and theca cells (TCs) and its expression increased with follicle diameter, but was lower in atretic follicles than in healthy follicles. The proliferation rate of LGCs was significantly higher in the Vit D3-treated groups than in the control group, with the highest proliferation rate observed in the 10nM group; this was accompanied by changes in the expression of cell cycle-related genes. These data indicate that Vit D3 affects LGC proliferation in a dose-dependent manner. Contrary to the VDR knockdown effects, its overexpression upregulated and downregulated cell cycle- and apoptosis-related genes respectively; moreover, supplementation with 10nM of Vit D3 significantly enhanced these effects. These results suggest that changes in VDR expression patterns in LGCs may be associated with follicular development by regulation of cell proliferation and apoptosis. These findings will enhance the understanding of the roles of Vit D3 and VDR in goat ovarian follicular development.

Download Full-text

Metformin attenuates steroidogenesis in ovarian follicles of the broiler breeder hen

Reproduction ◽

10.1530/rep-20-0066 ◽

2020 ◽

Vol 160 (5) ◽

pp. 659-672

Author(s):

Evelyn A Weaver ◽

Ramesh Ramachandran

Keyword(s):

Granulosa Cells ◽

Dependent Manner ◽

Metformin Treatment ◽

Broiler Breeder ◽

Breeder Hens ◽

Progesterone Secretion ◽

Expression Of Genes ◽

Broiler Breeder Hens ◽

Chicken Ovary ◽

Dose Dependent

The follicular hierarchy in broiler breeder chicken ovary is often deranged due to excessive ovarian follicular recruitment, resulting in a condition that resembles polycystic ovary syndrome (PCOS) in women. Metformin is widely prescribed to correct PCOS and has been shown to affect granulosa cell functions in humans and rodent models. The objectives of this study are to determine the effects of metformin on signal transduction pathways, gene expression related to steroidogenesis, and progesterone secretion from granulosa cells isolated from the most recently recruited preovulatory and prehierarchical follicles of broiler breeder chickens. Granulosa cells were treated with 0, 1, 10, or 20 mM of metformin in the presence of FSH. The abundance of pAMPK, pACC, pERK, and pAkt was determined by Western blotting. The expression of genes related to progesterone biosynthesis was quantified by qPCR. Progesterone concentrations in culture media were quantified by ELISA. Metformin treatment did not have an effect on the abundance of pAMPK and pACC in prehierarchical follicles but significantly decreased the abundance of pERK and pAkt in a dose-dependent manner in preovulatory and prehierarchical follicles. The expression of genes related to steroidogenesis such as FSHR, STAR, CYP11A1, HSD3B, and progesterone secretion was significantly decreased in response to metformin treatment in a dose-dependent manner. Our data suggest that metformin treatment attenuates progesterone secretion via AMPK-independent pathways in granulosa cells of prehierarchical and preovulatory follicles of broiler breeder hens. Further studies are required to determine if metformin administration could ameliorate ovarian dysfunction in obese broiler breeder hens.

Download Full-text

Changes in cAMP-dependent protein kinase (PKA) and progesterone secretion in luteinizing human granulosa cells

Journal of Endocrinology ◽

10.1677/joe.1.05549 ◽

2004 ◽

Vol 183 (1) ◽

pp. 39-50 ◽

Cited By ~ 9

Author(s):

E C Chin ◽

T E Harris ◽

D R E Abayasekara

Keyword(s):

Protein Kinase ◽

Granulosa Cells ◽

Gradient Centrifugation ◽

Dependent Manner ◽

Dependent Protein Kinase ◽

Human Granulosa Cells ◽

Camp Dependent Protein Kinase ◽

Progesterone Secretion ◽

Dependent Protein ◽

Dose Dependent

Luteinization of follicular granulosa cells leads to an increase in progesterone secretion that is regulated by luteinizing hormone (LH). LH acts mainly by elevating intracellular cyclic 3′,5′-adenosine monophosphate (cAMP) and activating cAMP-dependent protein kinase (PKA). In this study, we have examined the role of PKA in relation to progesterone output by luteinizing human granulosa cells. Human granulosa cells were obtained by percoll gradient centrifugation of follicular aspirates of patients undergoing oocyte retrieval for assisted conception. Cells were cultured in serum-supplemented medium for up to 3 days in the presence and/or absence of human (h)LH and other cAMP-elevating agents. Spent medium was assayed for cAMP and progesterone content by specific RIA. Cell lysates were collected and assessed for PKA regulatory (R)IIα/catalytic (C)α expression by Western blotting. Although basal progesterone secretion increased progressively throughout culture, cAMP levels remained unchanged. Under basal conditions, PKA RIIα/Cα expression appeared to increase throughout the 3-day culture period. In the presence of hLH and other cAMP-elevating agents, progesterone secretion increased in a dose-dependent manner coincident with an increase in cAMP. However, despite the increase in both progesterone secretion and cAMP accumulation, there was a dose-dependent decrease in both PKA RIIα and Cα expression. Thus, data presented in this study show that increases in progesterone secretion in luteinizing human granulosa cells can be dissociated from increases in PKA expression. This notion implies that progesterone secretion may be regulated by PKA-dependent as well as PKA-independent mechanisms.

Download Full-text

Effect of Synchronous Versus Sequential Regimens on the Pharmacokinetics and Biodistribution of Regorafenib with Irradiation

Pharmaceutics ◽

10.3390/pharmaceutics13030386 ◽

2021 ◽

Vol 13 (3) ◽

pp. 386

Author(s):

Tung-Hu Tsai ◽

Yu-Jen Chen ◽

Li-Ying Wang ◽

Chen-Hsi Hsieh

Keyword(s):

Stereotactic Body Radiation Therapy ◽

In Vivo Studies ◽

Control Group ◽

High Dose ◽

Dependent Manner ◽

Hep G2 ◽

Time Curve ◽

Dose Dependent

This study was performed to evaluate the interaction between conventional or high-dose radiotherapy (RT) and the pharmacokinetics (PK) of regorafenib in concurrent or sequential regimens for the treatment of hepatocellular carcinoma. Concurrent and sequential in vitro and in vivo studies of irradiation and regorafenib were designed. The interactions of RT and regorafenib in vitro were examined in the human hepatoma Huh-7, HA22T and Hep G2 cell lines. The RT–PK phenomenon and biodistribution of regorafenib under RT were confirmed in a free-moving rat model. Regorafenib inhibited the viability of Huh-7 cells in a dose-dependent manner. Apoptosis in Huh-7 cells was enhanced by RT followed by regorafenib treatment. In the concurrent regimen, RT decreased the area under the concentration versus time curve (AUC)regorafenib by 74% (p = 0.001) in the RT2 Gy × 3 fraction (f’x) group and by 69% (p = 0.001) in the RT9 Gy × 3 f’x group. The AUCregorafenib was increased by 182.8% (p = 0.011) in the sequential RT2Gy × 1 f’x group and by 213.2% (p = 0.016) in the sequential RT9Gy × 1 f’x group. Both concurrent regimens, RT2Gy × 3 f’x and RT9Gy × 3 f’x, clearly decreased the biodistribution of regorafenib in the heart, liver, lung, spleen and kidneys, compared to the control (regorafenib × 3 d) group. The concurrent regimens, both RT2Gy × 3 f’x and RT9Gy × 3 f’x, significantly decreased the biodistribution of regorafenib, compared with the control group. The PK of regorafenib can be modulated both by off-target irradiation and stereotactic body radiation therapy (SBRT).

Download Full-text

Effect of lemon peel flavonoids on anti-fatigue and anti-oxidation capacities of exhaustive exercise mice

Applied Biological Chemistry ◽

10.1186/s13765-020-00573-3 ◽

2020 ◽

Vol 63 (1) ◽

Author(s):

Guili Bao ◽

Yinglong Zhang ◽

Xiaoguang Yang

Keyword(s):

Skeletal Muscle ◽

Superoxide Dismutase ◽

Manganese Superoxide Dismutase ◽

Fatty Acid Content ◽

Hepatic Tissue ◽

Control Group ◽

Dependent Manner ◽

Lemon Peel ◽

Tnf Α ◽

Dose Dependent

AbstractIn this study, lemon peel flavonoids (LPF) were administered to investigate its effect on the anti-fatigue and antioxidant capacity of mice that undergo exercise until exhaustion. LPF (88.36 min in LPFH group mice) significantly increased the exhaustion swimming time compare to the untreated mice (40.36 min), increased the liver glycogen and free fatty acid content in mice and reduce lactic acid and BUN content in a dose-dependent manner. As the concentration of lemon peel flavonoids increased, the serum creatine kinase, aspartate aminotransferase, and alanine aminotransferase levels of mice gradually decreased. LPF increases superoxide dismutase (SOD) and catalase (CAT) levels in mice and reduces malondialdehyde levels in a dose-dependent manner. And LPF raises hepatic tissue SOD, CAT activities and reduces skeletal muscle tissue iNOS, TNF-α levels of mice compared to the control group. LPF also enhanced the expression of copper/zinc-superoxide dismutase (Cu/Zn-SOD), manganese-superoxide dismutase (Mn-SOD), and CAT mRNA in mouse liver tissue. LPF also enhanced the expression of alanine/serine/cysteine/threonine transporter 1 (ASCT1) mRNA and attenuate the expression of syncytin-1, inducible nitric oxide synthase (iNOS), and tumor necrosis factor (TNF)-α in mouse skeletal muscle. According to high-performance liquid chromatography (HPLC) analysis, it was found that LPF contains flavonoids such as rutin, astragalin, isomangiferin, naringin, and quercetin. Our experimental data show that LPF has good anti-fatigue effects and anti-oxidation ability. In summary, LPF has high prospects to be developed and added to nutritional supplements.

Download Full-text

Toxicopathological effects of lambda-cyhalothrin in female rabbits (Oryctolagus cuniculus)

Human & Experimental Toxicology ◽

10.1177/0960327110376550 ◽

2010 ◽

Vol 30 (7) ◽

pp. 591-602 ◽

Cited By ~ 11

Author(s):

Abdul Basir ◽

Ahrar Khan ◽

Riaz Mustafa ◽

Muhammad Zargham Khan ◽

Farzana Rizvi ◽

...

Keyword(s):

Blood Cell ◽

Oryctolagus Cuniculus ◽

Hemoglobin Concentration ◽

Control Group ◽

Mean Corpuscular Hemoglobin ◽

Dependent Manner ◽

Cell Counts ◽

Lambda Cyhalothrin ◽

Group A ◽

Dose Dependent

The aim of this study was to investigate effects of lambda-cyhalothrin (LCT) on clinical, hematological, biochemical and pathological alterations in rabbits (Oryctolagus cuniculus). New Zealand white female rabbits (n = 24) of 4-5 months age having 997.92 ± 32.83 g weight were divided into four equal groups. Group A (control) received normal saline intraperitoneally (ip). Animals in groups B, C and D were treated with LCT 1.0, 4.0 and 8.0 mg/kg bw ip. Each group received seven consecutive doses at an interval of 48 hours. Blood and serum samples were collected at an interval of 96 hours. Blood analysis revealed a significant (p < 0.05) decrease in red blood cell and white blood cell counts, hemoglobin concentration and lymphocytes, while mean corpuscular hemoglobin concentration, mean corpuscular volume, neutrophils, monocytes and eosinophils were increased. Serum biochemical analysis revealed significant (p < 0.05) decrease in serum total proteins and serum albumin, while an increase was seen in serum alanine aminotransferase and aspartate aminotransferase activities compared with the control group. Serum globulin values varied non-significantly in all treatment groups as compared to control group. A dose-dependent increase in the incidence of micronucleated polychromatic erythrocyte was observed. All gross and histopathological lesions observed in LCT-treated rabbits were dose-dependent. Liver of the treated rabbits exhibited extensive perihepatitis, hyperplasia of bile duct, necrosis, hemorrhages and congestion. In lungs, there were hemorrhages, thickened alveolar walls, congestion, emphysema, collapsed alveoli and accumulation of extensive inflammatory cells. Kidneys were congested and hemorrhagic whereas renal parenchyma and stroma were normal. Microscopically, heart showed congestion of blood vessels and nuclear pyknosis, myodegeneration. It was concluded from the study that LCT produced toxicopathological alterations in rabbits in a dose-dependent manner. On the basis of the results, it can be suggested that overdosing of LCT be avoided while treating animals for ectoparasites.

Download Full-text

Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on bovine granulosa cell steroidogenesis in vitro

Journal of Endocrinology ◽

10.1677/joe.0.1430157 ◽

1994 ◽

Vol 143 (1) ◽

pp. 157-164 ◽

Cited By ~ 59

Author(s):

J G Gong ◽

D McBride ◽

T A Bramley ◽

R Webb

Keyword(s):

Granulosa Cell ◽

Granulosa Cells ◽

Dependent Manner ◽

Serum Free ◽

Factor I ◽

Igf I ◽

Serum Free Conditions ◽

Dose Dependent ◽

Bovine Somatotrophin ◽

Cells Cultured

Abstract Our previous studies have demonstrated that physiological concentrations of metabolic hormones, including recombinant bovine somatotrophin (BST), insulin-like growth factor-I (IGF-I) and insulin, can significantly stimulate the proliferation of bovine granulosa cells cultured under serum-free conditions. In this study we investigated the effects of these factors on bovine granulosa cell steroidogenesis using the same culture system. Bovine granulosa cells were obtained from antral follicles classified into three size classes: small, <5 mm; medium-sized, 5–10 mm and large, >10 mm in diameter. Whilst not affecting steroidogenesis by granulosa cells from small and medium-sized follicles, BST (10–1000 ng/ml) stimulated the secretion of both oestradiol and progesterone by granulosa cells from large follicles in a dose-dependent manner. Insulin (1–1000 ng/ml) and IGF-I (10–1000 ng/ml) stimulated the secretion of oestradiol and progesterone by granulosa cells from all three size categories of follicles in a dose-dependent manner. FSH (200 ng/ml) alone increased progesterone secretion by granulosa cells from all three size classes of follicles, but had no effect on oestradiol secretion by granulosa cells. Both IGF-I (200 ng/ml) and insulin (30 ng/ml) acted in synergy with FSH (200 ng/ml) to stimulate steroidogenesis by granulosa cells from all three size categories of follicles, but no such interaction was observed between BST (50 ng/ml) and FSH (200 ng/ml). In conclusion, BST, IGF-I and insulin significantly influence the steroidogenic activity of bovine granulosa cells cultured under serum-free conditions. However, unlike their effects on cell proliferation, the minimal effective concentrations of these factors required to stimulate granulosa cell steroidogenesis were higher than those observed in our previous studies in vivo. Journal of Endocrinology (1994) 143, 157–164

Download Full-text

Adenosine 5′-Monophosphate-Activated Protein Kinase Regulates Progesterone Secretion in Rat Granulosa Cells

Endocrinology ◽

10.1210/en.2005-0301 ◽

2005 ◽

Vol 146 (10) ◽

pp. 4500-4513 ◽

Cited By ~ 87

Author(s):

Lucie Tosca ◽

Pascal Froment ◽

Patricia Solnais ◽

Pascal Ferré ◽

Fabienne Foufelle ◽

...

Keyword(s):

Protein Kinase ◽

Granulosa Cells ◽

Dominant Negative ◽

Detectable Effect ◽

Mrna Levels ◽

Progesterone Secretion ◽

Igf I ◽

Amp Activated Protein Kinase ◽

Acetyl Coenzyme A Carboxylase ◽

Dose Dependent

The AMP-activated protein kinase (AMPK) is a major regulator of energy metabolism involved in fatty acid and cholesterol synthesis. In the ovary, cholesterol plays a key role in steroid production. We report the presence of AMPK in rat ovaries, and we have investigated its role in granulosa cells. We show using RT-PCR and Western blot that the mRNAs for the α1/2 and β1/2 subunits and the proteins are found in the ovaries. Immunohistochemistry localized the α1 AMPK subunit in granulosa cells, corpus luteum, and oocyte and less abundantly in theca cells. Treatment with 1 mm 5-amino-imidazole-4-carboxyamide-1-β-d-ribofuranoside (AICAR), an activator of AMPK, increased dose-dependent and time-dependent phosphorylation of AMPKα1 on Thr172 in primary granulosa cells. Simultaneously, phosphorylation of acetyl-coenzyme A carboxylase at Ser79 was also increased. AICAR treatment for 48 h halved progesterone secretion, 3β-HSD protein and mRNA levels, and phosphorylation of both basal MAPK ERK1/2 and p38 and in response to IGF-I and/or FSH in granulosa cells. AICAR treatment (1 mm) had no detectable effect on basal and FSH- and/or IGF-I-induced estradiol production and on granulosa cell proliferation or viability. Adenovirus-mediated expression of dominant negative AMPK totally abolished the effects of AICAR on progesterone secretion, 3β-HSD protein production, and MAPK ERK1/2 and p38 phosphorylation. Moreover, we showed using specific in- hibitors of ERK1/2 and p38 MAPK that the MAPK ERK1/2 and not p38 is involved in progesterone secretion and 3β-HSD expression, strongly suggesting that the activation of AMPK in response to AICAR reduces progesterone production through the MAPK ERK1/2 signaling pathway in rat granulosa cells.

Download Full-text

Thrombospondin-1 Inhibits Angiogenesis and Promotes Follicular Atresia in a Novel in Vitro Angiogenesis Assay

Endocrinology ◽

10.1210/en.2009-0686 ◽

2010 ◽

Vol 151 (3) ◽

pp. 1280-1289 ◽

Cited By ~ 30

Author(s):

Samantha A. Garside ◽

Christopher R. Harlow ◽

Stephen G. Hillier ◽

Hamish M. Fraser ◽

Fiona H. Thomas

Keyword(s):

Granulosa Cells ◽

Caspase 3 ◽

Polycystic Ovary ◽

Dependent Manner ◽

Ovary Syndrome ◽

Angiogenesis Assay ◽

Thrombospondin 1 ◽

In Vitro Angiogenesis ◽

Dose Dependent

Thrombospondin-1 (TSP-1) is a putative antiangiogenic factor, but its role in regulating physiological angiogenesis is unclear. We have developed a novel in vitro angiogenesis assay to study the effect of TSP-1 on follicular angiogenesis and development. Intact preantral/early antral follicles dissected from 21-d-old rat ovaries were cultured for 6 d in the presence or absence of TSP-1. At the end of the culture period, angiogenic sprouting from the follicles was quantified using image analysis. Follicles were fixed and sectioned, and follicular apoptosis was assessed by immunohistochemistry for activated caspase-3 in granulosa cells. The results showed that TSP-1 inhibited follicular angiogenesis (P < 0.01) and promoted follicular apoptosis (P < 0.001) in a dose-dependent manner. To determine whether the proapoptotic activity of TSP-1 is mediated by direct effects on granulosa cells, isolated granulosa cells were cultured with TSP-1 (0, 10, 100, and 1000 ng/ml) for 48 h. Apoptosis was quantified using a luminescent caspase-3/7 assay. TSP-1 promoted apoptosis of granulosa cells in a dose-dependent manner (P < 0.05), suggesting that TSP-1 can act independently of the angiogenesis pathway to promote follicular apoptosis. These results show that TSP-1 can both inhibit follicular angiogenesis and directly induce apoptosis of granulosa cells. As such, it may have potential as a therapeutic for abnormal ovarian angiogenesis and could facilitate the destruction of abnormal follicles observed in polycystic ovary syndrome.

Download Full-text