Razvoj medicinskih nauka baziran je na istraživanju koje krajnjoj instanci delimično mora da se obavi na ljudima.

Biomedicinsko istraživanje na ljudima deli se na:

istraživanje čiji je cilj poboljšanje dijagnostičkihi terapijskih postupaka istraživanje sa isključivo naučnim ciljem, bez direktne dobrobiti za dijagnostiku odnosno terapijusubjekta istraživanja.

Cilj biomedicinskog istraživanja na ljudima jeste poboljšanje postojećih dijagnostičkih, terapijskih i profilaktičkih metoda, kao i rasvetljenje etiologije i patofiziologije oboljenja.

Cilj biomedicinskog istraživanja na ljudima jeste poboljšanje postojećih dijagnostičkih, terapijskih i profilaktičkih metoda, kao i rasvetljenje etiologije i patofiziologije oboljenja.

Vrste kliničkih istraživanja

•Opservaciona•Intervenciona

Vrste kliničkih istraživanjaVrste kliničkih istraživanja

•Razjašnjavanje etiologije i patogeneze

• Procena i optimizacija dijagnostike

• Procena efikasnosti terapije

Vrste kliničkih istraživanjaVrste kliničkih istraživanja

– Kontrolnu grupu– Eksperimentalne metode– Cilj istraživanja (primarni,

sekundarni)

– Istraživače uključene u studiju– Eksperimentalnu grupu

Šta je najbitnije definisati pre otpočinjanja istraživanja?

Šta je najbitnije definisati pre otpočinjanja istraživanja?

Odabrati odgovarajuću grupu ispitanika

Odabrati odgovarajuću grupu ispitanika

Kriterijumi za uključenje u studiju

Kriterijumi za uključenje u studiju

• Slučajevi koji ne mogu da se procene (ergotest kod ispitanika bez noge)

• Ne-uključenje iz bezbednosnih razloga (trudnoća)

• Motivisanost bolesnika (non compliat)

Kriterijumi koje sprečavaju uključenje (ekskluzioni)

Kriterijumi koje sprečavaju uključenje (ekskluzioni)

•Paralelne grupe•Ukršteni tip (jedan pacijent)

Eksperimentalni dizajnEksperimentalni dizajn

Konsultacija eksperta i definisanje metoda se radi

pre otpočinjanja studije!

Analiza podataka: Analiza podataka:

• J Steroid Biochem. 1988 Dec;31(6):995-9.•

Androgen levels during sequential insulin euglycemic clamp studies in patients with polycystic ovary disease.

Micic D, Popovic V, Nesovic M, Sumarac M, Dragasevic M, Kendereski A, Markovic D, Djordjevic P, Manojlovic D, Micic J.

Clinic for Endocrinology, Diabetes and Diseases of Metabolism, University Clinical Center, Yugoslavia.

It is postulated that insulin may play a role in the regulation of ovarian androgen production. In order to test the possible interrelation between serum insulin levels and androgen production, sequential euglycemic insulin clamp (Mode 9:1 on Biostator, insulin infusion rate: 0.1; 0.2 and 0.4 U/kg b. wt/h, each rate for 90 min, BC = 80 mg/dl) was done in 6 patients with polycystic ovary disease and normal glucose tolerance. Insulin, C-Peptide, testosterone and dehydroepiandrosterone-sulphate were measured in 0, 70, 80, 90, 160, 170, 180, 250, 260 and 270 min. Significant suppression of C-Peptide levels were achieved (0 min vs 270 min = 0.81 + 0.25 vs 0.15 + 0.20 nmol/l; P less than 0.05). Basal insulin as well as the mean plateau for each insulin infusion rate were as follows: 28 + 9; 248 + 119; 427 + 69 and 524 + 77 microU/l. There was significant testosterone increase at the end of insulin infusion (0 vs 270 min = 4.8 + 1.2 vs 8.1 + 1.7 nmol/l; P less than 0.05). There were no significant changes in dehydroepiandrosterone-sulphate levels during clamp studies (0 vs 270 min = 1055 + 133 vs 913 + 114 ng/ml; P greater than 0.05). It is concluded that acute insulin infusion under the condition of sequential euglycemic clamp could increase androgen production in the ovaries of patients with PCO.

•It is postulated that insulin may play a role in the regulation of ovarian androgen production.

Teorijske postavkeTeorijske postavke

Le virilisme pilaire et son association a l’insufficance glycolitique

(diabete des femmes a barb) Achard C., Thiers J.Bull Acad Natl Med 1921; 86: 51-64

Correlation of hyperandrogenism with

hyperinsulinism in polycystic ovarian disease

Burgen G.A., Givens R.J., Kitabchi A.E., J. Clin. Endocrinol. Metab. 1980; 50: 113-116

Revised 2003 consensus on diagnostic criteria of PCOS

Fauser B., Human Reproduction 19: 41-47, 2004.

Salehi M. et al., Metabolism 2004; 53: 358-376

Theories of the Pathogenesis of PCOS

How common is it ?

• Common endocrine disorder in pre-menopausal women: 5-20 %Hoeger K, Obstet Gynecol Clin North Am 2001; 28: 85-97

• 50 % of PCOS women are overweightGambineri A et al., Int J Obes Relat Metab Disord 2002; 26:883-896

The role of Obesity in PCOS

•Enhancement of hyperinsulinemia

•The role of leptin

•The enzymatic activity of adipose tissue in relation to steroid hormone metabolism

Syndrome X

•Resistance to insulin stimulated glucose uptake•Glucose intolerance•Hyperinsulinaemia•Increased very-low density lipoprotein triglycerides•Decreased high-density lipoprotein cholesterol•Hypertension

Fauser B., Human Reproduction 19: 41-47, 2004.

Criteria for the Metabolic Syndrome in PCOS

MARKERS OF THE RISK OF CORONARY HEART DISEASE

HYPERINSULINEMIC WOMEN WITH POLYCYSTIC OVARY SYNDROME MAY REPRESENT THE FEMALE COMPONENT OF REAVEN’S SYNDROME X

Jacobs H.S.: Polycystic Ovary Syndrome: the

present positionGynecol Endocrinol 1996;10:427-433.

Health consequences of PCOS

• Syndrome X: Elevated VLDL triglyceridesDecreased HDL cholesterolHypertensionInsulin resistanceHyperinsulinemiaGlucose intolerance

• Syndrome XX:PCOSEndometrial cancerBreast cancer (?)

Kazer R.R., Seminars in Reproductive Endocrinology, 1997; 15:193-194.

Zaključci hipoteze Sy PCO “ DUAL DEFECT “ (Poretsky & Piper, 1994)

• Dva nezavisna genetička defekta:Dva nezavisna genetička defekta:•Povećanje LH sekrecijePovećanje LH sekrecije•Insulinska rezistencijaInsulinska rezistencija

• Razvoj Sy PCO je rezultat:Razvoj Sy PCO je rezultat:Sinergističkog delovanja povišenih LH Sinergističkog delovanja povišenih LH nivoa i hiperinsulinemije na jajnik.nivoa i hiperinsulinemije na jajnik.

HIPOFIZA

JAJNIK

NADBUBREG

Insulin

Androgeni

LH

Mišić

Periferna insulinskarezistencija

Serumski insulin

Serumski IGFBP-1Slobodni IGF-1

Povisena sekrecija LH

Folikularni IGFBPs

Izostanak FSH efekta

Povecano stvaranjeandrogena u teki Defektna folikularna

maturacija

Aciklicno stvaranjeestrogena

HIPERANDROGENIZAM ANOVULACIJA

PATOFIZIOLOGIJA Sy PCO

Tscichorozidou T et al.., Clin Endocrinol 60: 1-17, 2004

PATHWAYS LEADING TO ANDROGEN EXCESS IN PCOS

•The aim of the study was to test the possible interrelation between serum insulin levels and androgen production.

Definisanje ciljevaDefinisanje ciljeva

Insulin Effects Related to Ovarian Function

Salehi M. et al., Metabolism 2004; 53: 358-376

Dve karakteristike Sy PCO

• Hiperinsulinemijska insulinska rezistencijaHiperinsulinemijska insulinska rezistencija• Povećana aktivnost ovarijalnog citohroma Povećana aktivnost ovarijalnog citohroma

P450c17 P450c17• HiperinsulinemijaHiperinsulinemija stimuliše ovaj enzim: stimuliše ovaj enzim:

•direktnodirektno•indirektno, povećavajući sekreciju indirektno, povećavajući sekreciju

gonadotropinagonadotropina• Urodjena abnormalnost ?Urodjena abnormalnost ?

Insulin and Cytochrome P450c17

• Cytochrome P450c17 : key enzyme in the biosynthesis of ovarian androgens

• Bifunctional enzime :- 17-hydroxylase- 17, 20-lyase

• Many women with PCOS: increased ovarian cytohrome P450c17activity

• Hallmark: exaggerated serum 17-hydroxyprogesterone response to stimulation by GnRH agonist ( nafarelin; buserelin; leuprolide )

HipofizaHipofiza

ĆĆelija tekeelija teke

HOLESTEROL

PREGNENOLON

17 aHIDROKSIPROGESTERON

ANDROSTENEDION

TESTOSTERON

INSULIN

LH

+

+ ?

+ ?PROGESTERON

17 hidroksilaza

17, 20 - liaza{P450c17

17reduktaza

STIMULACIJA OVARIJALNOG STVARANJA ANDROGENA INSULINOM

Postulated role for insulin-sensitising agents

Harborne L et al.,Lancet2003; 361:1894-1901

• dehydroepiandrosterone-sulphat (nadbubreg vs. ovarijum)

• PCOS vs. zdrave zene

Značaj kontrolne grupeZnačaj kontrolne grupe

“PCOS gen hipoteza”

• Insulin nije dovoljno visok u normalnih ženInsulin nije dovoljno visok u normalnih ženaa ili ili insulin ne reguliše ovarijalne androgene pod insulin ne reguliše ovarijalne androgene pod fiziološkim uslovimafiziološkim uslovima

• Atraktivno objašnjenje je da normalne žene Atraktivno objašnjenje je da normalne žene nemaju genetsku predispoziciju za stimulatorno nemaju genetsku predispoziciju za stimulatorno delovanje insulina na ovarijalne androgenedelovanje insulina na ovarijalne androgene

Nestler JE: Insulin resistance effects on sex hormones and Nestler JE: Insulin resistance effects on sex hormones and ovulation in the Polycystic Ovary Syndrome,ovulation in the Polycystic Ovary Syndrome,U: Contemporary Endocrinology: Insulin Resistance, 1999: 347-U: Contemporary Endocrinology: Insulin Resistance, 1999: 347-365.365.

• Definisanje eksperiementa• Sequential euglycemic insulin clamp (Mode 9:1

on Biostator, insulin infusion rate: 0.1; 0.2 and 0.4 U/kg b. wt/h, each rate for 90 min,) was done in 6 patients with polycystic ovary disease and normal glucose tolerance. Insulin, C-Peptide, testosterone and dehydroepiandrosterone-sulphate were measured in 0, 70, 80, 90, 160, 170, 180, 250, 260 and 270 min.

• Sigurnost za pacijenta• BC = 80 mg/dl

Eksperiementalni protokolEksperiementalni protokol

0

1

2

3

4

5

6

7

8

9

10

TE

ST

OS

TE

RO

NE

(n

mol

/l)

t (min)

0.10.2

0.4Insulin (U/kg/h)

0 70 80 90 160 170 180 250 260 270

Micić D. et al.; J Steroid Biochem 1988; 31:995-999.

• It is concluded that acute insulin infusion under the condition of sequential euglycemic clamp could increase androgen production in the ovaries of patients with PCO.

ZakljucakZakljucak

2 Phenotypes

Low LH-High Insulin

High LH-Low Insulin

Barbieri R.,1988

Minimal model - IVGTT

-20 0 20 40 60 80 100 120 140 160 180 2000

50

100

150

200

250

300

Controls PCOS

Plams

a glu

cose (

mg/dl

)

time (min)-20 0 20 40 60 80 100 120 140 160 180 200

-50

0

50

100

150

200

250

300

350

400

450

Controls

PCOS

Plasm

a insul

in (mU

/l)

time (min)

Pla

sma g

luco

se (

mg/d

l)

Pla

sma insu

lin (m

U/l)

M. Sumarac-Dumanovic,, Insulin secretion and action in PCOS, PhD thesis, Belgrade, 2000

Insulin sensitivity in patients with PCOS and in controls

controls PCOS

0

2

4

6

8

10

Si

P < 0.05BMI p < 0.05IN-BMI +IN-WHR +

M. Sumarac-Dumanovic,, Insulin secretion and action in PCOS, PhD thesis, Belgrade, 2000

Korelacija testosterona i insulinske senzitivnsti (Si) u

SyPCO

Testosteron (nmol/l)Testosteron (nmol/l)

121086420

SISI

10

8

6

4

2

0

gojazne SyPCO

negojazne negojazne SyPCOSyPCO

Sve SyPCOSve SyPCO

r= -0,258, p<0,05r= -0,258, p<0,05