Artikel Penelitian

Prevention of Malignancy Following Hydatidiform Mole With Vitamin A

Andri Andrijono,* Muhammad Muhilal,** Emil Taufik,*** Meny Hartati,*** Ria Kodariah,*** Wan Leli Heffen****
*Department of Obstetrics and Gynecology, Faculty of Medicine University of Indonesia/ Dr. Cipto Mangunkusumo GeneralHospital, **Center for Research and Development of Nutrition, Ministry of Health, ***Department of Anatomic Pathology, Faculty of Medicine University of Indonesia, ****Center for Research and Development, Dharmais Cancer Hospital

Abstract: The aim of this study was to demonstrate the role of vitamin A as chemoprevention for malignancy following hydatidiform mole. This study was a series of trials. Two supporting studies, i.e. the study on the expression of RPB (retinol binding protein) receptor, the study on apoptosis activity in trophoblastic cells with the administration of retinoic has been conducted. The main study is clinical trial on the prevention of malignancy following hydatidiform mole with vitamin A. The study was a randomized double-blind clinical trial. Subjects of the study were patients with complete hydatidiform mole. Placebo and vitamin A were administered at 200,000 IU per day until patients were declared cured or MTD. Variables of outcome were the incidence of regression and MTD which were established based on WHO criteria. From as many as 21 specimens of with immunohistochemistry study, we found RBP receptor expression in trophoblastic cells. In the study on apoptosis activity in the culture of trophoblastic cells receiving retinoic, we found apoptosis activity. Apoptosis activity in the control was 60%, in ATRA of 50 g/ml was 89.54%, of 100 g/ml was 87.23%, of 150 g/ml was 94.63%, and of 200 g/ml was 94,83%. The clinical trial found 67 cases admitted to the study. As many as 2 cases were lost from observation, and 3 cases experienced pregnancy during observation. The incidence rate of MTD in the control group was 28.57%, and in the therapy group 6.25%. Keywords: vitamin A, Malignant trophoblastic Disease, trophoblastic cells, hydatidiform mole

Maj Kedokt Indon, Volum: 59, Nomor: 6, Juni 2009

251

Prevention of Malignancy Following Hydatidiform Mole With Vitamin A

Pencegahan Keganasan Pascamola Hidatidosa dengan Vitamin A Andri Andrijono,* Muhammad Muhilal,** Emil Taufik,*** Meny Hartati,*** Ria Kodariah,*** Wan Leli Heffen****
*Departemen Obstetri dan Ginekologi Fakultas Kedokteran Universitas Indonesia/ Rumah Sakit Umum Dr. Cipto Mangunkusumo **Pusat Penelitian dan Pengembangan Gizi Departemen Kesehatan *** Departemen Patologi Anatomi Fakultas Kedokteran Universitas Indonesia **** Pusat Penelitian dan Pengembangan Rumah Sakit Kanker Dharmais

Abstrak: Penelitian ini bertujuan membuktikan vitamin A sebagai kemoprevensi keganasan pascamola hidatidosa. Penelitian ini merupakan rangkaian penelitian. Dua penelitian yang mendukung penelitian yaitu penelitian ekspresi reseptor retinol binding protein dan penelitian aktivitas apoptosis pada pemberian asam retinoat pada sel trofoblas. Penelitian utama adalah penelitian pencegahan keganasan pascamola dengan vitamin A. Penelitian adalah uji klinik acak double-blind. Plasebo atau vitamin A dengan dosis 200 000 IU perhari sampai dinyatakan regresi atau PTG (penyakit trofoblas ganas). Variabel keluaran adalah regresi atau keganasan yang ditetapkan berdasarkan criteria WHO. Dengan imunohistokimia terdapat ekspresi reseptor RBP pada 21 sampel sel trofoblas. Aktivitas apoptosis dijumpai pada penelitian kultur sel trofoblas yang diberikan retinoat. Aktivitas apoptosis pada kontrol 60%, 89,54% pada dosis ATRA 50 g/ ml , 87,23% pada 100 g/ml, 94,63% pada dosis 150 g/ml, dan 94,83% pada dosis 200 g/ml. Sejumlah 67 kasus masuk dalam uji klinik. Dua kasus hilang pada pengamatan, 3 kasus hamil. Kejadian PTG pada kelompok control 28,57%, dan pada kelompok terapi 6,25%. Kata kunci: vitamin A, penyakit tropoblas ganas, se; trofoblas, mola hudatidosa.

Introduction Hydatidiform mole showed complaints and signs of pregnancy, with several more dominant symptoms, such as symptoms of vomiting, more rapid uterus enlargement. Frequently, it is accompanied by symptoms of bleeding, or symptoms of thyrotoxicosis. With the adjunct of ultrasonographic examination, hydatidiform mole could be diagnosed earlier.1-3 Histopathologically, datidiform mole is an abnormal pregnancy characterized by the proliferation of trophoblastic cells and hydrophic chorionic villi and with or without fetus.1,2 Diagnosis of hydatidiform mole could be made on the basis of clinical findings. The incidence of MTD following hydatidiform mole was approximately 15-28%. Several factors are implicated in the occurrence of MTD, i.e clinical factor and molecular factor. The clinical factors which are suspected as the risk factors for the occurrence of MTD include, among others, pre-evacuation HCG level and uterus size. Uterus size which is larger than the uterus of gestational age of 20 weeks is a risk factor. Uterus size is a factor that is easy to examine.4 Trophoblastic cells have several activities; however, two primary activities of trophoblastic cells are proliferation activity and apoptosis. If the proliferation following curettage continues, a malignancy degeneration will occur which

is known as persistent hydatidiform mole or malignant trophoblastic disease (MTD). If the activity of apoptosis is dominant, a spontaneous regression will occur. Etiology of hydatidiform mole is still unknown. Molecular factor, which induces cell cycle15 suspected as risk factor. Gene c-erbB2 is a receptor gene of epithelial growth factor (EGF). Disruption of c-erbB2 expression is suspected to have strong correlation with malignancy degeneration following hydatidiform mole.5 PCNA (proliferating cell nuclear antigen) is one of the genes that plays a role in metastasis. Malignancy following hydatidiform mole has potential to metastasize, particularly into the lungs. Manifestation of the PCNA expression is a marker of malignancy risk of trophoblastic cells.6 Expression of human telomerase reverse transcriptase (hTERT), ribonucleoprotein telomerase plays a role in the survival or carcinogenesis. Telomerase expression is discovered in hydatidiform mole and choriocarcinoma, and is not found in the partial hydatidiform mole or normal pregnancy.6,7 Activation of this enzyme is frequently found in malignancy. The role of telomerase in hydatidiform mole is still unclear yet. It was suspected that this enzyme plays a role in the possible occurrence of malignancy following hydatidiform mole.

252

Maj Kedokt Indon, Volum: 59, Nomor: 6, Juni 2009

Prevention of Malignancy Following Hydatidiform Mole With Vitamin A Apoptosis is controlled or stimulated by several genes, such as Bcl-2 and other genes that work to inhibit apoptosis. Expression of apoptosis gene proved to be higher in hydatidiform mole trophoblastic cells than in the trophoblastic cells of normal placenta.8 Vitamin A works to control cell proliferation and stimulate apoptosis. Vitamin A intake from food would be metabolized intro retinol. Inside the liver, retinol takes the form of retinyl ester. Retinol in the plasma is bound by receptor on the cell surface. Retinol would enter cytoplasm with the aid of receptor. In the cytoplasm, retinol is metabolized into retinoic acid. Retinoic acid in the cytoplasm would enter cell nucleus and form a complex of retinoic receptor.11 Retinoic acid plays a role in controlling cell cycle by arresting cell cycle at G1 phase and S phase. The cell arrest by retinoic is achieved through the activation of p53, p21, p27, and it inhibits cyclin.12, Retinoic acid also plays a role in inducing apoptosis. Apoptosis induction by retinoic occurs through the induction of caspase, dab and p53.13,14 Proliferation and apoptosis are the activities of trophoblastic cells and constitute the main activities of vitamin A. There might be a relationship between vitamin A intake and hydatidiform mole. This relationship was identified in the epidemiological study of vitamin A level in hydatidiform mole patients, which showed that level of vitamin A was lower in hydatidiform mole patients than in pregnant women. The risk for developing hydatidiform mole in women less than 24 years of age and with vitamin A deficiency was 6.29 times as high. This risk increased by 7 times if the pregnancy experienced was the first pregnancy.15 Based on this study, there were two questions raised: could vitamin A be one of the factors responsible for the occurrence of hydatidiform mole, and could therapy of vitamin A reduce the risk for developing MTD. Methods In order to demonstrate the benefits of vitamin A administration in reducing the incidence of MTD, it is necessary to conduct a series of studies. This series of studies needs to be performed since such studies have not yet been reported by previous investigators. Study on the expression of retinol receptor in trophoblastic cells. The presence of retinol receptor in trophoblastic cells is extremely important. This is because retinol could enter trophoblastic cell by active mechanism with the aid of receptor. Diffusion mechanism is difficult to demonstrate. Active mechanism could be demonstrated by the presence of receptor in the cell.16 The demonstration of retinol receptor in trophoblastic cells could be carried out by immunohistochemistry examination.17 With the absence of retinol receptor, the role of vitamin A in trophoblastic cells was relatively small. The presence of retinol receptor in trophoblas-

CRABP

RETINOIC + RAR
Cmyc,Cjun (+)

Apaff-1 (+) (+) (+) (+)

(-)

(-)

(+)

CYCLIN-D1, A, E

(+)

P21

(-) CASPASE-7 CASPASE-9 P53 (-) CYCLIN D3 (+) (-) + E2F (+) (-)

(+)

(+)

APOPTOSIS
(+)

(-) Bcl-2

G1
(-)

CELL CYCLE

G2

GATA6

dab2

(+)

(+) (-) P27

M
pRb-E2F

(+)

Figure 1. Activity of Cell Cycle Arrest and Apoptosis by Retinoic

Maj Kedokt Indon, Volum: 59, Nomor: 6, Juni 2009

253

Prevention of Malignancy Following Hydatidiform Mole With Vitamin A culture media. The cell culture was administered ATRA at doses of 50 g/ml, 100 g/ml, 150 g/ml, and 200 g/ml. The outcome variables evaluated were percentages of cells undergoing apoptosis. Evaluation of apoptosis was made with flowcytometry examination in 24 hours following the administration. The percentage of cells undergoing apoptosis was recorded in cytogram by flow-cytometry at the lower right quadrant.18 Study on the prevention of malignancy following hydatidiform mole with vitamin A. Vitamin A could be categorized as chemoprevention. As a medication, vitamin A was also a metabolite of natural substance, easily afforded, inexpensive with mild side effect, and worked at precancerous stage. The work mechanism of vitamin A in trophoblastic cells was demonstrated by laboratory studies. If vitamin A plays a role in trophoblastic cells, it is necessary to demonstrate that vitamin A is capable to work as chemoprevention in hydatidiform mole. Design of this study was randomized clinical trial, double blind study. Samples of the study were patients with complete hydatidiform mole who met the inclusion criteria. We performed treatment by administering placebo and vitamin A 200.000 IU per day until regression or degeneration of MTD was observed. Diagnosis of MTD and regression was established on the basis of WHO criteria.3 The interfering variables were age, education, gestational age, uterine size, and retinol deposit in the liver. The variables were dependent on the incidence of regression and MTD. Results Expression of retinol receptor in trophoblastic cells. We performed the examinations of receptors by indirect immunohistochemistry.

SERUM LEVEL OF VITAMIN A < VIT A DEFICIENCY & 24 YS (OR:6.29) VIT A DEFICIENCY & 24 YS + PARITY <1 (OR:7)
SERUM LEVEL OF RETINOL <N

THERAPY OF VITAMIN A

SERUM LEVEL OF VITAMIN A : N

PROLIFERATION PROLIFERATION (-)

APOPTOSIS
HYDATIDIFORM MOLE

MALIGNANT OF TROPHOBLASTIC DISEASE

Figure 2. Background of Study

PROBLEM SOLVING -----------------------------------VITAMIN A HYDATIDIFORM MOLE

EPIDEMIOLOGICAL STUDY ---------------------------------------LEVEL OF RETINOL IN HYDATIDIFORM MOLE

CLINICAL TRIAL ---------------------------------------CHEMOPREVENTION BY VITAMIN A

LABORATORY STUDY -----------------------------------------EXPRESSION OF RBP RECEPTOR AT HYDATIDIFORM MOLE TROFOBLASTIC CELL

LABORATORY STUDY --------------------------------------------------------APOPTOSIS INDUCTION OF TROFOBLASTIC CELL BY RETINOIC ACID

Figure 3. Problem Solution, Study Series

tic cells should be demonstrated because such a study has not yet been reported by previous investigators. Materials of the study were fixed and paraffinized specimens. The immunohistochemistry examination was indirect. We used secondary antibody of Retinol Binding Protein, and it was evaluated by anatomic pathologist at the Department of Anatomic Pathology, Faculty of Medicine University of Indonesia. The variables evaluated were the presence of RBP expression, the strength and position of RBP receptor expression in trophoblastic cells. Study on apoptosis signals by retinoic acid in trophoblastic cells. Apoptosis signals were better in identifying the activity of medication used as chemoprevention. Apoptosis was considered to be better because it would occur if the arrest of cell cycle took place. The presence of retinol receptor in trophoblastic cells showed that retinol could enter the cell. The study on various cells demonstrated that the activity of retinoic could cause apoptosis. In trophoblastic cells, the activity of retinoic in trophoblastic cells has not yet been reported by previous investigators. Samples of the study were trophoblastic cells. We performed the culture of trophoblastic cells, the presence of trophoblastic cells in the cell culture was demonstrated by hCG examination of
254

Figure 4. Expression of RPB Receptor in Trophoblastic Cells

Figure 5. Expression of RBP Receptor in Cell Membrane, Cytoplasm of Hydatidiform Mole Trophoblastic Cells

Maj Kedokt Indon, Volum: 59, Nomor: 6, Juni 2009

Prevention of Malignancy Following Hydatidiform Mole With Vitamin A As many as 21 specimens of paraffin blocks were obtained. Expression of RBP receptor was found in all specimens. The expression of RBP receptor in syncytiotrophoblast was stronger than the expression of RBP receptor in cytotrophoblast. The expression of RBP was found on the cell membrane, and in cytoplasm of trophoblastic cells. Apoptosis Signals by Retinoic Acid in Trophoblastic Cells Culture of trophoblastic cells was done at the following stages: Mole cell was obtained through curettage evacuation. Specimens were taken from the mole bubbles with disposable syringe. The fluid bubbles were injected into the tubes containing RPMI media of 10% (FBS). Mole cells were washed two times with PBS, and cultured for 24 hours at 370C in 5 % CO2 incubator. After 24 hours, the cells appeared to proliferate, the medium was disposed from tissue culture flask and washed with PBS 2 times of 10 ml. ATRA was administered at doses of 50 g/ml, 100 g/ml, 150 g/ml, 200 g/ ml within the well. ATRA was administered at doses of 50 g/ ml, 100 g/ml, 150 g/ml, 200 g/ml within the well. Incubation for 24 hours in CO2 incubator. Washed with cold PBS and centrifugation. Add 1 mL of the medium and calculate the cell number reaching 4-6 x 107/mL with hematocytometry. Cells were ready to be analyzed with flow cytometry. Cytogram Produced by Flowcytometry Examination: The results of cytogram examination showed that in the control apoptosis reached 60.64%. Apoptosis at ATRA administration of 50 g/mL was 89.45%. Apoptosis at ATRA administration of 100 g/ml was 87.23%. Apoptosis at ATRA administration of 150 g/ml was 94.63%. Apoptosis at ATRA administration of 200 g/ml was 94.83%. Percentage of trophoblastic cells increased with the increased doses of ATRA (see Figure 6). Prevention of Malignancy Following Hydatidiform Mole with Vitamin A Analysis of the study variables against the incidence of regression and malignancy following hydatidiform mole showed that there was a disparity of malignancy incidence following hydatidiform mole between the control group and the therapy group. Distribution of Subjects Characteristics This test was performed to see the distribution of numberical variables in the both groups of study based on the median and mean values. The test results of distribution with equality of populations (Kruskal-Wallis test) and two-

Figure 6. Cytogram Diagram A: Control (DMSO), B: ATRA 50, C: ATRA 150, D:ATRA 200

Maj Kedokt Indon, Volum: 59, Nomor: 6, Juni 2009

255

Prevention of Malignancy Following Hydatidiform Mole With Vitamin A

Table 1. Distribution of Median and Mean Values in Each Group of Intervention According to Characteristic Variables Control (n=35) Median Mean (25-75 pct) (95% IK) 25 (21;30) 1 (0;2) 9 (6;12) 9 (6;12) 12 (0;16) 16 (12;19) 27,03 (24.42;29.64) 1.23 (0.62;1.84) 8.63 (7.40;9.86) 9.40 (8.20;10.60) 11.06 (8.48;13.63) 14.86 (12.95;16.76) Therapy (n=32) Median Mean (25-75 pct) (95% IK) 26 (23;33) 1 (0;3,5) 8.5 (6;10,5) 9 (6;12) 12.5 (4,5;16) 16 (12;20) 28.31 (25.63;31.00) 2.06 (1.18;2.95) 8.00 (6.71;9.29) 9.31 (7.88;10.75) 11.38 (8.73;14.02) 16.00 (14.36;17.64)

Characteristics

P value

Age Parity Education Husband education Gestational age Sounding

0.488 0.113 0.475 0.924 0.863 0.363

Note :

P value of test results of Equality of populations (Kruskal-Wallis test) P value of test restuls of two-sample t test with equal variances

sample t test with equal variances, and the results obtained showed an equal distribution of numerical variables in the both groups of study (see Table 1). Distribution Sparty of Normal Variable The test of proportion disparity of nominal variables was performed to see the distribution of nominal varialbes in the both groups of study by using the test of proportion disparity. The test results of the distribution of uterine fundus height using Pearson chi test (meeting the requirements of chi square test) showed an equal distribution of the results (see Table 2).
Table 2. Distribution of Proportion in the Control and Therapy Groups According to Characteristic Variables Characteristic Control (N=35) n % Therapy (N=32) n % P value

Correlation of the Incidence of MTD and the Time of Incidence

1.00

Kaplan-Meier survival estimates, by randomization

0.50

0.75

P logrank test : 0,0146

0.00 0

0.25

10

15

20 randomization : therapy

25

analysis time
Randomization : control

Figure 7.

Survival Chart of MTD Incidence of Each Group of Intervention

Fundus height <20 weeks >20 weeks Retinol deposit in the liver No sample Sufficient Insufficient End results Regression MTD Loss to follow up Pregnancy Note :

0.587 23 12 65.71 34.29 23 9 71.88 28.13 0.759 3 7 25 24 10 0 1 8.57 20 71.43 68.57 28.57 0.00 2.86 1 7 24 26 2 2 2 3.13 21.88 75 0.029 81.25 6.25 6.25 6.25

This was designed to understand the correlation of MTD incidence and the time the survival test based on KaplanMeier test was done. The table of survival analysis was designed to identify the time of MTD occurrence, number or percentage of patients who developed into MTD associated with time unit in the control group and therapy group (see Figure 7). Side Effects Mean values of SGOT and SGPT before intervention in the control group were not different from those in the therapy group. Mean values of SGOT and SGPT after intervention in the control group was not different from those in the therapy group

P value of test results of proportion disparity with Pearson chi 2 test. P value of the test results of proportion disparity with two direction Fishers exact test

256

Maj Kedokt Indon, Volum: 59, Nomor: 6, Juni 2009

Prevention of Malignancy Following Hydatidiform Mole With Vitamin A No significant difference was found in the changes of mean values of SGOT and SGPT before and after internvetion in the control group. No significant difference was found in the changes of mean values of SGPT before and after intervention. However, a difference was found in the changes of mean values of SGOT before and after intervention in the therapy group (p=0.0092). Discussion A series of three studies on the prevention of malignancy following hydatidiform mole with vitamin A. Vitamin A is metabolized into retinoic acid, and this acid plays a role in controlling proliferation, increasing cell differentiation and increasing apoptosis. The correlation between hydatidiform mole was first demonstrated in epidemiological studies that discovered that vitamin A level in the blood of patients with hydatidiform mole was lower than in the normal pregnant women. However, the results of these studies were relatively difficult and they required an enormous amount of time and cost. Low retinol level in the blood was corroborated by the data showing the less incidence rates of retinol deposit in the liver of hydatidiform mole patients (73.13%). These data showed for a long period of vitamin A deficiency. In order to demonstrate the correlation between vitamin A and hydatidiform mole, the previous study was continued with the study on the prevention of malignancy following hydatidiform mole with vitamin A. The rationale of this study was the activity of proliferation of trophoblastic cells following the evacuation. Malignancy following hydatidiform mole was a continuation of the proliferation of hydatidiform mole trophoblastic cells. Vitamin A that plays a role in controlling the proliferation and increasing apoptosis may prevent the process of the continued proliferation of trophoblastic cells. Retinol which was metabolized into retinoic acid constituted an important part of vitamin A activity. Retinoic acid was an active substance of vitamin A. Vitamin A would be metabolized into retinoic acid if vitamin A could enter the trophoblastic cell. A substance may enter a cell through active mechanism with the aid of receptor. It was relatively easier to demonstrate the entry of vitamin A into trophoblastic cells in an active way with the aid of receptor. By demonstrating the presence of retinol receptor in the membrane of trophoblastic cells and cytoplasm, it showed that retinol could enter a trophoblastic cell. The entry of retinol into trophoblastic cells would be continued with the metabolization of retinol into retinoic acid. Study on the expression of RBP receptor in trophoblastic cells demonstrated that trophoblastic cells have retinol receptor. The presence of retinol receptor in the membrane of trophoblastic cells and cytoplasm goes to show retinol could enter trophoblastic cells with the aid of receptor.
Maj Kedokt Indon, Volum: 59, Nomor: 6, Juni 2009

Retinoic acid was also produced by other cells, to the extent that retinoic acid could be found in the blood. Retinoic acid could enter a cell either through an active way. The entry of retinoic acid into the cell in an active way could be demonstrated by examining the receptor of retinoic acid in trophoblastic cells. The examination of the expression of retinoic acid receptor could not be performed because the receptor antibody of retinoic acid was not available. The administration of retinoic acid in trophoblastic cells suggested that retinoic acid could enter trophoblastic cells. The entry of retinoic acid into trophoblastic cells could be demonstrated by the presence of cell cycle arrest and the arrest of apoptosis activity. Laboratory studies showed the presence of apoptosis activity in trophoblastic cells after the administration of retinoic acid. The percentage of this apoptosis activity increased with the increased doses of retinoic acid administered. Laboratory studies, administration of retinoic acid in trophoblastic cells showed an increase in apoptosis activity. The roles of vitamin A, retinol and retinoic acid in controlling proliferation and increasing apoptosis would provide benefits in preventing the occurrence of malignancy following hydatidiform mole.
RETINOL DEPOSIT IN THE LIVER 71,43% < N THERAPY VITAMIN A

SERUM RETINOL <N SERUM RETINOL > PROLIFERATION RETINOL RECEPTOR AT TROPHOBLASTIC CELL

HYDATIDIFORM MOLE RETINOIC

MALIGNANT TROPHOBLASTIC DISEASE 28,57 % VS 6,25%

APOPTOSIS 60,64% VS 94,83%

Figure 8. The Series of Studies on Vitamin A and Hydatidi form Mole Note: Less retinol deposit was reponsible for less than normal ret inol level in the serum. Retinol deficiency caused proliferation, and this proliferation was responsible for the occurrence of hydatidiform mole and MTD. Vitamin A therapy increased retinol level. Trophoblastic cells had retinol receptor. The retinol entering trophoblastic cells impeded the occurrence of MTD.

Retinoic acid controlled cell proliferation by impeding cell cycle. Cell cycle was impeded through p53, p21, p27, and through the inhibiting effect of cyclin activity to the extent that cell proliferation was impeded. (see Figure 1). Retinoic acid stimulated or induced apoptosis through the stimulation of p53, p21, caspase, and dab.
257

Prevention of Malignancy Following Hydatidiform Mole With Vitamin A Trophoblastic cells had a relatively high level of apoptosis activity. This spontaneous regression process may be due to the fact that trophoblastic cells had apoptosis activity. In laboratory studies the apoptosis activity that was observed in trophoblastic cells was relatively high, i.e. 60.64%. Hydatidiform mole has two main activities, i.e. proliferation and apoptosis. The increase of cell proliferation and decrease of apoptosis constituted the risk for continued proliferation of trophoblastic cells which clinically known as MTD. Vitamin A has two main activities: i.e. controlling and arresting cell proliferation and inducing apoptosis. These two main activities of vitamin A constituted the rationale for administering preventive therapy for malignancy following hydatidiform mole with vitamin A. The administration of vitamin A would increase retinol level in the serum. The increase of retinol level in the serum would increase the amount of retinol entering trophoblastic cells. The increase of retinol in cytoplasm of trophoblastic cells would enhance the metabolism of retinoic acid. The increase of retinoic acid would enhance the signals controlling cell proliferation and increase apoptosis activity. Clinically, the arrest of cell cycle and increase of apoptosis was considered as the increased incidence of regression following hydatidiform mole. The increased incidence of regression by vitamin A was demonstrated by identifying the decreased incidence of malignancy following hydatidiform mole during the administration of vitamin A. The incidence rates of malignancy following hydatidiform mole in the control group was 28.57%, and in the group receiving the therapy of vitamin A was 6.25%. These findings were nearly the same as those obtained at the study on chemoprevention following hydatidiform mole with actinomycin (the control group was 29% and the therapy group 6.9%).19 The risk for developing malignancy following hydatidiform mole when vitamin A was not administered was 8.4 times as high as when hydatidiform mole patients received therapy of vitamin A. In addition, the administration of vitamin A therapy did not result in different side effects as when vitamin A was not administered. However, the administration of vitamin A improved SGOT level of hydatidiform mole patients. Randomized clinical trial, double blind study (clinical trial) showed that the incidence rates of malignancy following hydatidiform mole receiving vitamin A therapy were lower than hydatidiform mole that did not receive vitamin A. Conclusions The study showed existed a receptor of retinol binding in trophoblastic cells. The laboratory study showed that
258

trophoblastic cells of hydatidiform mole had apoptosis activity of 60.64% and retinoic acid increased apoptosis activity of trophoblastic cells. The clinical trial showed that the incidence rates of malignancy following hydatidiform mole (MTD) receiving vitamin A was 6.25%, and in the control group was 28.57%. Recommendations Further studies should be performed on the doses of vitamin A, correlation of vitamin A and ovulation disorder, correlation of vitamin A and ovum abnormalities, correlation of vitamin A and invasive mole, and correlation of vitamin A and choriocarcinoma. References
1. Kurman RJ. The Morphology, Biology, and Pathology of Intermediate Trophoblast: A Look Back to the Present. Human Pathology. 1991;22(9):847-55. Kurowski K, Yakoub N. Staying Alert for Gestational Trophoblastic Disease. Womens Health in Primary Care. 2003;6(1):3945. Tham KF, Ratnam SS. Current views on the management of trophoblastic tumors. Int J Gynecol Obstet. 1995;49:S77-89. Sasaki S. Clinical presentation and management of molar pregnancy. Best Practice and Research Clinical Obstetrics and Gynaecology. 2003;17(6):885-92. Yang X, Zhang Z, Jia C, Li J, Yin L, Jiang S. The Relationship Between Expresion of c-ras, c-erbB-2, nm23, and p53 Gene Products and Development of Trophoblastic Tumor and Their Predictive Significance for the Malignant Transformation of Complete Hydatidiform Mole. Gynecol Oncol. 2002;85:43844. Amezcua CA, Bahador A, Naidu YM, Felix JC. : Expression of human telomerase reverse transcriptase, the catalytic subunit of telomerase, is associated with the development of persistent disease in complete. Am J Obstet Gynecol. 2001;184:1441-6. Bae SN, Kim SJ. Telomerase activity in complete hydatidiform mole. Am J Obstet Gynecol. 1999;180(2);328-33. Halperin R, Peller S, Sandbank J, Bukovsky I and Schneider D.: Expression of the p53 Gene and Apoptosis in Gestational Trophoblastic Disease. Placenta. 2000;21:58-62. Ross AC, Zolfaghari R. Regulation of Hepatic Retinol Metabolism: Perspectives from Studies on Vitamin A Status. J Nutr. 2004;134:269S-75S. Sundaram M, Sivaprasadarao A, DeSousa MM, Findlay JBC. The Transfer of Retinol from Serum Retinol-binding Protein to Cellular Retinol-binding Protein Is Mediated by a Membrane Receptor. J Biol Chem. 1998;273:3336-42. Chen H, Howald WN, Juchau MR. Biosynthesis of All-transretinoic acid from All-trans-retinol : catalysis of All-trans-retinol oxidation by human P-450 cytochromes. Drug Metabolism and Disposition. 1999;28(3):315-22. Budhu AS, Noy N. Direct Chanelling of Retinoic Acid between Cellular retinoic Acid-Binding Protein II and Retinoic Acid receptor Sensitizes Mammary Carcinoma Cells to Retinoic AcidInduced Growth Arrest. Mol and Cell Biology. 2002;22:2632-41. Zhang Y, Rishi AK, Dawson MI, Tschang R, Farhana L. S-phase Arrest and Apoptosis Induced in Normal Mammary Epithelial Cells by a Novel Retinoid. Cancer Res. 2000;60:2025-32. Donato LJ, Noy N. Suppression of mammary Carcinoma Growth by Retinoic Acid : Proapoptotic Genes Are Targets for Retinoic Acid Receptor and Cellular Retinoic Acid-Binding Protein II Signaling. Cancer Res. 2005;65:8193-9.

2.

3. 4.

5.

6.

7. 8.

9.

10.

11.

12.

13.

14.

Maj Kedokt Indon, Volum: 59, Nomor: 6, Juni 2009

Prevention of Malignancy Following Hydatidiform Mole With Vitamin A

15. Andrijono, Kurnia K, Asikin N. A Case-control Study of Vitamin A Level in Hydatidiform Mole. Med J Indones. 1997; 6(3):1537. 16. Sundaram M, Sivaprasadarao A, DeSousa MM, Findlay JBC. The Transfer of Retinol from Serum Retinol-binding Protein to Cellular Retinol-binding Protein Is Mediated by a Membrane Receptor. J Biol Chem. 1998;273:3336-42. 17. Johansson S Denker L, Dantzer V. Immunohistochemical Localization of Retinoid Binding Proteins at the Materno-Fetal Interface of the Porcine Epitheliochorial Placenta. Biol Reprod. 2001;64:60-8. 18. Kravtsov VD, Daniel TO, Koury MJ. Comparative Analysis of Different Methodological Approaches to the inVitro Study of Drug-Induced Apoptosis. Am J Pathol. 1999;155:1327-39. 19. Uberti EMH, Diestel MCF, Guimaraes FE, De Napoli G, Schmid H. Single-dose actinomycin D: Efficacy in the prophylaxis of postmolar gestational trophoblastic neoplasia in adolescents with high-risk hydatidiform mole. Gyn Oncol 2006;102:325-32.

MS

Maj Kedokt Indon, Volum: 59, Nomor: 6, Juni 2009

259