Within 24 hours of life the infant showed no sign of viscous obstructionnor perforation.

The fetus was found to have an enlarged scrotumcontaining a large fluid collection with

densities of echogenicity, highly suggestive of calcifications.

EDITORIAL BOARD

INDONESIAN JOURNAL OF OBSTETRICS AND GYNECOLOGYMajalah Obstetri dan Ginekologi Indonesia

Chief Editor Dr. dr. Laila Nuranna, SpOG(K)

Vice Chief Editor Dr. dr. Dwiana Ocviyanti, SpOG(K) Dr. dr. Junita Indarti, SpOG

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Dr. Med. Damar Prasmusinto, SpOG(K)dr. Kartiwa Hadi Nuryanto, SpOGdr. Herbert Situmorang, SpOGdr. Yuditya Purwosunu, SpOG

Peer Reviewer Prof. dr. Delfi Luthan, SpOG(K) (Medan, Endokrinologi Imunologi Reproduksi)Prof. dr. A. Kurdi Syamsuri, SpOG(K) (Palembang, FetomatemaltProf. Dr. dr. Andrijono, SpOG(K) (Jakarta, Onkologi Ginekologi)Prof. Dr. dr. Djamhoer Martaadisoebrata, SpOG(K), MSPH (Bandung, Obstetri Ginekologi Sosial)Prof. Dr. dr. Johanes C. Mose, SpOG(K) (Bandung, Fetomaternal)Dr. dr. Tono Djuwantono, SpOG(K), M.Kes. (Bandung, Endokrinologi Imunologi Reproduksi)dr. Samodra Suparman, SpOG (Malang, Obstetri Ginekologi Sosial)Prof. dr. Samsulhadi, SpOG(K) (Surabaya, Endokrinologi Imunologi Reproduksi)dr. Amru Sofian, SpOG(K) (Pekanbaru, Onkologi Ginekologi) -dr. Pribakti Budinurdjaja, SpOG(K) (Banjarmasin, Uroginekologi)Prof. dr. Eddy Suparman, SpOG(K) (Manado, Endokrinologi Imunologi Reproduksi)dr. Maria Flavia Loho, SpOG(K) (Manado, Endokrinologi Imunologi Reproduksi)

International Peer Reviewer Prof. Togas Tulandi, MD, MHCM (Milton Leong Chair in Reproductive Medicine,McGill University, Montreal Canada)

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First Published July 1st 1974

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214 Marriska et al

Research Report I

Indones JObstet Gynecol

The Comparison of Expression of Cyclin D and Retinoblastoma MutantProtein in Hydatidiform Mole and in Normal Placenta

Perbandingan Ekspresi Cyclin D dan Protein Retinoblastoma Mutanpada Mola Hidatidosa dan Iaringan Plasenta Normal

Keika Marriska, udi M. Hidayat Tono DjuwantonoDepartment of Obstetrics and Gynecology

Faculty of Medicine University of Padjadjaran/Dr. Hasan Sadikin Hospital

Bandung

Abstract Abstrak

Objective: To know the expression of cyclin Oland mutant re-tinoblastoma in hydatidiform mole and to know the pathogenesis ofhydatidiform mole pregnancy. .

Method: Research specimens were taken from hydatidiform mo-le trophoblastic tissue in Or. Hasan Sadikin Hospital and network-ing. Specimen preparations were stained with Immunohistochemis-try and examined under a light microscope without knowing thestatus of the patient. Significance of the result was tested throughthe Mann-Whitney test, Fisher exact, and McNeman square test.

Result: Significant differences in the expression of cyclin 01between the hydatidiform mole study groups and normal placentaltissue research groups (p<O.OOI). There were significant differencesin the expression of mutant retinoblastoma between the hydatidi-form mole study groups and normal placental tissue research groups(p<0.05). There were significant differences between the domina-tion of cyclin 01 and mutant retinoblastoma in hydatidiform moletissues.

Conclusion: The expression of retinoblastoma was found diffe-rent from normal histopathologically and it was suspected as mutantretinoblastoma. Expression of cyclin 0 I as well as mutant retino-blastoma in hydatidiform mole trophoblast tissue increased, withmutant retinoblastoma being more dominant.

[Indones J Obstet Gynecol 2011; 35-4:214-7]Keywords: expression of cyclin 01, retinoblastoma mutant, and

hydatidiform mole

Tujuan: Untuk niengetahui ekspresi protein Cycliti OJ dau re-tinoblastoma inutan pada mola hidatidosa, serta mengetahui pato-genesis terjadinya kehamilan inola hidatidosa.

Metode: Bahan penelitian diambil dari jaringau trofoblas molahidatidosa di Rumah Sakit Dr. Hasan Sadikin dan Rumah Sakit Je-jaring, lalu dibuat sediaan deugan pewarnaan linunohistokimia.sediaan penelitian diperiksa dengaii niikroskop cahaya tanpa me-ngetaliui status pasien. Uji keinaknaan melalui uji Mann- Whitney,Eksak Fisher, dan Uji square McNeman.

Hasil: Didapatkan perbedaan bermakna pada ekspresi cyclinOf antara kelompok penelitianmola hidatidosa don kelotnpok kOI/-

trol jaringan plasenta normal (p<O,OOI). Terdapat perbedaau yangbermakna pada ekspresi retinoblastoma inutan antara kelompok pe-nelitian mota liidatidosa dan kelotnpok kontrol jaringan plasentanormal (p<0,05). Terdapat perbedaan yal/g bermakna antara domi-nasi ekspresi cyclin 01 dan retinoblastoma inutan pada jaringanmola hidatidosa.

Kesimpulan: Ditemukan ekspresi retinoblastoma yang berbedasecara histopatologi yang diduga sebagai retinoblastoma niutan.Kedua ekspresi Cyclin Of dan retinoblastoma niutan tersebut me-ningkat pada jariugan trofoblas inola hidatidosa, dengan retina-blastoma tnutan Iebih dominan.

[Maj Obstet Ginekol Indones 201 I; 35-4:214-7]Kata kunci: ekspresi cyclin 01, retinoblastoma nuttau, inola hi-

datidosa

Correspondence: Keika Marriska, Department of Obstetrics and Gynecology, Faculty of Medicine University of Padjadjaran, Bandung.Telephone: 08112296135, Emai I:keikaicha@yahoo.com

INTRODUCTION

Gestational trophoblastic disease is a group of disea-ses related to chorialis villous trophoblast cells in par-ticular, consist of complete hydatidiform mole andpartial hydatidiform mole which are benign and inva-sive mole, choriocarcinoma, placental site trophoblas-tic tumors which are malignant.1-5

Incidence of diseases both benign and malignanttrophoblast in Indonesia and several other developingcountries is still high compared with developed coun-tries. Based on research data from WHO, in develo-ped countries there were hydatidiform mole incidenceof between 1:1450 to 1:2000 pregnancies, whereas inJapan there were 2 cases per 1000 pregnancies inwhich 3 times higher than Europe and North Americawhich was about 0.6 to 1 and, 1 per 1000 pregnancies.When compared with developed countries and otherdeveloping countries, it appears that the incidence ofhydatidiform mole in Bandung was quite high, 1: 427.6

Several factors may affect the occurrence of molaare age, parity, previous molar pregnancy, twin preg-nancy, ethnic, and genetica.Lv Me Connell et al exa-mined the role of gene abnormalities during tropho-blast cell proliferation resulting in a failure of preg-nancy that produces a form of malignancy. An imba-lance between the tumor supressor gene and growthfactor in trophoblast cell cycle, makes excessive pro-liferation toward malignancy/d'l

The cause of the normal development of tropho-blast cells to become abnormal until now is still stu-died, suspecting the role of genetic factors. This be-came the basic for researchers to find the cause ofhydatidiform mole associated with a factor of geneticchanges through an initial examination of immuno-histochemical staining in hydatidiform mole tropho-blast tissue that contained the expression of tumorsuppressor genes and growth factor is excessive com-pared with normal placental tissue.

Vol 35, No 4October 2011 Cyclin D and retinoblastoma mutant pro ein

METHODSTumor suppressor genes (TSG) is a protein thatplays a role to control and stop the cell cycle. TSGwhich sorts hydatidiform moles contained in them isRetinoblastoma, p53, GAP (GTPase activating pro-tein). Retinoblastoma (Rb), a protein that functions asa suppressor gene was first discovered in patients withretinoblastoma. Mutations of this gene can be foundin patients with breast cancer, lung cancer, renal can-cer, bone cancer. This gene in normal circumstances,binds to the transcription factor E2F, the complex islocated on the GO and Gl phase. Rb-E2F complexessplit, regardless E2F-free. E2F transcription factorthat escapes becomes active, and is instrumental instimulating the cell cycle to enter the stage of S phaseand E2F transcription factors are also required forDNA replication. The entry of the viral genes for ex-ample the SV40 virus infection, adenovirus and viralinfection7,IO,12, may also inactivated Rb.

If the function of TSG is missing then the cell cy-cle and cell proliferation are not controlled, and ma-kes an abnormal condition such as hydatidiform mole.Studies on the expression of mutant retinoblastomaand p53 mutant showed an increase in hydatidiformmole, which means that the control mechanisms oftrophoblast cells from placental trophoblast tissue candevelop into a pregnancy failure, in this case a hyda-tidiform mole. But if not found expression of retino-blastoma mutant or mutant p53, then the risk of de-generating into the abnormality is low. Protein is veryimportant to suppress the cell cycle, which is the pri-mary control mechanism of cell proliferation.

Growth Factor is a protein that has a role to acti-vate and stimulate the cell cycle and cell proliferation.One of the trophoblast tissue growth factor is a cyclin,a protein which levels fluctuate at each phase of thecell cycle. Cyclin forms a complex with cyclin-De-pendent Protein Kinase (CDK proteins) which are ac-tive, and playa role in the process of phosphorylationof specific protein substrates (Rb ).2,8,9 There are sev-enteen of cyclin proteins that have been identified,working on the phases of the cell cycle. Cyclin-de-pendent protein kinase (CDK) is an enzyme that playsa role in catalyzing the process of protein phospho-rylation. Phosphorylation process is an important partin the activation or inactivation of a number of pro-teins that playa role in cell cycle. Each CDK formsa complex with cyclin to become active. CDK4 andcyclin Dl, D2, and D3 catalyze the phosphorylationof Rb protein, thus E2F previously is not active be-cause it binds to the Rb. E2F is released and becomesactive. Some also binds to cyclin CDK2, CDK5 andCDK6. CDK2 complexes with cyclin El or E2, willstimulate the regulatory cells from G 1 phase to Sphase. CDKI complexes with cyclin Bl or B2 wouldregulate the G2 phase to phase M.lO,11

The central theme in this study is the difference in theexpression of cyelin proteins (growth factors) and mutantretinoblastoma (a tumor suppressor gene) on trophoblastictissue, as a cause of hydatidiform mole. Cyelin D andretinoblastoma mutants are factors that playa role in tro-phoblast tissue cell cyele. Excessive expression of bothcauses excessive proliferation of trophoblast tissue whichresulted in a hydatidiform mole. Immunohistochemicalexamination of an initial examination was done to deter-mine the expression of both proteins.

Analytics observational study aimed to determinewhether there are differences in protein expre iolevels of cyclin Dl and Retinoblastoma mutants inhydatidiform mole trophoblastic tissue compared withnormal placenta.

Research subjects were patients with hydatidiformmole at Dr. Hasan Sadikin hospital Bandung and net-working during the study period. Sampling of thisstudy was based on the order of arrival of patientsincluding exclusion and inclusion criteria and theminimum sample size. Test of significance was doneusing the Manu-Whitney test, Fisher Exact, andMcNeman square test.

RESULT

Table 1. Comparison of cyelin D I expression in patients withhydatidiform mole and normal placenta.

VariableCydin DI~

Category

Mo!a HydatLdifoim (1!=15) .

Cylin DIPositiveNegative

< 0.001"2 (6.7)

13 (43.3)0(0)

15 (50)

**: Value p < 0.05 Fisher exact test

Based on statistical tests showed there were signifi-cant differences in the expression of cyclin Dl in thetwo groups. In the study group with hydatidiformmole the obtained expression of cyclin D 1 is 2, whilein the control group did not obtain the expression ofcyclin D 1, so it can be said that there were statisticallysignificant differences in cyclin D 1 expression bet-ween patients with hydatidiform mole and those withnormal placenta tissue.

Table 2. Comparison of Mutant Retinoblastoma expression inpatients with hydatidiform mole and those with normal pla-centa.

VariableMutant

Retinoblastoma

Mutant Retino-blastoma

PositiveNegative

< 0.017"

6 (20)9 (30.00)

0(0)15 (50)

**: Value p<0.05 based on Fisher exact test

Based on statistical tests showed there were signi-ficant differences in expression between the two gro-ups of mutant retinoblastoma. In the study group withhydatidiform mole the obtained expression of mutantretinoblastoma is 6, while in the control group wasfound no expression of retinoblastoma. In this stud;was found statistically significant differences in ex-pression of mutant retinoblastoma between patienwith hydatidiform mole and those with normal ~-centa tissue. Based on histopathology, the expressi -of retinoblastoma protein are found in patie - -hydatidiform mole which is a mutant rerinoblast -

216 Marriska et at

Table 3. Comparison of expression of cyclin D 1 and MutantRetinoblastoma in hydatidiform mole

Cyclin D 1 (6.7)

5 (33.3)

1 (6.7)

8 (53.3)

+ 2 (1.3)

13 (86.7)0.012*

*p < 0.05 based an test of square McNemar

Based on statistical tests showed there were signi-ficant differences (p = 0012) between the expressionof cyelin Dl and retinoblastoma mutants in t~e hyda-tidiform mole. In hydatidiform mole the obtained theexpression of cyelin D1 is 2, whereas expression ofmutant retinoblastoma is 6, so it can be said that thereare differences between the expression of cyelin D 1and retinoblastoma mutant in patients with hydatidi-form mole statistically. .

DISCUSSION

Comparison of cyclin Dl expression in patientswith hydatidiform mole and in those with normalplacenta

Involvement of cyelin D in cell cycle control andzrowth factors of both normal and malignant cells,~akes the cyelin D as an oncogene (wild type). Ac-tually this form are also on normal cells but. in thiscondition the cell is not normal, because there IS phos-phorylated retinoblastoma that suppre~s the prol~fera-tion. As a result of excessive production of cyclin D,G 1 phase duration becomes shorter, resulting in gr~w-th rezulation of cancer cells. Uncontrolled productionof c~elin D affects the amount of cyelin D-CDK4complexes, leading to excessive proliferation of tro-phoblast cells into mala hydatidiform.ll,12

Mas Rizky Research (2009) showed that there wasincreased expression of cyelin D in hydatidiform mo-le. Cyclin D cause excessive loss of function of TSG(Tumor Supressor Gene) on the phase of the cell cyclecheckpoint that took place without any control andoccurs excessive proliferation .13-15

Figure 1. Immunohistochemistry staining of cyclin DI in nor-mal placenta (400x)

lndones JObstet Gynecol

Figure 2. Immunohistochemistry staning of cyclin DI in hyda-tidiform mole trophoblast (400x)

Comparison of Mutant Retinoblastoma expres-sion in patients with hydatidiform mole and inthose with normal placenta

Retinoblastoma (Rb), a protein that functions as asuppressor protein was first disc,overed in p~tientswith retinoblastoma. This gene In normal CIrcum-stances, binds to the transcription factor E2F, thecomplex is located on the GO and G 1 phase. Rb- E~Fcomplexes split, regardless E2F-fre~. E2F t~a~scnp-tion factor that escapes becomes actrve, and IS instru-mental in stimulating the cell cyele to enter the stageof S phase and E2F transcription factors are also .re-quired for DNA replication. The entry of the viralgenes may also inactivates retinoblastoma Rb and ma-kina a mutant virus infection for example the SV40,ade~ovirus and viral infection 7-12.

If the function of TSG is missing then the cell cy-ele and cell proliferation will not to be well control-led, and makes an abnormal condition such as hyda-tidiform mole. Studies on the expression of mutantretinoblastoma and p53 mutant showed an increase inhydatidiform mole, which means that the control me-chanisms of trophoblast cells from placental tropho-blast tissue can develop into a pregnancy failure, inthis case a hydatidiform mole.

Figure 3. Immunohistochemistry staining of RetinoblastomaMutant in Normal Placenta (400x)

Vol 35, No 4October 2011 Cyclin D and retinoblastoma mutant protein 217

CONCLUSIONSComparison of expression of eyelin 01 and Mu-tant Retinoblastoma in hydatidiform mole

In normal placenta, cyelin D 1 is a dominant gene onthe cell cyele so that cells can move on to the nextphase of the cycle. However, if the function of cyelinD1 is missing then there is the business of compen-sation by increasing regulation of cyelin D expressionof the other so that the cell cyele can take place. Thisdoes not occur in hydatidiform mole, the study byGarnier 0 stated that there was decreased gene ex-pression of cyelin D 1, D2, and D3 in hydatidiformmole, so that the compensation process did not occurand resulted in trophoblast cells turning into hydatidi-form mole.

Several studies have shown that the expression ofretinoblastoma being increased in hydatidiform moleis a mutant retinohlastoma. Retinoblastoma is usuallyfound in patients with mutant breast cancer, lung can-cer, renal cancer, bone cancer. 16,17 If the mutant wasfound in hydatidiform mole it was suspected that thefunction of retinoblastoma is disappeared so that cellproliferation and cell cyele became unwell controlledwell, and created an abnormal condition which washydatidiform mole. Expression of retinoblastoma mu-tant showed an increase in hydatidiform mole, whichmeans that the control mechanisms of trophoblastcells from placental trophoblast tissue is not goingwell. If mutant retinoblastoma expression was notfound, the risk of degenerating into the abnormalityis low.

Expression differences between them can be prov-ed by examination of immunohistochemical-stainingintensity with the view shown in trophoblast cell nu-elei, which role is suspected of cyelin D wild-typeand mutant retinoblastoma, but until now there hasbeen no research that ensures the type of cyelin Dand retinoblastoma mutant so that the necessary fur-ther tests to determine the type of the two are needed.

Figure 4. Retinoblastoma Immunohistochemistry Staining in hy-datidiform mole trophoblast (400x)

Cyclin D1 expression increased in hydatidiform moletrophoblast tissue, expression of mutant retinoblasto-ma was found increased in hydatidiform mole tropho-blast tissue and more dominant than cyelin D1.

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