stable archaeal tetraether lipid liposomes for ... 25, no 4 (2016...nafrialdi. editorial

Nafrialdi.editorial

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Medical Journal of Indonesia, under managerial transition

pISSN: 0853-1773 • eISSN: 2252-8083 • http://dx.doi.org/10.13181/mji.v25i4.1735 • Med J Indones. 2016;25:195

Corresponding author: Nafrialdi, [email protected]

Copyright @ 2016 Authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original author and source are properly cited.

NafrialdiDepartment of Pharmacology and Traumatology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Editorial

Medical Journal of Indonesia

Two years ago, in the editorial of the Medical Journal of Indonesia (MJI) No. 1 Volume 23 (2014) I wrote about the year of transition. The main transition at that time was a great change of MJI working system from conventional fto web-based system with Open Journal System (OJS), from paper based to paper less system. Everyone, including the authors, editors, reviewers, secretary staffs, should adapt to the new system. These changes were unavoidable to harmonize our path with international system. And this is in line with the target of MJI to be indexed in an international indexing company.

Indeed, on December 2015 we received announcement from Scopus stating that our journal was accepted to be indexed in the Scopus. For us in Indonesia, this indexation means that the MJI is acknowledged as an international journal by the Indonesian Ministry of Education and Research.

As has been predicted, the change of this status will certainly be followed by significant “shock” perceived by all editorial team as submission of articles to MJI will be enormous. And indeed, now we receive article submission in 2016 that almost double that of the last year.

On the one hand, these changes give us the possibility to increase the quality of our journal, since more selected articles are available. On the other hand, we have to make much greater efforts

since numerous article should be published in each edition, or alternatively we should increase the frequency of yearly publication. It never close the possibility that both, the increase in frequency and number of articles have to be done. On the author point of view, it means heavier competition to be published in MJI, since more articles will be subjected to rejection from the first or subsequent round of review process.

In order to cope with all these changes, the management system of MJI undoubtedly should change, by making adaptations in many aspects of our work. In response to this situation, the Dean of the Faculty of Medicine, Universitas Indonesia has appointed dr. Agus Rizal Hamid, a potential man power, to reinforce our editorial team. In the first time, he will take the position as deputy chief editor for giving him sufficient time to adapt with MJI’s working environment. And in long run he will be projected as editor-in-chief. Of course significant restructuration should be implemented by recruiting more editorial board members and administrative staffs, along with regular training for capacity building. Financial, equipment, and other facilities should be made available.

We hope that dr. Agus Rizal Hamid will bring great innovations for the better future of MJI. Hopefully, this will give good impacts for Indonesian researchers and will heighten the rank of the Faculty of Medicine Universitas Indonesia amongst research universities in the world.

http://dx.doi.org/10.13181/mji.v25i4.1735

196 Med J Indones, Vol. 25, No. 4December 2016

Stable archaeal tetraether lipid liposomes for photodynamic application: transfer of carboxyfluorescein to cultured T84 tumor cells

Keywords: archaea, archaeosomes, carboxyfluorescein, liposomes, T84 colon carcinoma cells, tetraether lipid, Thermoplasma

pISSN: 0853-1773 • eISSN: 2252-8083 • http://dx.doi.org/10.13181/mji.v25i4.1506 • Med J Indones. 2016;25:196–206• Received 20 Jul 2016 • Accepted 25 Sep 2016

Corresponding author: Hans-Joachim Freisleben, [email protected]


Anton Oertl,1,2 Emmanouil Antonopoulos,1 Seruni U. Freisleben,3 Hans-Joachim Freisleben1,4

1 Faculty of Medicine, Johann-Wolfgang-Goethe University, Frankfurt am Main, Germany2 Center of Urology, Asklepios Paulinen Klinik Wiesbaden, Germany3 Department of Physics, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok, Indonesia4 Medical Research Unit, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Basic Medical Research


ABSTRAK

Latar belakang: Membran arkea memiliki lipid fitanil eter yang berbeda dengan ester asam lemak-gliserol dari sel bakteri dan sel eukariotik. Spesies Sulfolobus dan spesies Thermoplasma memiliki tetraeter lipid (TEL) unik sepanjang membran yang membentuk liposom stabil. Spesies Thermoplasma dari Tangkuban Perahu sudah berhasil dikultur dan TEL diisolasi. Pada penelitian ini, kami mengamati secara in vitro transfer carboxyfluorescein (CF) dari liposom TEL yang stabil ke sel karsinoma kolon dalam kultur.

Metode: TEL diekstraksi dari kultur sel Thermoplasma dengan kloroform-metanol (1:1), kemudian difraksinasi dan dimurnikan melalui kolom dietilaminometil-selulose-asetat dan arang diaktivasi untuk pembentukan liposom yang stabil. Untuk menguji pertukaran fluoresensi, TEL liposom diisi dengan CF yang dapat larut dalam air. Pewarnaan diuji dengan berbagai kultur sel dan untuk percobaan utama dipilih sel karsinoma usus T84. Stabilitas liposom diuji melalui ukurannya dengan menggunakan light scattering particle sizer dan mikroskop elektron, serta melalui pelepasan CF non-spesifik pada pH rendah (6,0–7,4) dan suhu sangat tinggi (4–50°C/70°C).

Hasil: Liposom TEL mempunyai stabilitas tinggi dan permeabilitas proton yang sangat kecil pada pH rendah. Pewarnaan CF sel karsinoma usus T84 dengan liposom TEL terlihat lebih intensif daripada dengan liposom dipalmitoylphosphatidylcholine.

Kesimpulan: Hasil percobaan in vitro tersebut memperlihatkan transfer CF ke sel karsinoma kolon dan liposom TEL mempunyai stabilitas tinggi pada pH rendah, sesuai dengan kondisi di jalur gastro-intestinal dan di dalam sistem uro-genital. Oleh karena itu, penelitian in vivo tentang fotosensitizer fluoresensi liposomal untuk aplikasi lokal pada terapi fotodinamik kanker dalam usus besar dan kandung kemih sangat perlu dilakukan.

ABSTRACT

Background: Archaeal membranes have phytanyl ether lipids instead of common fatty acid-glycerol esters in bacterial and eukaryotic cells. Sulfolobus and Thermoplasma species have unique membrane-spanning tetraether lipids (TEL), which form stable liposomes. Recently, we cultured Thermoplasma species from the Indonesian volcano Tangkuban Perahu and isolated TEL. The purpose of this in vitro study is to investigate the transfer of fluorescent dye from stable TEL liposomes to cultured colon carcinoma cells.

Methods: TEL was extracted from cultured cells with chloroform-methanol (1:1), then it was fractionated and purified via diethylaminoethyl-cellulose-acetate columns and activated charcoal for the formation of stable liposomes. For the fluorescence exchange assay, TEL liposomes were loaded with water-soluble carboxyfluorescein (CF). Staining experiments were conducted with various cell cultures, and T84 colon carcinoma cells were chosen for the main experiments. Liposome stability was tested by light scattering and electron microscopic size determinations as well as by unspecific CF release at low pH (6.0–7.4) and increased temperature (4–50°C/70°C).

Results: TEL liposomes exhibit high stability and extremely low proton permeability at low pH. CF staining of cultured T84 colon carcinoma cells appeares more intensive from TEL liposomes than from dipalmitoylphosphatidylcholine liposomes.

Conclusion: The results of this in vitro study demonstrate CF staining of colon carcinoma cells and high stability of TEL liposomes at low pH, matching the condition in the gastro-intestinal (GI) route and in the urogentital (UG) tract. For this reason, in vivo studies on liposomal fluorescent photosensitizers for topical application of photodynamic cancer therapy in the GI and UG tracts should be carried out.


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Recently, we reported about the isolation of Sufolobus species1 and the culture and identification of Thermoplasma acidophilum and Thermoplasma volcanium from Tangkuban Perahu.2 Sulfolobales and Thermoplasmatales produce high amounts of unique tetraether lipids to build their cell membranes.3 Whereas Sulfolobales have a cell wall, Thermoplasmatales do not; their lipid membrane is naked and easily accessible. Therefore, we especially focus on Thermoplasma species because their membrane-spanning tetraether lipids (TEL) form stable liposomes.4 In the laboratory, heterotrophic growth optimum of Th. acidophilum strain DSM 1728 (DSM, Deutsche Sammlung von Mikroorganismen, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures) is at pH 2 and 59°C in a sulfuric acid-containing Freundt’s medium.5 During the last 20 years, archaeal tetraether lipid technology was transferred from Frankfurt am Main, Germany by one of us to Indonesia. We isolated TEL from these cultures and showed the same lipid pattern as in Th. acidophilum strain DSM 1728 and purified the main polar glycophospholipid (MPL) both in our laboratories in Germany and in Indonesia.6 Research on the biomedical application of TEL liposomes have been started 20 years ago in Germany and is now continued by our working group in Indonesian laboratories. Experimental laboratory data were also confirmed by our computerized simulation of a tetraether lipid membrane model.7 Because of the chemical stability of tetraether lipids, we are especially interested in the application of stable liposomes of this lipid in the gastrointestinal (GI) and urogenital (UG) tracts. The purpose of this study is to examine the transfer of fluorescent dyes to carcinoma cells. This is particularly suitable to topical tumor treatment in the GI tract via oral administration and in the UG tract via trans-urethral cystoscopic administration. More specifically, we want to find out whether TEL liposomes can be used for liposomal photodynamic diagnosis (PDD) and therapy (PDT), which have attracted great attention during the last 10 years. The benefits of 5-aminolevulinic acid (5-ALA) PDD in colorectal oncology – including abdominal metastases – have been reported by Kondo et al.8 Furthermore, 5-Ala PDD is widely performed in urological oncology with transurethral fluorescence cystoscopy, especially for the detection of small bladder tumors9 and urothelial premalignant lesions,10 as well as in prostatectomy.11 Whereas the endogenous

metabolic pathway supports 5-ALA PDD and PDT,12 the way of administration of 5-ALA to the target tissue still needs improvement; liposomal delivery in the GI or UG tracts could provide solutions. In urology our study aims to the intravesical application with acid-stable archaeosomes via trans-urethral cystoscopic equipment.

METHODS

Materials, instruments, and human cell culturesPhosphatidylcholine (PCL)= dipalmitoyl-phosphatidylcholine (DPPC), carboxyfluorescein (CF), Nile red, and Triton X-100 were purchased from Sigma (Deisenhofen, Germany); bromobimanes from Calbiochem, Bad Soden, Germany; Sephadex G-50 fine from Pharmacia, Freiburg, Germany; all other substances, solutions and buffers were obtained from Merck, Darmstadt, Germany or from their subsidiaries in Jakarta or Singapore.

Two different types of fluorometers were used in our investigations: a filter fluorometer model 112, Sequoia Turner Corp. California, USA with excitation filter 7.60 (λ=315-385 nm) and emission filter 2A (λ>415 nm) and a spectrofluorometer RF-5001 PC, Shimadzu Corp. Darmstadt, Germany. The two inverse phase contrast fluorescence microscopes used in this study were Type BH2-RFC-1, Olympus Optical Co. Hamburg, Germany and from Olympus subsidiary in Jakarta, Indonesia.

Human T84 colon carcinoma cells were kindly provided by the Center of Physiology, JHW Goethe-University of Frankfurt/ Main, Germany and grown at 37°C under an atmosphere containing 5% CO2 in Nunc Lab-TekTM culture discs (Nunc, Wiesbaden, Germany). Culture medium consisted of 500 mL of Dulbecco’s Modified Eagle Medium containing NaHCO3 1.85 g; HEPES 3.9045 g; penicillin (10,000 U) / streptomycin (10,000 µg/mL); Ham’s F12 5.3 g; fetal calf serum (FCS) 5%. Nunc Lab-Tek discs can be directly used as microscope slides for adherent cell colonies.

Thermoplasma TEL extraction, purification and physical characterizationThermoplasma species were cultured according to Freisleben et al5 or Malik et al.2 Extraction of


the TEL fractions and purification of the main phospho-glyco-tetraether lipid (MPL) were accomplished according to Antonopoulos et al.6

Preparation of liposomes and archaeosomesArchaeosomes, tetraether lipid (TEL) and DPPC (PCL) liposomes were generally (if not indicated differently) prepared according to the classical methods of liposome technology as compiled in New.13 The polar lipid fraction or purified tetraether lipid was dissolved in organic solvent (chloroform-methanol mixture), which was subsequently removed by evaporation in a rotavapor (Büchi, Switzerland) at 45°C. For liposome or archaeosome preparation, the dry lipid film was suspended in phosphate-buffered saline (PBS) and was shaken by hand at room temperature (RT) to disperse the lipid film until a homogenous multilamellar vesicle (LMV) suspension was formed with a lipid concentration of 20 mg/mL. For carboxyfluorescein (CF)-stained liposomes, carboxyfluorescein (CF) was added to PBS at 20 µM concentration. Hand-shaken liposomes or archaeosomes were sonicated with a Branson Sonifier at 20 kHz for five minutes on ice before applied to filter extrusion by means of a LiposoFast™Extruder using 100 nm pore-sized polycarbonate filters (Avestin Inc., Ottawa, Canada). After preparation of the liposomes, non-liposomal material was removed through open column chromatography utilizing Sephadex G 50 fine (Pharmacia, Freiburg, Germany).

Although TEL liposomes are heat stable and can be autoclaved or even heat sterilized,14 we used sterile filtration, especially, since the latter can be applied equally to any other lipids (in our case PCL, DPPC) and can be done in one run with the extrusion method for liposome preparation if polycarbonate filters with 100 nm pores are used. The influence of protons, temperature, alcohols and detergents on swelling and long-term shelf stability of the liposomes was determined according to Freisleben.3,4

Intermembrane exchange assayIn order to test the release of lipophilic compounds to membranes of intact target cells from TEL liposomes intermembrane exchange assay to erythrocyte ghosts was carried out.15,16 Nile red or MBB was mixed with TEL at 20 µM concentration as described in 2.3 and co-evaporated. Lipid film formation incorporated the lipophilic dyes

almost quantitatively at the low concentrations was used for our experiments. Liposomes were then prepared as described and passed through Sephadex G50 columns to remove non-entrapped dyes. Control experiments for the demonstration of membrane fusion between liposomes and erythrocyte ghosts were carried out by the incorporation of cholesteroyl-anthracen-9-carboxylate (CA9C) into erythrocyte ghosts and of dihexadecylamino-7-nitrobenzo-2-oxa-1,3-diazole (DH-NBD) into the liposomal membrane.

RESULTS

Physicochemical characterization of liposomesOur preparation method yielded uniform TEL liposomes with narrow size distribution of 150.0±22.1 nm. Besides the method of preparation, the grade of purity of the lipid was important; particle size decreased in parallel with higher purity of tetraether lipid, primarily with the removal of pigments and lipopolysaccharide complexes (not shown). The zeta potential of TEL liposomes was -58.2±0.2mV at pH 7.4 in diluted PBS as determined in a Malvern Zeta Sizer. For comparison, DPPC liposomes were slightly smaller (143.5±41.0 nm) than TEL liposomes.

TEL liposomes were stable for more than 100 weeks when stored in PBS (10 mg lipid/mL) at 37°C, RT, and at 4–8°C, as measured by changes in size distribution (swelling). For comparison, liposomes from DPPC were stable only up to four weeks in the refrigerator (4–8°C), at RT and 37°C only for a few days. At high temperatures (>60°C), TEL liposomes were stable for 10 weeks.

Swelling of TEL liposomes by addition of glycerol was less than 5% at RT. The liposomes also resisted several-fold higher detergent and alcohol concentrations than DPPC liposomes. Proton leakiness of common liposomal membranes was about 50-times higher than that of TEL membranes.

Loading of liposomes (incorporation of lipophilic or hydrophilic compounds)Most of CF was found dissolved in the outer volume and some of it was attached to the outer surface of the liposomes. Non-liposomal CF (i.e., not associated with, entrapped or attached

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Temperature pH Time % release from TEL liposomes

% release from PCL liposomes

RT 6.0 15 min 0 31.4RT 6.6 15 min 2.1 23.3RT 7.0 15 min 4.2 17.5RT 7.4 15 min 5.8 19.44°C 7.4 24 h 2.5 9.030°C 7.4 24 h 3.0 41.050°C 7.4 24 h 8.0 100.0

Table 1. Control experiments: comparison of non-specific CF release (%)

At the end of each experiment, 2% Triton X-100 was added to destroy the liposomes and the resulting fluorescence set to 100% CF release. TEL= tetraether lipid; PCL: phosphati-dylcholine; DPPC: diphosphatidylcholine; RT: room temper-ature

to liposomes) was removed by open column separation with Sepadex G 50 fine (principle of “Penefsky” columns). On the other hand, the experimental incorporation of lipid-soluble compounds, i.e. Nile red and thiolyte MBB into liposomes of archaeal lipids showed that they were almost quantitatively taken up into the liposomal membrane.

The incorporation rates of water-soluble compounds, such as CF can be explained as follows. Hydrophilic CF is dissolved in the buffer, entrapped into the liposomes when they form closed spherical particles, and thus distributed between the inner volume of the liposomal lumen and the outer volume. The amount of lipid was 20 mg/mL and the outer volume exceeded the inner volume. Hence, most of CF was not entrapped and subsequently removed by open column chromatography. The remaining liposomal CF was sufficient for the staining experiments. Lipophilic compounds (Nile red or thiolyte MBB) were taken up into the liposomal membrane by approximately 100%.

Table 1 at the end of each experiment, 2% Triton X-100 was added to destroy the liposomes and the resulting fluorescence set to 100% CF release; TEL, tetraether lipid; PCL, phosphatidylcholine = DPPC, diphosphatidylcholine.

Preliminary experiments: interaction with cell membranes and cellsThe study for CF staining from liposomal TEL was performed by using various cell cultures

available in the laboratories of the Medical Faculty in Frankfurt, e.g., human colon carcinoma HT-29 and T84 cell lines, human hepatocellular carcinoma cells (HepG2), cystic fibrosis human ductal pancreatic adenocarcinoma cells (CFpac), Chinese hamster ovary cells (CHO), baby hamster kidney cells (BHK), and primary normal human dermal fibroblasts (NHDF). At liposomal lipid concentration of 200 µg/mL in the medium, cell-bound fluorescence was detected at gradual intensity in the sequence: T84>HT29>HepG2=CHO>CFpac>NHDF>BHK. The observed differences in staining experiments depended on the amount of liposomal lipid applied in the experiments; doubling the lipid concentration to 400 µg/mL modified the fluorescence intensity to CHO>>HepG2≥HT-29>>CFpac (not shown).

The interaction with isolated red cell membranes (erythrocyte ghosts) showed that Nile red and MBB were taken up quantitatively into the erythrocyte membranes by intermembrane exchange within 60 minutes. Membrane fusion between liposomal TEL membrane and erythrocyte membrane could not be proven.

Fluo

resc

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50

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Time [minutes]5 10 15 20 25 30 35 40 45 50 55 60

Figure 1. Transmembrane exchange of monobromobimane (MBB) from TEL liposomes to isolated erythrocyte mem-branes (ghosts); x= negative control, fluorescence of TEL liposomes labeled with MBB, no ghosts present (excitation at 380 nm, emission at 480 nm); += positive control, ghosts labeled with MBB, no liposomes present; the time course of increasing fluorescence emission denotes the reaction kinet-ics between MBB and sulfhydryl groups in the ghosts; *= TEL liposomes labeled with MBB in the presence of ghosts; the time course denotes the intermembrane exchange of MBB from liposomes to the erythrocyte membrane and its reac-tion with sulfhydryl groups in the cell membrane. Ghosts were incubated with 20 µM concentration of MBB for 2 min-utes before measurement of fluorescence was started. Lipo-somes with MBB were passed through a Sephadex column before added to ghosts


As demonstrated in this experiment lipophilic dyes (e.g., MBB and Nile red) are transferred from liposomes to cell membranes via intermembrane exchange. The comparable mechanism with hydrophilic dyes like CF is called contact release, i.e., release of CF from liposomes and uptake into cells if loaded liposomes touch the cell membrane or are attached to it.15

Our experiments with erythrocyte ghosts containing fluorescent dye cholesteroyl-anthracene-9-carboxylate (CA9C) and TEL liposomes containing dihexadecylamino-7-nitrobenzo-2-oxa-1,3-diazole (DH-NBD) showed that almost no fusion between liposomal and cell membranes occurred, i.e., excitation at λ 370 nm only slightly increased emission at 550 nm and decreased emission at 450 nm (not shown) through direct interaction and energy transfer between CA9C and DH-NBD.13 Although membrane fusion and uptake of liposomes into cultured T84 tumor cells may occur, our experiments confirmed that intermembrane exchange is possible without

membrane fusion and/or the uptake of liposomes into cells.16 Liposome–cell interactions and uptake into tumor cells were investigated, and effects of the liposomal protein corona extensively discussed.17 The latter phenomenon depends on the environment surrounding the liposomes and on the formation of a biomolecular corona at the liposomal surface, which differs between cell cultures and the biological systems in the body (e.g., GI or UG tracts or blood circulation). In general, formation of a biomolecular corona at the liposomal surface appears to enhance uptake into tumor cells.17 These phenomena will be investigated with our TEL liposomes in the future.

As a first approach, incorporation and release of CF without the presence of cell membranes or intact cells was studied with sonicated TEL liposomes and compared to liposomes made of PCL (DPPC) and egg yolk lecithin (EYL). At the end of each experiment, 2% Triton X-100 were added for 100% release of CF. Temperature- and pH-dependent release was 5–10 times lower from

Figure 2. Control experiments with trypsinated T84 cells: A, B) treated with carboxyfluorescein (CF, incubation 30 minutes with-out liposomes, magnification 200x); C, D) treated with CF-loaded dipalmitoylphosphatidylcholine (DPPC) liposomes (no incuba-tion time, magnification 400x); A, C) phase contrast microscope; B, D) fluorescence microscope

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TEL than from common liposomes with greatest differences at low pH and high temperature (Table 1). The concentrations of alcohols and detergents necessary for 50% CF release were considerably higher with TEL than with common liposomes. These experiments demonstrate that the leakiness of common PCL (DPPC) liposomes is considerably higher than that of TEL liposomes or archaeosomes.3

Main experiments: interaction with cultured T84 colon carcinoma cellsFrom the orientation in our preliminary experiments, we used the liposomal lipid concentration of 200 µg/mL in the main experiments of cellular CF staining. Now, we report on the promising interaction with T84 colon carcinoma cells. Cell cultures were incubated up to four hours. After more than four hours incubation, fluorescence became diffuse, seemed less associated with cells and differences between cell cultures blurred.

Figure 3. Experiments with T84 cell colonies (magnification 200x): A, B) incubation with carboxyfluorescein (CF)-loaded dipal-mitoylphosphatidylcholine (DPPC) liposomes for one hour; C, D) incubation with CF-loaded tetraether lipid (TEL) liposomes for one hour; A, C) phase contrast microscope; B, D) fluorescence microscope

Cultured T84 colon carcinoma cells were incubated with CF (Figure 2A,B) or liposomal CF (Figures 2C,D through 5), either after trypsination or as adherent colonies. The latter experiments were conducted in order to rule out mere effects of trypsination on our results. Figure 2A,B shows control experiments with trypsinated T84 cells incubated for 30 minutes with non-liposomal CF. No cell-bound fluorescence can be seen. Figure 2C,D shows control experiments with CF-loaded DPPC (PCL) liposomes immediately after addition, i.e., without incubation (incubation time= 0). Almost no cell-bound fluorescence can be seen. Figure 3A,B shows T84 cell colonies incubated with CF-loaded DPPC (PCL) liposomes for one hour. Figure 3A is the picture in the phase contrast microscope and 3B is the fluorescence picture with weak diffuse CF staining. Figure 3C,D shows T84 cell colonies incubated with CF-loaded TEL for one hour. Figure 3C is the phase contrast picture, and figure 3D is the fluorescence picture with weak CF staining.

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Figure 4A,B shows trypsinated T84 cells incubated with CF-loaded DPPC (PCL) liposomes for one hour. Figure 4A is the phase contrast picture, and figure 4B is the fluorescence picture with slightly stronger CF staining than in figure 3B. Figure 4C,D shows trypsinated T84 cells incubated with CF-loaded TEL liposomes for two hours. Figure 4C is the phase contrast picture, and figure 4D is the fluorescence picture with very strong CF staining.

Figure 5A,B shows T84 cell colonies incubated with CF-loaded DPPC (PCL) liposomes for four hours. Figure 5A is the phase contrast picture, and figure 5B is the fluorescence picture with diffuse CF staining. Figure 5C,D shows T84 cell colonies incubated with CF-loaded TEL liposomes for two hours. Figure 5C is the phase contrast picture, and figure 5D is the fluorescence picture with strong and clear CF staining. Over a time course of up to four hours, cell-bound fluorescence did not strongly increase with DPPC

Figure 4. Experiments with trypsinated T84 cells (magnification 400x): A, B) incubation with carboxyfluorescein (CF)-loaded dipalmitoylphosphatidylcholine (DPPC) liposomes for one hour; C, D) incubation with CF-loaded tetraether lipid (TEL) liposomes for two hours; A, C) phase contrast microscope; B, D) fluorescence microscope

(PCL) liposomes (Figures 3B, 4B, 5B). On the other hand, cell-bound fluorescence strongly increased from TEL liposomes within two hours (Figures 3D, 4D, 5D). The most relevant result is shown in figure 4D with the strongest cell-bound fluorescence in trypsinated cells after two hours of incubation with stable TEL liposomes. Hence, all other pictures should serve for comparison with the result in figures 4D and 5D as the confirmation of the result also in non-trypsinated adherent cell colonies.

DISCUSSION

Physicochemical characterization of TEL membranes and liposomesThis study describes the potential of tetraether lipid (TEL) extracted from the membranes of Thermoplasma species, laboratory stem DSM 1728 and cultured wildtype isolates from the Indonesian volcano Tangkuban Perahu. In

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archaeal membranes, TEL forms monolayers in similar thickness as bilayer membranes.7 This is a major reason for mechanical stability of TEL membranes under extreme environmental and experimental conditions18 since they do not break in the middle of the membrane as bilayers do.3,4 Mechanical and physicochemical stability are important for the administration of liposomes via GI or UG routes. The phytanyl chains of TEL are fully saturated and contain methyl side groups, which provide membrane fluidity on one hand.3,4 On the other hand, they are (different from unsaturated fatty acids) highly resistant towards oxidation and peroxidation.14 Membrane fluidity is regulated through these methyl side groups and pentacyclization within the phytanyl chains, which vary depending on growth temperature and external pH.7,19 Resistance of these membranes to acidic pH and their impermeability for protons (i.e., extremely low leakiness from TEL liposomes) are important for the stability of TEL liposomes

Figure 5. Experiments with T84 cell colonies: A, B) incubation with carboxyfluorescein (CF)-loaded dipalmitoylphosphatidylcho-line (DPPC) liposomes for four hours (magnification 400x); C, D) incubation with CF-loaded tetraether lipid (TEL) liposomes for two hours (magnification 200x); A, C) phase contrast microscope; B, C) fluorescence microscope

in the acidic environment of the gastric fluid or urine.

Cytotoxicity, mutagenicity, toxicity and biodistribution in mice The influence of liposomes from highly purified TEL (MPL) on a wide range of cell cultures was investigated in cytotoxicity screening including tests on mutagenicity and antimutagenic efficacy. None of the testing showed cytotoxic or mutagenic properties of TEL (MPL) or TEL liposomes.20 No toxicity was detected in mice in central nervous screening and the survival rates. The MPL-fed mice lived even longer than the controls.21 Biological stability of TEL-containing liposomes and biodegradation was investigated in the liver of mice.22 Although biotransformation of TEL needs further clarification, it is concluded that TEL liposomes can be safely applied also to humans at amounts needed for therapeutic purposes.

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Release of 6-carboxyfluorescein (CF) and lipophilic dyes Lipophilic dyes (Nile red and MBB) used in this study are transferred via intermembrane exchange from the liposomal membrane into the cell membrane. Membrane fusion and the uptake of liposomes into cells could not be proven in our experiments.

If the liposomes touch the cell surface or get attached to it, lipophilic dyes follow the pattern of ‘intermembrane exchange’ and in case of water-soluble CF, uptake into the cells occurs via ‘contact release’ from the liposomes at the cell surface. Our preliminary experiments with thiol-reactive MBB showed that it was readily transferred from liposomal TEL membranes into isolated erythrocyte membranes. The latter have thiol groups whereas liposomal TEL membranes do not. Hence, MBB exerted very low fluorescence emission in liposomal membranes. As soon as erythrocyte ghosts are present, MBB penetrates into the red cell membrane, reacts with thiols, and its fluorescence emission increases strongly.

The application of TEL to form more stable liposomes than common DPPC (PCL) appears advantageous, because the former are less leaky than the latter, especially at low pH (Table 1). Hence, the non-specific loss of CF from unstable and leaky DPPC (PCL) liposomes leads to diffuse fluorescence whereas the more cell-specific contact release from TEL liposomes leads to more intensive cell-bound fluorescence.

Biomedical application of archaeosomes and TEL liposomesThe liposomes used in this study were around 150 nm in diameter. Liposome technology with archaeal lipids does not differ in principle from the technology with common lipids. Archaeal TEL can even be used to stabilize liposomes of common lipids.22 The hydrolytic stability of TEL (MPL) liposomes and archaeosomes towards acidity and the low permeability of their membranes for protons3,4 provide wide possibilities for gastro-intestinal applications, in the first place for oral vaccination, especially since bacterial and archaeal membrane lipids seem to exert adjuvant properties in the GI mucosa.23,24 Further applications were shown by González-Paredes et al25 suggesting ‘archaeosomal hydrogels’ as novel delivery system for antioxidants.

After the intensive interaction of TEL liposomes with cultured T84 colon carcinoma cells seen in our experiments, we can add another possible application in the GI tract as antitumor delivery systems in the therapy of colon carcinomas. It is, of course, a long way from our in vitro experiments to in vivo animal studies and eventually to clinical application. For toxicological reasons, TEL liposomes and archaeosomes from archaeal membrane fractions must be purified from pigments and lipopolysaccharide complexes and from any other possibly toxic components. Lipid purification, in general, is very time-consuming and cost-intensive.6 Hence, for pharmaceutical application in oral drug delivery or oral vaccination, the use of sufficiently purified membrane fractions may be advantageous over highly purified single lipids. Purified membrane fractions both from Thermoplasma and Sulfolobus species form stable archaeosomes.26 Another possibility to reduce costs is the stabilization of (generally less expensive but instable) liposomes made of common bilayer-forming phospholipids with TEL at 2.5 mol% concentrations.22

Photodynamic therapy (PDT) using 5-ALA and its derivatives is based on heme biosynthesis and has been approved for treatment of various cancers. Treatment of cells with 5-ALA induces high bioactivity of the heme pathway and accumulation of porphyrins, mainly protoporphyrin IX (PpIX). Ferrochelatase, which is known to be less active in cancer cells, incorporates Fe2+ into PpIX and converts it into the photochemically inactive heme. Hence, PpIX mainly accumulates in cancer cells treated with 5-ALA. Subsequent illumination of these tumor cells at wavelength of 454 to 545 nm induces fluorescence of PpIX and thus differentiation from non-cancerous tissues. Since the fluorescent dye is also a photosensitizer, illumination will also induce the formation of singlet oxygen and other radicals which kill the respective tumor cells, but are less harmful to the surrounding healthy tissue.12 The technique, which is already successfully used for tumor diagnostics has already been approved for photodynamic cancer therapy.

Whereas the endogenous metabolic pathways support PDD and PDT, the way of administration of 5-ALA to the target tissue needs improvement and our idea comes in with liposomal systems for the delivery of fluorescent or photosensitizing

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dyes for diagnosis and treatment of colorectal cancers und urocyst transition cell carcinomas. Liposomal application in the UG tract will provide solutions aiming to intravesical administration of acid-stable archaeosomes via trans-urethral cystoscopic equipment. In the GI tract, the oral administration of acid-stable archaeosomes is particularly suitable to topical tumor treatment.

In conclusion, although stability of liposomes from archaeal TEL is much higher and leakiness much lower than that of liposomes from common phospholipids, the transfer of fluorescent dyes is more effective from these than from common DPPC liposomes, both for lipophilic dyes (Nile red, MBB) and hydrophilic CF. One reason may be that common liposomes already release part of their load unspecifically whereas stable liposomes from archaeal lipids keep their load and release it more specifically when they get in contact with target cells. A weakness of our study is that our fluorescence measurements could not be standardized for quantification. This should be the aim of following studies.

Acknowledgment This study was financially supported by the Deutsche Forschungsgemeinschaft in Germany and by URGE in Indonesia.

Conflict of interestThe authors affirm no conflict of interest in this study.

REFERENCES

1. Handayani S, Santoso I, Freisleben HJ, Huber H, Andi, Ardiansyah F, et al. Archaeal life on Tangkuban Perahu - sampling and culture growth in Indonesian laboratories. Hayati J Biosci. 2012;19(3):150–4.

2. Malik A, Santoso I, Yehuda A, Freisleben SKU, Wanandi SI, Huber H, et al. Characterization of thermoplasma species cultured from sampling on Tangkuban Perahu, Indonesia. Microbiol Indones. 2014;8(1):16–23.

3. Freisleben HJ. Tetraether lipid liposomes. In: Zimmer G, editor. Membrane structure in disease and drug therapy. New York: M. Dekker; 2000. p. 127–52.

4. Freisleben HJ. Archaeosomes and tetraether lipid liposomes. Pharm Sci Res. 2012;9(1):53–65.

5. Freisleben HJ, Henkel L, Gutermann R, Rudolph P, John G, Sternberg B, et al. Fermentor cultivation of Thermoplasma acidophilum for the production of cell mass and of the main phospholipid fraction. Appl Microbiol Biotechnol. 1994;40:745–52.

6. Antonopoulos E, Freisleben HJ, Krisnamurti DGB, Estuningtyas A, Mulyanto C, Ridwan R, et al. Fractionation and purification of membrane lipids from the archaeon Thermoplasma acidophilum DSM 1728/10217. Separ Purific Technol. 2013;110:119–26.

7. Luthfa Z, Freisleben HJ, Saleh R. Temperature and pH-dependent molecular dynamics of Thermoplasma acidophilum tetraether lipid membrane in a computer-simulated model. Int J Mat Engin Technol. 2014;13(2):161–85.

8. Kondo Y, Murayama Y, Konishi H, Morimura R, Komatsu S, Shiozaki A, et al. Fluorescent detection of peritoneal metastasis in human colorectal cancer using 5-aminolevulinic acid. Int J Oncol. 2014;45(1):41–6.

9. Al-Naieb Z. New advances in aminolevulinic acid in urology. Med Surg Urol. 2014;3:e110.

10. Zaak D, Hungerhuber E, Schneede P, Stepp H, Frimberger D, Corvin S, et al. Role of 5-aminolevulinic acid in the detection of urothelial premalignant lesions. Cancer. 2002;95(6):1234–8.

11. Fukuhara H, Inoue K, Kurabayashi A, Furihata M, Shuin T. Performance of 5-aminolevulinic-acid-based photodynamic diagnosis for radical prostatectomy. BMC Urol. 2015;15:78–83.

12. Berg K. Photosensitizers in medicine [Internet]. Norway: The Norwegian Radium Hospital; 2009 [cited 2015 Nov 2]. Available from: http://photobiology.info/Berg.html

13. New RRC. Liposomes - a practical Approach. United Kingdom: IRL Press; 1990. p. 33–105.

14. Choquet CG, Patel GB, Sprott GD. Heat sterilization of archaeal liposomes. Can J Microbiol. 1996;42(2):183–6.

15. Oertl A, Freisleben HJ. Intermembrane exchange from liposomes of archaebacterial tetraether lipid into ghosts. Biol Chem Hoppe-Seyler. 1993;374:145.

16. Antonopoulos E, Oertl A, Henkel L, Freisleben HJ. Liposomes of the main phospholipid from Thermoplasma acidophilum. Fluorescence determinations of interactions with cell membranes. Freiburg, Germany:, Fourth Liposome Research Days Conference University of Freiburg, Aug. 30 – Sept. 2, Book of Abstracts; 1995. P. 69.

17. Corbo C, Molinaro R, Taraballi F, Toledano Furman NE, Sherman MB, Parodi A, et al. Effects of the protein corona on liposome–liposome and liposome–cell interactions. Int J Nanomedicine. 2016;11:3049–63.

18. Vidawati S, Sitterberg J, Bakowsky U, Rothe U. AFM and ellipsometric studies on LB films of natural asymmetric and symmetric bolaamphiphilic archaebacterial tetraether lipids on silicon wafers. Colloids Surf B Biointerfaces. 2010;78(2):303–9.

19. Shimada H, Nemoto N, Shida Y, Oshima T, Yamagishi A. Effect of pH and temperature on the composition of polar lipids in Thermoplasma acidophilum HO-62. J Bacteriol. 2008;190(15):5404–11.

20. Freisleben HJ, Neisser C, Hartmann M, Rudolph P, Geck P, Ring K, et al. Influence of the main phospholipid (MPL) from Thermoplasma acidophilum and of liposomes from MPL on living cells: cytotoxicity and mutagenicity. J Liposome Res. 1993;3(3):817–33.

21. Freisleben HJ, Bormann J, Litzinger DC, Lehr F, Rudolph P, Schatton W, et al. Toxicity and biodistribution of liposomes of the main phospholipid from the archaebacterium Thermoplasma acidophilum in mice. J Liposome Res. 1995;5(1):215–23.



22. Purwaningsih EH, Arozal W, Jusman SWA. Uji stabilitas fisik, kimia dan biologik terhadap formulasi terbaru liposom tetra eter lipid (EPC-TEL 2,5) sebagai pembawa obat (drug carrier). Makara Kesehatan. 2007;11(2):84–9. Indonesian.

23. Krishnan L, Sprott GD. Archaeosome adjuvants: immunological capabilities and mechanism(s) of action. Vaccine. 2008;26(17):2043–55.

24. Li Z, Zhang L, Sun W, Ding Q, Hou Y, Xu Y. Archaeosomes with encapsulated antigens for oral vaccine delivery. Vaccine. 2011;29(32):5260–6.

25. González-Paredes A, Clarés-Naveros B, Ruiz-Martínez MA, Durbán-Fornieles JJ, Ramos-Cormenzana A, Monteoliva-Sánchez M. Delivery systems for natural antioxidant compounds: Archaeosomes and archaeosomal hydrogels characterization and release study. Intern J Pharm. 2011;421(2):321–31.

26. Brown DA, Venegas B, Cooke PH, English V, Chong PLG. Bipolar tetraether archaeosomes exhibit unusual stability against autoclaving as studied by dynamic light scattering and electron microscopy. Chem Phys Lipids 2009;159:95–103.

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Paramita, et al.Vimentin in endoxifen-induced MCF-7

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Increased vimentin mRNA expression in MCF-7 breast cancer cell line after repeated endoxifen-treatment

Keywords: endoxifen, EMT, vimentin

pISSN: 0853-1773 • eISSN: 2252-8083 • http://dx.doi.org/10.13181/mji.v25i4.1397 • Med J Indones. 2016;25:207–13• Received 1 Mar 2016 • Accepted 19 Dec 2016

Corresponding author: Melva Louisa, [email protected]


Paramita,1 Melva Louisa,2 Nafrialdi21 Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia2 Departement of Pharmacology and Therapeutics, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Basic Medical Research


ABSTRAK

Latar belakang: Epithelial mesenchymal transition (EMT) adalah suatu mekanisme yang terlibat dalam terjadinya resistensi sel kanker. Vimentin, protein filamen intermedia tipe III, merupakan salah satu penanda EMT. Ekspresinya yang tinggi berhubungan dengan meningkatnya pertumbuhan tumor, perubahan bentuk sel dan semakin memburuknya prognosis kanker. Endoksifen sebagai metabolit tamoksifen telah menjadi agen baru yang poten pada terapi kanker payudara. Studi ini merupakan studi yang bertujuan melihat pengaruh endoksifen dalam kombinasi dengan β-estradiol terhadap ekspresi mRNA vimentin setelah pemaparan jangka pendek (4 minggu).

Metode: Endoksifen 100 nM, 1.000 nM, dengan atau tanpa beta estradiol dipaparkan secara berulang pada sel MCF-7. Sel yang dipaparkan dengan dimethyl sulfoxide (DMSO) 0,001% digunakan sebagai kontrol. Setelah 4 minggu paparan, dilakukan hitung sel, analisis ekspresi mRNA vimentin dan pengamatan morfologi sel.

Hasil: Sel yang diberikan endoksifen saja maupun dengan kombinasi β-estradiol terjadi penurunan ekspresi mRNA vimentin pada minggu kedua dibandingkan dengan kontrol, yang kemudian pada minggu keempat terjadi peningkatan ekspresi vimentin. Pada pengamatan pada morfologi sel MCF-7, tidak terlihat adanya perubahan morfologi.

Kesimpulan: Paparan berulang endoksifen dapat menginduksi aktivasi jalur EMT melalui peningkatan regulasi ekspresi mRNA vimentin.

ABSTRACT

Background: Epithelial mesenchymal transition (EMT) plays a significant role in the development of cancer cell resistance to drugs. Vimentin, a type III intermediate filament protein, is a marker of EMT. Vimentin's over-expression in cancer correlates well with increased tumor growth, change in cell shape and poor prognosis. Endoxifen is an active metabolite of tamoxifen and has become a new potent agent in the treatment of breast cancer. This is a study that aimed to investigate the effect of endoxifen exposure with or without estradiol on cell viability, cell morphology and EMT progression through the analysis of vimentin mRNA expression after 4-week treatment.

Methods: Endoxifen, 100 nM or 1,000 nM, with or without beta-estradiol were given repeatedly to MCF-7 cells. Cells treated with dimethyl sulfoxide (DMSO) 0.001% were used as control. After 2- and 4-week exposure, the cells were counted, analyzed for mRNA vimentin expression, and observed for morphological changes.

Results: Compared to control, there were significant decreases in vimentin mRNA expressions in endoxifen and endoxifen+β-estradiol treated cells after 2-weeks, which then significantly increased after 4-week compared with the 2-week exposure. We found no change in morphology of MCF-7 cells.

Conclusion: Repeated exposure of endoxifen might induce EMT progression through increased expression of vimentin in MCF-7 breast cancer cell line.



Tamoxifen, a prodrug, is commonly given to women diagnosed with estrogen receptor (ER)-positive breast cancer. In human body, tamoxifen is metabolized into 4-hydroxy tamoxifen (4HT) and N-desmethyl-tamoxifen (NDT) by the cytochrome P-450 enzyme system, followed by secondary metabolism to 4-hydroxy-N-desmethyl-tamoxifen (endoxifen).1,2

4HT is assumed as the primary means by which tamoxifen exerts its anti-breast cancer effect. However, current studies suggest that endoxifen is equipotent to 4HT and significantly more potent than tamoxifen in its capability to bind to estrogen receptor a and b (ERa and ERb), and also in suppressing ER-dependent breast cancer proliferation.1

Currently, endoxifen is being developed as a novel drug for the treatment of endocrine responsive breast cancer patients.3 Hawse et al2 showed that endoxifen had similar actions to selective estrogen receptor antagonist (fulvestrant) with regard to its ability to degrade ERα through proteasome degradation system, to block ERα-mediated transcriptional activation and to prevent estrogen-induced breast cancer cell proliferation.2 A study was conducted by Wu et al1 and showed that cells treated with high endoxifen (E) concentrations, i.e. 100–1,000 nmol/L resulted in significant decreases in ERα protein levels. Otherwise, low endoxifen concentrations (20-40 nmol/L) did not induce degradation of ERα protein.

Cancer cell resistance to drugs is one of major issues that developed in the use of therapeutic drugs for breast cancer treatment. There are some categories of mechanisms that promote direct or indirect drug resistance in human cancer cells such as inactivation of drug, alteration of drug target, drug efflux, deoxyribonucleic acid (DNA) damage repair, inhibition of cell death and epithelial-mesenchymal transition (EMT).4 A recent study showed that exposure of endoxifen 1,000 nmol/L for 15 months induced epithelial-mesenchymal transition (EMT) in Michigan Cancer Foundation (MCF-7) cells.3

Epithelial-mesenchymal transition is a cellular reprogramming process in which cells change their epithelial towards mesenchymal phenotype that lead the cells to alter their shape and show increased motility. During EMT, there are

reorganization of cytoskeletal elements including replacement of peripheral actin cytoskeleton and cytokeratin intermediate filaments with stress fibers and vimentin, respectively. Because of this dramatic changes in intermediate filament (IF) composition, vimentin expression has become a canonical marker of the EMT.5–11

Vimentin, a type III IF protein, is found in normal mesenchymal cells and is known to maintain integrity of cells and provide resistance against stress.5 Mendez et al6 showed that during epithelial to mesenchymal transition process, vimentin can cause alteration in cell shape, motility, and adhesion.

The positive correlation between increased migration and invasion of cancer cells and elevated vimentin expression has been found.12,13 In the MCF-7 cells, vimentin gene transfection caused increased invasiveness.13 A previous study demonstrated that invasive breast cancer cell lines expressed more vimentin, suggesting its important role in identifying cases with worse prognosis.12

Our study was aimed to investigate the effect of repeated exposure of endoxifen with or without β-estradiol to MCF-7 breast cancer cell line on cell viability, morphology and EMT progression through the analysis of vimentin mRNA expression.

METHODS

Study design This was an in vitro experimental study in a commercially available breast carcinoma cell line, MCF-7; therefore ethical clearance is exempted. This study was conducted in Pharmacokinetics Laboratory, Department of Pharmacology and Therapeutics, Faculty of Medicine, Universitas Indonesia, from March to September 2015.

Cell culture MCF-7 human breast carcinoma cell line was kindly provided by Makmal Terpadu Laboratory, Faculty of Medicine, Universitas Indonesia. MCF-7 cell morphology was characterized by cobblestone-like appearance and strong cell–cell adhesion.14 The cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in Dulbecco’s

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Modified Eagle Medium (DMEM)-high glucose supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/mL streptomycin, and 2.5 µg/ml fungizone. Cells were counted, then 10,000 cells were seeded into triplicate wells of 6-well plates and growth was assessed at about 90% confluency, following trypsinisation and resuspension in medium, by direct cell counting using a hemocytometer.

Doubling time and growth curve analysis Cells were harvested at day 1, 2, 3, 4, 5, 6, 7 and 9. Afterwards the cells were counted and plotted in a growth curve. Cell doubling time were calculated using the doubling time calculation provided in http://www.doubling-time.com.

Drug treatmentMCF-7 cells were plated at 10,000 cells and were treated with endoxifen (100 nmol/L and 1,000 nmol/L) with or without b-estradiol 1 nmol/L in 6-well plates. Endoxifen and estradiol were dissolved in dimethyl sulfoxide (DMSO) 0.001%. Therefore, the control used was DMSO 0.001%. Every seven days, the cells were trypsinized, counted and 10,000 cells were re-plated in complete medium. Drug treatments were continuously given for four weeks, three times per week along with medium changes. Cell viability assays were done every week. At the end of second and fourth week of drug treatment, cells were harvested and isolated for ribonucleic acid (RNA) and further synthesized to complementary DNA (cDNA) for quantitative realtime polymerase chain reaction (PCR) analysis.

Cell viability assayCell viability assay was performed by using trypan blue dye exclusion assay. The numbers of viable cells were counted by trypan blue dye exclusion using a hemocytometer. The results were expressed as percentage of viable cells in given drug treatments relative to the viable cells in DMSO group.

Quantitative realtime PCR analysisTotal cellular RNA was extracted from 1,000,000 cells after trypsinized from six well plates using High Pure RNA isolation kit (Roche, USA) according to the manufacturer’s protocol. Further, 1 µg (in 20 µl) was converted to cDNA using a Transcriptor First Strand cDNA Synthesis kit (Roche, USA). Quantitative realtime PCR

was performed on 100 ng cDNA using a FastStart DNA master SYBR Green I kit (Roche, USA), according to manufacturer’s protocol. As reference, β-actin, a housekeeping gene, was used. Primer sequences used for vimentin: forward primer was 5’-GACAATGCGTCTCTGGCACGTCTT-3’ and reverse primer:was 5’-TCCTCCGCCTCCTGCAGGTTCTT-3’; for β-actin: forward primer was 5’-GCTGGAAGGTGGACAGCGA-3’ and reverse primer was 5’-GGCATCGTGATGGACTCCG-3’.

A total of 20 µl reactions were performed in a 32-well plate on an qRT-PCR Light Cycler Nano RocheTM by incubation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 20 seconds and 65°C for one minute. The raw quantification cycle (Cq) values were analysed by relative quantification using Livak method to determine normalized expression ratios of vimentin to b-actin.

Statistical analysisData collected were cell viability, morphology and vimentin expression. Cell morphologies were presented as figures, and MCF-7 cell viabilities and vimentin mRNA expressions were presented as mean ± standard deviation (SD) of three experiments. Statistical analyses were performed with one-way ANOVA. p<0.05 was taken to be statistically significant difference. If one-way ANOVA showed significant difference, then we proceeded with Tukey’s multiple comparison method.

RESULTS

Optimization of cell cultureThe growth curve showed that MCF-7 cells had an exponential growth pattern with a doubling time of 24.51 hours (Figure 1).

The effects of endoxifen on MCF-7 cell viabilityViabilities were expressed as percentage relative to the control (cells exposed to DMSO 0.001%). Results are presented as means of three experiments (Figure 2).

Observation of MCF-7 cell morphology after four weeks of treatments

Observation of cell morphology through inverted microscope with 40x magnification did not show any morphological changes in MCF-7 cells after four weeks.



2nd Week

4th Week

Figure 3. Morphology of MCF-7 cells after seeding at a cell density of 104 viable cells/mL into 6-well plates and grown for four weeks. There was no difference in morphology af-ter four weeks of drug treatment at 40x magnification using inverted microscope. A2, A4= endoxifen 1,000 nM, B2, B4= endoxifen 100 nM, C2, C4= endoxifen 1,000 nM+β-estradiol 1 nM, D2, D4= endoxifen 100 nM+ β-estradiol 1 nM, E2, E4= β-estradiol 1 nM, F2, F4= control (DMSO)

Figure 1. MCF-7 cell growth curve

Figure 2. Effect of endoxifen with or without β-estradiol on MCF-7 cells viability. E1000= endoxifen 1,000 nM, E100= en-doxifen 100 nM. E1000+BE= endoxifen 1,000 nM+ β-estradiol 1 nM, E100+BE= endoxifen 100 nM+ β-estradiol 1 nM, BE= β-estradiol 1 nM.

The effect of endoxifen on vimentin mRNA expressionThe quantitative data of vimentin was normalized to β-actin as reference gene was shown in Figure 4. The results are presented as mean±SD of six experiments in three trials.

Vimentin mRNA expression analysis showed that both endoxifen 1,000 nM and endoxifen100 nM were able to decrease vimentin mRNA expression to 0.325 and 0.529 fold compared to control group after 2-week of treatment, whereas vimentin mRNA expression level on endoxifen 1,000 nM + β-estradiol and endoxifen 100 nM + β-estradiol were higher than endoxifen treatment without estradiol.

Analysis of vimentin mRNA expression after 4-week of treatment showed that endoxifen 1000 nM and endoxifen100 nM decreased vimentin expression to 0.650 and 0.871 respectively, relative to control, whereas endoxifen 1000 nM + β-estradiol and endoxifen100 nM + β-estradiol showed vimentin mRNA expression levels of 0.990 and 1.251 respectively.

DISCUSSION

This study was aimed to analyze the effect of endoxifen with or without β-estradiol on the mechanisms of EMT in terms of vimentin mRNA expression. The addition of β-estradiol in this study was intended to resemble estradiol levels in breast cancer patient.15

We used very low concentrations of DMSO to dissolve endoxifen and b-estradiol, this is the reason why we chose DMSO as control. DMSO might have toxic and modulatory effects on cells,

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however, a previous study on MCF-7, RAW-264.7 and HUVEC cells suggested that DMSO up to 0.5% had little or no toxicity.16

One of common methods of establishing resistant cell lines is to use low-dosages intermittent incremental inducement with various and inconsistent dosages.17 However, this method requires a relatively long time. Louie et al18 required six months to make tamoxifen resistant MCF-7 breast cancer cell lines, while Hawse et al3 required 15 months to make endoxifen resistant MCF-7 breast cancer cell lines. In this study, we would like to observe whether short-term exposure of endoxifen, which in this study was four weeks could result in a similar result as the longer one. In this study we did not use increment dosage of endoxifen. A study on drug resistance showed that antibiotic treatment in subtherapeutic levels in the long term would lead to resistance.19

We used high doses of endoxifen (1,000 nM) in our study, based on the result given by Wu et al,1 which showed that low concentrations of endoxifen (20-100 nM) did not affect cell viability in the presence of estradiol in MCF-7 cells.

Analysis of cell viability after 4-weeks of treatment demonstrated that endoxifen 1000 nM was more effective than endoxifen 100 nM in decreasing cell viability. As shown by Wu et al,1 endoxifen 1000 nM could degrade ERα more than

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endoxifen 100nM. Cells that received estradiol showed an increase in viability compared to the endoxifen only. In our study, increase in cell viability occurred after 2-weeks of endoxifen 100 nM + β-estradiol exposures as compared to DMSO. This result indicated that endoxifen 100 nM was unable to suppress cell viability inducing effect of β-estradiol.

Vimentin mRNA expression analysis showed that endoxifen only (endoxifen100 nM and endoxifen 1000 nM) decreased the expression stronger than endoxifen + β-estradiol. Previous results had shown that addition of β-estradiol could increase vimentin expression. Experiments conducted by Dong et al20 demonstrated that estrogen could induce metastatic potential of thyroid cancer cells by increasing vimentin mRNA expression. Jimenez-Salazar et al21 showed that 1 nM β-estradiol elicit c-Src activation after 15 minutes. The p-Src/ZO-1 complex caused disruption of ZO-1 and ZONAB at the tight junction. In addition, these changes were correlated with the reduced expression of epithelial markers.21 Figure 4 shows that endoxifen 100 nM given concurrently with β-estradiol increased mRNA expressions of vimentin compared to endoxifen 100 nM only. However, the mechanism of this increase has not been clearly elucidated.

Analysis of the mRNA expression of vimentin after 4-weeks of treatment showed that endoxifen


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with or without β-estradiol relatively increased vimentin expression approximately 2-fold compared with 2-weeks of treatment. Results showed that longer time of exposure had a role in enhancing the expression of vimentin mRNA. Another study reported that administration of endoxifen 1,000 nM for 15 months could induce EMT, which was characterized by the increasing expression of vimentin.

EMT is a process where epithelial cells change into mesenchymal cells that are characterized by increased vimentin expression. Vimentin transfection to epithelial cell, which was conducted by Mendez et al6 showed a change in the shape of cuboidal epithelial cells into mesenchymal cells. These results prove that vimentin has a role in the change in cell shape during EMT. Observations of cell morphology through inverted microscope with a magnification of 40 times showed that there were no morphological changes in MCF-7 cells for four weeks.

Based on the results of this study after four weeks, repeated endoxifen exposure was supposed to induce EMT, which was characterized by a relative increase in vimentin mRNA expression and the addition of β-estradiol could augment mRNA vimentin expression due endoxifen exposure. However, in this study, morphological changes in MCF-7 cells did not occur. This might be due to a relatively short time of drug exposure, as compared to the study by Hawse et al3 (15 months). The vimentin expression in our sudy was still below the control, except for the combination of endoxifen 100 nM + β-estradiol, while the study by Zhao et al22 showed that increase in vimentin expression was about five times in cells treated with estradiol that resulted in morphological changes from cobblestone appearance to spindle-shaped cells.

In conclusion, four week endoxifen 1,000 nM exposure was the most effective in suppressing breast cancer cell proliferation. Repeated endoxifen exposure up to four weeks might induce EMT as characterized by the relative increase in vimentin mRNA expressions, while the addition of β-estradiol could augment the expression of vimentin.

Therefore, we suggest that in long-term treatment of endoxifen in breast cancer, regular asessment

of resistance marker such as vimentin is needed, so that the treatment can be optimized.

AcknowledgmentThis research was supported by Postgraduate Research Grant of DRPM UI 2015.


REFERENCES

1. Wu X, Hawse JR, Subramaniam M, Goetz MP, Ingle JN, Spelsberg TC. The tamoxifen metabolite, endoxifen, is a potent antiestrogen that targets estrogen receptor alpha for degradation in breast cancer cells. Cancer Res. 2009;69(5):1722–7.

2. Hawse JR, Subramaniam M, Cicek M, Wu X, Gingery A, Grygo SB, et al. Endoxifen’s molecular mechanism of action are concentration dependent and different than that of other anti-estrogens. PLoS One. 2013;8(1):e54613.

3. Hawse JR, Subramaniam M, Wu X, Negron V, Muzaffer C, Lingle WL, et al. Abstract PD05-11: development, characterization, and effective in vitro treatment of an endoxifen resistant breast cancer cell line. Cancer Res. 2010;70(24):1.

4. Housman G, Byler S, Heerboth S, Lapinska K, Longacre M, Snyder N, et al. Drug resistance in cancer: an overview. Cancers. 2014;6(3):1769–92.

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6. Mendez MG, Kojima S, Goldman RD. Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition. FASEB J. 2010;24(6):1838–51.

7. Micalizzi DS, Farabaugh SM, Ford HL. Epithelial-mesenchymal transition in cancer: parallels between normal development and tumor progression. J Mammary Gland Biol Neoplasia. 2010;15(2):117–34.

8. Savagner P. The epithelial-mesenchymal transition (EMT) phenomenon. Ann Oncol. 2010;21(7):vii89–92.

9. Kalluri R, Weinberg RA. The basic of epithelial-mesenchymal transition. J Clin Invest. 2009;119(6):1420–8.

10. Wang Y, Zhou BP. Epithelial-mesenchymal transition--a hallmark of breast cancer metastasis. Cancer Hallm. 2013;1(1):38–49.

11. Voulgari A, Pintzas A. Epithelial-mesenchymal transition in cancer metastasis: mechanisms, markers, and strategies to overcome drug resistance in the clinic. Biochim Biophys Acta. 2009;1796(2):75–90.

12. Kusinska RU, Kordek R, Pluciennik E, Bednarek AK, Piekarski JH, Potemski P. Does vimentin help to delineate the so-called ‘basal type breast cancer’? J Exp Clin Cancer Res. 2009;28(1):118.

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13. Kidd ME, Shumaker DK, Ridge KM. The role of vimentin intermediate filaments in the progression of lung cancer. Am J Respir Cell Mol Biol. 2014;50(1):1–6.

14. Yang J, Bielenberg DR, Rodig SJ, Doiron R, Clifton MC, Kung AL, et al. Lipocalin 2 promotes breast cancer progression. Proc Natl Acad Sci USA. 2009;106(10):3913–8.

15. Folkerd EJ, Lønning PE, Dowsett M. Interpreting plasma estrogen levels in breast cancer: caution needed. J Clin Oncol. 2014;32(14):1396–400.

16. Jamalzadeh L, Ghafoori H, Sariri R, Rabuti H, Nasirzade J, Hasani H, et al. Cytotoxic effects of some common organic solvents on MCF-7, RAW-264.7 and human umbilical vein endothelial cells. Avicenna J Med Biochem. 2016;4(1):e33453.

17. Yan XD, Li M, Yuan Y, Mao N, Pan LY. Biological comparison of ovarian cancer resistant cell lines to cisplatin and Taxol by two different administrations. Oncol Rep. 2007;17(5):1163–9.

18. Louie MC, McClellan A, Siewit C, Kawabata L. Estrogen receptor regulates E2F1 expression to

mediate tamoxifen resistance. Mol Cancer Res. 2010;8(3):343–52.

19. Carlet J, Jarlier V, Harbarth S, Voss A, Goosens H, Pittet D, et al. Ready for a world without antibiotics? The pensières antibiotic resistance call to action. Antimicrob Resist Infect Control. 2012;1(1):11.

20. Dong W, Zhang H, Li J, Guan H, He L, Wang Z, et al. Estrogen induces metastatic potential of papillary thyroid cancer cells through estrogen receptor α and β. Int J Endocrinol. 2013;2013(941568):1–6.

21. Jiménez-Salazar JE, Posadas-Rodríguez P, Lazzarini-Lechuga RC, Luna-López A, Zentella-Dehesa A, Gómez-Quiroz LE, et al. Membrane-initiated estradiol signaling of epithelial-mesenchymal transition-associated mechanisms through regulation of tight junctions in human breast cancer cells. Horm Cancer. 2014;5(3):161–73.

22. Zhao G, Nie Y, Lv M, He L, Wang T, Hou Y. ERβ-mediated estradiol enhances epithelial mesenchymal transition of lung adenocarcinoma through increasing transcription of midkine. Mol Endocrinol. 2012;26(8):1304–15.



Prevalence of Helicobacter pylori among patients with different gastrointestinal disorders in Saudi Arabia

Keywords: duodenal ulcer, gastric ulcer, H. pylori, PCR, ureA

pISSN: 0853-1773 • eISSN: 2252-8083 • http://dx.doi.org/10.13181/mji.v25i4.1442 • Med J Indones. 2016;25:214–20• Received 09 May 2016 • Accepted 21 Jul 2016

Corresponding author: Mohammed S. Alhussaini, [email protected]


Mohammed S. AlhussainiDepartment of Clinical Laboratory Sciences, College of Applied Medical Sciences, Shaqra University, Saudi Arabia

Clinical Research

ABSTRAK

Latar belakang: Helicobacter pylori adalah patogen gastrointestinal yang penting yang berkaitan dengan gastritis, ulkus peptikum, dan peningkatan risiko kanker lambung. Studi ini bertujuan mencari hubungan patogen ini dengan berbagai kelainan gastrointestinal.

Metode: 150 pasien rawat jalan yang dirujuk ke Saudi Arabian Medical City, Riyadh, Arab Saudi diikutsertakan pada bulan Januari sampai Juni 2015. Setiap pasien menjalani endoskopi dan biopsi diambil spesimennya untuk pemeriksaan urease dan kultur. Koloni yang dicurigai sebagai H. pylori dilakukan identifikasi morfologi, pemeriksaan mikroskopik dan biokimia. Dilakukan juga pemeriksaan PCR untuk mendeteksi gen urease subunit ureA.

Hasil: Hasil endoskopi bervariasi dari normal, ulkus gaster, ulkus duodenum, gastritis, dan kanker lambung dengan prevalensi masing-masing 20.7%, 20%, 24%, 33,3%, dan 2%. Pemeriksaan pewarnaan langsung menunjukkan 52% pasien positif H. pylori, sedangkan kultur dan uji cepat menghasilkan prevalensi 71,33%. Lima puluh empat biopsi (36%) menunjukkan urease positif setelah satu jam pada suhu ruang, 39 spesimen (62%) setelah satu jam inkubasi pada 37°C, dan 14 spesimen (71,33%) setelah inkubasi 24 jam. Isolat H. pylori memperlihatkan hasil positif katalase, oksidase, dan urease. Hasil PCR menunjukkan fragment 411-bp, yang sesuai dengan gen urease subunit ureA.

Kesimpulan: Prevalensi H. pylori cukup tinggi pada populasi yang diteliti. Terdapat hubungan kuat antara H. pyori dengan ulkus duodenum. Fragmen 411-bp merupakan indikator adanya gen urease subunit ureA.

ABSTRACT

Background: Helicobacter pylori is an important gastrointestinal pathogen associated with gastritis, peptic ulcers, and an increased risk of gastric carcinoma. The present study was carried out to determine the relationship between this organism with different gastrointestinal ailments.

Methods: 150 outpatients referrals to Saudi Arabian Medical City, Riyadh, Kingdom of Saudi Arabia was recruited in January to June 2015. Each patient was subjected to endoscopic examination. Biopsy specimens were taken from the stomach for rapid urease test and culture. Suspected H. pylori colonies were subjected to colony morphology identification, microscopical examination and biochemical reactions. The samples were also subjected to PCR to detect ureA subunit of urease gene.

Results: The endoscopic examination of patients revealed normal, gastric ulcer, duodenal ulcer, gastritis, and gastric cancer with a rate of 20.7%, 20%, 24%, 33.3%, and 2%, respectively. Direct smear exam revealed that 52% of patients were H. pylori positive while culture and rapid urease test showed a prevalence of 71.33%. Fifty four biopsies (36%) were urease positive after 1 hour at room temperature, 39 (62%) after 1 hour incubation at 37°C and 14 (71.33%) after 24 hours incubation. Isolated H. pylori showed that they were catalase, oxidase, and urease positive. PCR results showed 411-bp fragment, which is indicative for the ureA subunit of urease gene.

Conclusion: The prevalence of H. pylori infection was high among tested population. Strong association between H. pylori and duodenal ulcer was noticed. A 411-bp fragment indicative of the ureA subunit of urease gene was detected in all the tested isolates.



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Helicobacter pylori (H. pylori) is one of the most common infections in the world. It presents in 70–90% of the population in developing countries and 35–40% in developed ones.1 H. pylori infection is associated with duodenal ulcer,2 gastric ulcer and gastric cancer.3 Approximately, 50% of the normal population across the world harbor H. pylori, though only 10–20% of them become symptomatic.4 There is a high prevalence of H. pylori infection in developing countries especially among children. In India, the prevalence of this infection is 22%, 56%, and 87% in the 0–4 years, 5–9 years and in the 10–19 years age group, respectively.5

A wide range of laboratory investigations are available for diagnosis of H. pylori. The tests are both non-invasive and invasive. Non-invasive tests include, urea breath test, serological immunoglobulin G (IgG), and immunoglobulin M (IgM) detection, saliva and urinary antibody test, and stool antigen test.6 Culture is also a successful method for diagnosis but it is time-consuming.7 Rapid urease test is a reliable method for presumptive identification of H. pylori8 and enzyme linked immunosorbant assay (ELISA) is the most common tool because of its speed, low costs, simplicity, and reproducibility.9

Polymerase chain reaction (PCR) techniques have also been used for diagnosis of H. pylori. It has widespread use in research as it holds great promise in the detection of genetic differences between H. pylori strains for epidemiological studies. Vinette et al10 showed that PCR is the method with potential for greatest sensitivity and specificity in the detection of H. pylori specific deoxyribonucleic acid DNA. Huang et al11 reported a PCR method based on H. pylori isocitrate dehydrogenase gene sequence that can rapidly and specifically detect the organism. Furthermore, Brooks et al7 developed a PCR assay, which is reportedly 100% sensitive and specific for detection of H. pylori infection in gastric mucosal biopsy specimens. The study aimed to determine the prevalence of H. pylori among 150 outpatients with different gastrointestinal disorders, validate different microbiological diagnostic techniques and detect a subunit of the urease gene (ureA) using PCR to confirm the identification of H. pylori.

METHODS

PatientsA total of 150 outpatients referrals to Saudi Arabian Medical City, Riyadh, Kingdom of Saudi Arabia with dyspeptic symptoms and clinically indicated for upper gastrointestinal endoscopy were selected. Informed consent mentioning that data from the diagnosis would be used for research purpose was obtained from all the patients. The research protocol was approved by institutional research committee College of Applied Medical Sciences, Shaqra University (53/27844. There were 105 male and 45 female participants with age between 15–75 years. The samples were collected from January to June, 2015. Personal history; name, age, sex, residence, and job were taken from each patient. The main complaints were dyspepsia, nausea, vomiting, abdominal pain and hematemesis. Patients taking antimicrobial drugs and/or bismuth salts within two weeks before endoscopy were excluded from the study.7

EndoscopyPatients were instructed by the gastroenterologist not to eat or drink for at least eight hours before endoscopy procedure. They received oropharyngeal local anesthetic (Xylocain spray 10%). The endoscope and biopsy forceps were cleansed with Savlon and then sterilized by soaking in Cidexin for 10 minutes followed by washing with sterile water. All endoscopic examinations were carried out by using video-endoscopes PENTAX EPM-3500. Three biopsy specimens were taken from antrum region along the greater curvature of stomach, one was biopsy specimen was used for urease test.12

CultureThe second biopsy specimen was directly smeared and examined for Gram negative curved bacilli. The third biopsy specimen was homogenized and streaked on fresh brain heart infusion agar supplemented with 10% sterile horse serum and on Colombia blood agar with selective supplement (Dent, Oxoid) and modified Columbia agar (MCUA) slant. The plates were incubated under microaerophilic condition using Gas Generating Kits Campylobacter system BR 0546A (Oxoid, Hampshire, England) at 37°C for 3–5 days. H. pylori suspected colonies


were identified by Gram stain and biochemical tests. Biopsies were transported in ice from the endoscopic unit to the microbiological laboratory in 1 ml horse serum supplemented brain heart infusion broth13 and processed within two hours.7

Primary diagnosis of H. pyloriThe suspected purified colonies were chosen according to the Gram staining and culture's characteristics.

Biochemical testsBiochemical tests include production of catalase, oxidase, urease, and H2S, nitrate reduction, growing in 3.5% NaCl, growing with 1% glycine, and growing at different temperatures.

Bacterial DNA extraction and purificationTwenty representative samples from pathological cases were subcultured on selective Colombia blood agar and subjected to DNA extraction and purification. The method is described in the protocol of QIAamp DNA Mini Kit® (QIAGENE Inc., Santo Clarita, Calif.), which contains Proteinase K and animal tissue lysis (ATL), cell lysis (AL), wash buffer 1 (AW1), wash buffer 2 (AW2) buffers. Three bacterial colonies were removed from each culture plate with an inoculating loop and suspended in ATL buffer by vigorous stirring. Twenty μl of Proteinase K was added for each sample and incubated at 56°C in incubator shaker until the sample was completely lysed. The tubes were centrifuged briefly to remove drops from the inside of the lid. Two hundred μl of AL buffer was added, mixed by pulse-vortexing for 15 seconds and incubated at 70°C for 10 minutes. The mixture of each sample was applied to the QIAamp spin column. Tubes were centrifuged at 8,000 rpm for one minute. QIAamp spin column was placed in a clean 1.5 ml microcentrifuge tube, carefully opened and 200 μl of AE buffer was added. The mixture was incubated at room temperature for one minute and centrifuged at 8,000 rpm for one minute.

Amplification of DNA by PCRThe oligonucleotide primers used for the amplification were:HPU1, (5'-GCC AAT GGT AAA TTA GTT–3') and HPU2, (5'- CTC CTT AAT TGT TTT TAG –3').

This target DNA sequence was used in developing the PCR assay according to Lu et al14 and He et al15 followed by agarose gel electrophoresis for PCR products. Gel was photographed using Polaroid Pand Camera with an orange lens filter on T 667 Polaroid film.

Statistical analysisThe obtained results were statistically analyzed using MINITAB statistical software, copyright 1992, release 8, MINITAB INC. Chi-square test was applied to calculate the significance difference between various gastrointestinal disorders groups (e.g. gastric ulcer, duodenal ulcer, gastritis, etc).

RESULTS

A total of 150 outpatients with dyspeptic symptoms and clinically indicated for upper gastrointestinal endoscopy were selected. Among these examined patients, the endoscopic findings revealed that 20.7% (31/150) were normal, 20% (30/150) were suffering from gastric ulcer, 24% (36/150) with duodenal ulcer, 33.3% (50/150) with gastritis and only 2% (3/150) with gastric cancer. Table 2 and Figure 1 showed the distribution of these groups according to endoscopic examination. This result revealed that most prevalent finding was patients with gastritis and the lowest those suffering from gastric cancer as shown in Table 1 and Figure 1.

The culture and biochemical tests showed that 107 (71.33%) out of 150 gastric biopsy specimens were positive for H. pylori. The presence of H. pylori in gastric biopsy specimens, correlated

Variables DescriptionSex (male/female) 105/45Age years (range) (15–75)Endoscopic finding n (%)

Normal 31/150 (20.7%)Gastric ulcer 30/150 (20.0%)Duodenal ulcer 36/150 (24.0%)Gastritis 50/150 (33.3%)Gastric cancer 3/150 (2.0%)

Chief complaints Dyspepsia, nausea, vomiting, abdominal pain and hematemesis

Table 1. Demographic and clinical characteristics of pa-tients included in the study (n=150)

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Gastric biopsy specimens

H. pylori positive n (%)

H. pylori negative n (%)

Normal 13/31 (42 %) 18/31 (58%)Gastric ulcer 19/30 (63%) 11/30 (37%)Duodenal ulcer 33/36 (92%) 3/36 (8%)Gastritis 39/50 (78%) 11/50 (22%)Gastric cancer 3/3 (100%) 0 (0%)Total 107 (71%) 43 (29%)

Table 2. Frequency of H. pylori detection in gastric biopsy specimens by using rapid urease test and culture

Statistical significant at (p<0.01)

Figure 1. Endoscopic findings in gastric biopsy specimens of studied patients

Figure 1. Endoscopic findings in gastric biopsy specimens of studied patients

with the change in the color of the slant MCUA tube from orange to pink that occurred at the same time thus giving an additional evidence for

Figure 2. Change in color of slant MCUA tube, a) slant MCUA tube only; b) positive slant MCUA tube culture; c) negative slant MCUA tube culture

the presence of H. pylori in the samples (Figure 2).The colonies of the isolated H. pylori were small to middle in size, rounded, and creamy in color. All H. pylori isolates were Gram-negative spiral to coccobacilli and shared the characteristic catalase, urease, and oxidase production, but differed

Figure 3. Biochemical tests for identification of H. pylori isolates. *107 isolates were positive for H. pylori out of 150 isolates

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slightly with respect to other tests (Figure 3). Collectively, some isolates were being positive in nitrate reduction, some were able to grow at 42°C or both. All isolates were unable to grow on 3.5% NaCl, 1% glycin or at 25°C, respectively.

The culture and urease test revealed that 107 (71.33%) out of 150 gastric biopsy specimens were positive for H. pylori. The H. pylori was

Figure 5. Lane 1 shows molecular weight marker (ladder), lanes 2, 4 and 6 are negative controls and lanes 3 and 5 are PCR positive

positive in 42% (13/31) of the normal patients, in 63% (19/30), and in 92% (33/36) of patients with gastric and duodenal ulcer, respectively. Seventy eight percent (39/50) of patients with gastritis were H. pylori positive. All patients with gastric cancer were H. pylori positive. The relation between presence or absence of H. pylori and endoscopic findings are shown in Table 2.

Figure 4 shows similarity between culture and rapid urease test results, while highly significant difference between different gastrointestinal groups with Gram stain results.

Samples from 107 isolated H. pylori colonies were subjected to PCR. A 41l-bp fragment indicative of the urea subunit of urease gene was obtained with primers specific for this subunit from these isolated strains. This result confirmed that the isolated colonies were H. pylori. PCR results are shown in Figure 5. Lane 1 shows molecular weight marker (ladder), lanes 2, 4 and 6 are negative controls and lanes 3 and 5 are PCR positive. All of samples were positive for ureA gene by PCR.

DISCUSSION

The isolation rate of H. pylori among dyspeptic patients by culture and rapid urease test was inconsistence with Franzin et al16 who reported that 75.5% of dyspeptic patients were H. pylori positive. On the other hand, our findings were higher than the percentage in the study obtained by Matsukura et al.12 Studies performed at Ismailia (Egypt), 66% and 68%, respectively of dyspeptic patients were H. pylori positive.17,18 A possible explanation for these differences in isolation rates could be attributed to different sample size and methods used for isolation. The present study revealed that incidence of H. pylori among dyspeptic patients with normal endoscopic findings was 42%. This result is similar to the one reported by Suzan18 and Maii.17 The incidence of H pylori among gastric ulcer patients in this study was 63%. This result was relatively around the spectrum of results reported in different parts of the world, which averaged 70%,20 while it was significantly lower than that reported by Franzin16 and Suzan,18 who recorded 78.6% and 75%, respectively. This could be attributed to gastric reflux or due to the effect of analgesic and anti-inflammatory drugs.

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Figure 4. Prevalence of positive H. pylori among studied cases

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Furthermore, host characteristics and strain viability may play a role in the pathogenesis of peptic ulcer disease.

Among the duodenal ulcer patients, 92% were H. pylori positive by culture and urease test (Table 1 and Figure 1). This finding is in agreement with Calvet et al20 who reported 95% of duodenal ulcer patients with positive H. pylori. In a similar study, Li et al21 recorded a wide range of positive cases (73-100%). On contrary, Laura et al22 found that duodenal ulcer patients were positive only in 74.3% of studied cases. A strong association between H. pylori and duodenal ulcer was observed in our study.

The present work revealed that incidence of H. pylori among gastritis patients was 78%. This is close to those reported by Suzan18 where isolation rate was 67.5%. Moreover, we detected the organism in all patients with gastric cancer. This result is relatively similar to a previous study which reported 98% of gastric cancer patients were H. pylori positive.11

The results of rapid urease test showed that 71.33% of dyspeptic patients were positive for H. pylori. This finding was in close agreement with previous observations of Suzan18 and Maii17 who reported 66% and 68%, respectively. On contrary, Oyedeji et al23 and Brooks et al7 reported 42.1% and 43.3% positive for H. pylori by urease test. These variations in rapid urease test result may be attributed to certain limitations23 such as the presence of some isogenic urease negative mutants of H. pylori24 or false positive results owing to presence of other urease positive bacteria in the sample.23 Other possible explanation for conflicting results of rapid urease test could be due to reflux of alkaline bile into the stomach that gave false positive result.7 Results of rapid urease test were optimized at different environments. Positive results (36%) were obtained at room temperature after one hour. At 37°C after one hour and 24 hours incubation, 62% and 71.33% positive results, respectively were obtained. The results of this study indicate a good correlation between culture and rapid urease test, as all biopsy specimens diagnosed positive by culture were also positive by rapid urease test. Similarly, the previous findings of Suzan18 and Brooks et al7 showed complete agreement between culture and rapid urease test results.

We used the ure A gene as a PCR target. A PCR product of the anticipated size (411 bp) was obtained from all of samples. Our result is in agreement with He et al15 and Vinette et al10 who reported that ureA gene is conserved and specific to H. pylori. On the other hand, Lu et al14 showed that ureA gene PCR is specific but with low sensitivity (75%), this result was reported when they compared five commonly used primer sets targeting different segments of H. pylori genome. They attributed this low sensitivity to sequence polymorphism in this locus. Brooks et al7 attributed the sensitivity of PCR to the size of the amplified region. They showed that the smaller 294 bp glmM product provides a more accurate result than 411 bp ureA product. These controversial findings in the sensitivity of the target gene may be attributed to different samples as Lu et al14 and Brooks et al7 used gastric biopsy samples while in the current study we used H. pylori colonies directly. Maii17 supposed that different PCR protocols, sampling techniques, and different extraction methods may be a possible explanation for these controversial findings.

To conclude, the prevalence of the infection by H. pylori is high with strong association between H. pylori and duodenal ulcer is noticed. The UreA gene is a strong confirmatory and relevant to H. pylori presence in gastroduodenal diseases.

AcknowledgmentThe author is thankful to all the people who were helpful in collection of data.

Conflict of interest The authors affirm no conflict of interest in this study.

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10. Vinette KM, Gibney KM, Proujansky R, Fawcett PT. Comparison of PCR and clinical laboratory tests for diagnosing H. pylori infection in pediatric patients. BMC Microbiol. 2004;4:5.

11. Huang JQ, Sridhar S, Chen Y, Hunt RH. Meta-analysis of the relationship between Helicobacter pylori seropositivity and gastric cancer. Gastroenterology. 1998;114(6):1169–79.

12. Matsukura N, Tajiri T, Kato S, Togashi A, Masuda G, Tokunaga A, et al. Diagnostic value of culture, histology and PCR for Helicobacter pylori in the remnant stomach after surgery. Aliment Pharmacol Ther. 2004;20Suppl1:33–8.

13. Han SW, Flamm R, Hachem CY, Kim HY, Clarridge JE, Evans DG, et al. Transport and storage of Helicobacter pylori from gastric mucosal biopsies and clinical isolates. Eur J Clin Microbiol Infect Dis. 1995;14(4):349–52.

14. Lu JJ, Perng CL, Shyu RY, Chen CH, Lou Q, Chong SK, et al. Comparison of five PCR methods for detection of Helicobacter pylori DNA in gastric tissues. J Clin Microbiol. 1999;37(3):772–4.

15. He Q, Wang JP, Osato M, Lachman LB. Real-time quantitative PCR for detection of Helicobacter pylori. J Clin Microbiol. 2002;40(10):3720–8.

16. Franzin L, Pennazio M, Cabodi D, Paolo Rossini F, Gioannini P. Clarithromycin and amoxicillin susceptibility of Helicobacter pylori strains isolated from adult patients with gastric or duodenal ulcer in Italy. Curr Microbiol. 2000;40(2):96–100.

17. Maii AMA. Prevalence of Helicobacter pylori in saliva of patients with chronic gastritis disease as detected by PCR [thesis]. Egypt: Suez Canal University; 2003. p. 85–96.

18. Suzan GW. Comparison of serological detection of IgG, culture and rapid urease test for detection of Helicobacter pylori [thesis]. Egypt: Suez Canal University; 2000. p. 45–74.

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Saekhu, et al.Tigecycline did not reduce MMP-9, edema and LOS

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MMP-9, brain edema, and length of hospital stay of patients with spontaneous supratentorial intracerebral hemorrhage after hematoma evacuation along with the administration of tigecycline

Keywords: LOS, MMP-9, SICH, Tigecycline

pISSN: 0853-1773 • eISSN: 2252-8083 • http://dx.doi.org/10.13181/mji.v25i4.1520 • Med J Indones. 2016;25:221–7• Received 09 Aug 2016 • Accepted 25 Sep 2016

Corresponding author: Mohamad Saekhu, [email protected]


Mohamad Saekhu,1 Nurhadi Ibrahim,2 Ina S. Timan,3 Amir S. Madjid,4 Zainal Muttaqin,5 Teguh A.S. Ronokusumo,6 Sudigdo Sastroasmoro,7,8 Hilman Mahyuddin1

1 Department of Neurosurgery, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia2 Department of Physiology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia3 Department of Clinical Pathology, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia4 Department of Anasthesiology and Intensive care, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo

Hospital, Jakarta, Indonesia5 Department of Neurosurgery, Faculty of Medicine, Universitas Diponegoro, dr. Kariadi Hospital, Semarang, Indonesia6 Department of Neurology, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia7 Clinical Epidemiology and Evidence-Based Medicine, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo

Hospital, Jakarta, Indonesia8 Department of Pediatric, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia

Cl inical Research


ABSTRAK

Latar belakang: Kadar matrix metalloproteinase–9 (MMP-9) yang tinggi diyakini merusak sawar darah otak (SDO) sehingga terjadi edema serebri dan menambah lama rawat inap pasien. Penelitian pada binatang menunjukkan tigesiklin menurunkan kadar MMP-9. Kemampuan tigesiklin menurunkan kadar MMP-9 yang berdampak pada pengurangan edema serebri dan lama rawat inap pasien perdarahan intraserebral spontan supratentorial (PISS), belum diketahui.

Metode: Randomized clinical trial (RCT) dilakukan pada 72 pasien PISS yang menjalani evakuasi hematoma di sebelas rumah sakit di Jakarta; tigesillin 100 mg (n=35) atau fosfomisin 2 g (n=37) diberikan intravena sebelum insisi kulit sebagai antibiotik profilaksis infeksi pascabedah. Seluruh subjek diukur kadar plasma darah MMP-9 sebelum pembedahan, serta pada hari ke-1 dan ke-7 pascabedah. Pengurangan edema serebri dinilai dengan membandingkan edema serebri pada computed tomography scan (CT scan) prabedah dengan edema serebri pada CT scan pascabedah. Saat pasien keluar dari rumah sakit; hidup atau meninggal, dicatat lama rawatnya. Data dianalisis dengan uji Mann-Whitney atau uji Chi square.

Hasil: Didapatkan perbedaan tidak bermakna pada proporsi subjek yang mengalami penurunan kadar MMP-9 pada hari pertama (48% vs 50%; p=0,902; OR=1,1) dan hari ke-7 (33% vs 48%; p=0,296; OR=1,9); proporsi pengurangan edema serebri (68% vs 80%; p=0,58); LOS (median 12 hari vs 13 hari p=0,256; proporsi subjek dengan LOS ≥15 hari 40% vs 27% p=0,243; OR=1,81; NNT=8).

Kesimpulan: Pada pasien PISS yang dilakukan evakuasi hematoma, tigesiklin tidak menurunkan kadar MMP-9 dan derajat edema serebri, serta tidak memperpendek LOS.

ABSTRACT

Background: The high plasma level of matrix metalloproteinses–9 (MMP-9) is believed to disrupt the blood-brain barrier (BBB) and cause brain edema, as well as increase patient’s length of hospital stay (LOS). Tigecycline showed ability to reduce the MMP-9 level on study in animals. This study aimed to evaluate whether tigecycline can reduce the plasma levels of MMP-9; brain edema; and LOS of patients with supratentorial spontaneous intracerebral hemorrhage (SSICH).

Methods: A randomized clinical trial (RCT) was conducted on 72 SSICH patients who underwent hematoma evacuation in eleven hospitals in Jakarta; 100 mg tigecycline (n=35) or 2 g fosfomycine (n=37) administered intravenously before skin incision as an prophylactic antibiotics to avoid post-operative infections. Plasma levels of MMP-9 were measured in all subjects before and on the first and seventh day after the surgery. Reduction of brain edema was assessed by comparing the extent of brain edema on computed tomography scan (CT scan) before and CT scan after surgery. The length of stay (LOS) was recorded at the time of hospital discharge either survive or death. Data were analyzed using Mann-Whitney and Chi-Square test.

Results: There were non-significant statistical differences between two groups in the proportion of subjects with reduced MMP-9 levels on the first day (48% vs 50%; p=0.902; OR=1.1) and seventh day after the surgery (33% vs 48%; p=0.296; OR=1.9); proportion of the subjects with brain edema reduction (86% vs 80%, p=0.58); LOS (median 12 days vs 13 days, p=0.256; LOS ≥15 days 40% vs 27%; p=0.243; OR=1.81; NNT=8).

Conclusion: On SSICH patients who underwent hematoma evacuation, tigecycline did not either reduce MMP-9 levels and brain edema or shorthen LOS.



Early mortality following spontaneous intracerebral hemorrhage (SICH) was related to transtentorial herniation due to hematoma with or without perihematomal brain edema mass effects.1 Surgical evacuation of the hematoma intracerebral is a reasonable choice to reduce the hematoma volume and the mass effect, as well as potentially reduce the production of neurotoxic substances triggered by the hematoma degradation products.2 However, surgical procedure was accompanied by additional injury.3,4 Neurological outcome after SICH remains unsatisfactory.5 One of several markers of brain injury either related to ICH6 or brain surgery4 is matrix metalloproteinase-9 (MMP-9). High level of MMP-9 is believed to be a predictor of hematoma growth, extent of brain edema and neurological deterioration.6 In traumatic brain injury, high levels of MMP-9 was to be a predictor of length of stay (LOS) and death in intensive care unit (ICU).7 On the other hand, MMP-9 inhibition is neuroprotective,8,9 as well as tigecycline reduce the MMP-9 level in animal study.10 This study aimed to confirm the changes of plasma level of MMP-9, brain edema, and LOS of pateints with supratentorial SICH (SSICH) after hematoma evacuation along with administration of single dose 100 mg tigecycline.

METHODS

This article is the second part of a randomized clinical trial (RCT) to assess the effects of tigecycline on patients with SSICH.11 Seventy two patients at eleven hospitals in Jakarta - Indonesia who prepared for hematoma evacuation were randomized into either receiving 100 mg tigecycline or 2 g fosfomycine intravenously as prophylactic antibiotics. The protocol of the study has been approved by Health Research Ethics Committee, Faculty of Medicine, Universitas Indonesia – Cipto Mangunkusumo Hospital (No.493/PT02.FK/ETIK/2012).

The diagnosis of SSICH was based on clinical examinations and confirmed by computed tomography (CT) scan imaging. The indication for hematoma evacuation by surgery were SSICH patients with clinical deterioration as measured by GCS score less than 14 and hematoma valume ≥30 mL. The volume of hematoma was calculated with 1/2 (AxBxC) formula, where A is the greatest diameter of hematoma on CT scan image (on centimeter); B

is the vertical diameter (90° to A (on centimeter); C is the amaount of slices on CT scan image (on centimeter).12 The evacuation of hematoma performed with craniotomy or craniectomy. Sample size was calculated by following formula:13

n1=n2= (Zα√2PQ+Zβ√P1Q1+P2Q2)2/ (P1Q1)2

n1 = n2 = (Zα√2PQ + Zβ√P1Q1 + P2Q2)2/ (P1Q1)2

By taking P1=0.4 as the proportion of the effect of standar treatment, and P2=0.7 as proportion of the effect of tigecycline, α=0.05 and power 90%, a minimum os 22 subjects in each group were required.

Subject characteristics and outcomes were recorded as numeric and, or categorical data. The MMP-9 plasma level on postoperative day one and day seven changes the brain edema level on CT scan, and LOS at the time of hospital discharge were recorded as outcomes data. The MMP-9 plasma level was measured using the enzyme-linked immunosorbent assay (ELISA), the reagent produced by Boster Biological in the United States. According to the previous study,14 normal value of MMP-9 was determined at 265 ng/mL.

The brain edema level was measured with blinded by main researcher or author (MS) based on perihematoma hypodensity thickness on the CT scan before surgery and hypodensity thickness at the former hematoma area in millimeter; hipodensity thickness ≤5 mm was set as mild degree of brain edema, 6 mm to 10 mm was set as moderate degree, and thickness more than 10 mm was set as severe degree. The difference of relative edema on CT scan before and after surgery was recorded as the changes of brain edema levels.

Data from all of the subjects were included in the analysis. Numeric data was presented as mean value and data distribution while categorical data was presented as proportion (%). Hypothetical testing for numeric data was made using Mann–Whitney U test while categorical data were tested using Chi Square test. The statistical level of significance was set at p≤0.05. Relative clinical effectiveness was calculated using relative risk (RR), odds ratio (OR), and number needed to treat (NNT).

RESULTS

The number of samples collected during two years

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periode of study was 35 subjects in the treatment group (tigecycline) and 37 subjects in the control group (fosfomycine), the minimum number of sample required for this study exceeded.

Subject characteristicsAge mean, Glasgow coma scale (GCS), and hematoma volume of subjects in both groups

Experimental groupTigecycline Control

Subject amount (n%) 35 (49) 37 (51)Gender

Male (n%) 19 (54) 26 (70)Female (n%) 16 (46) 11 (30)

AgeMean (SD) 52.8 (9.1) 51.8 (8.9)>60 years (n%) 9 (26) 5 (14)

Poor Risk FactorsFemale (n%) 16 (46) 11 (30)Elderly (Age >60 years) 9 (26) 5 (14)Abnormal MAP (n%) 32 (91) 29 (78)Hypertension (n%) 32 (91) 32 (86)

GCSMedian (min–max) 9 (6–13) 9 (5–14)

Hematome Volume •Before surgery

Median (min–max) mL 45 (30–90) 50 (30–90)Volume ≥60 mL (n%) 21 (60) 22 (59)

•After surgeryVolume ≥30 mL (n%) 1 (4) 4 (16)

Midline ShiftMedian (min–max) mm 5 (0–15) 5 (0–20)≥10 mm (n%) 7 (20) 12 (32)

Brain Edema Moderate degree (n%) 17 (48) 15 (40)

MMP-9 Level Before interventionMedian (min–max) ng/mL 216.2 (107.9–653.2) 250.8 (79.2–629.1)Normal Level (n%) 19 (56) 11 (37)

Leukocyte LevelMean (SD) /μL 14.17 (3.48) 15.48 (5.54)

Surgical Onset Less than 24 hour (n%) 22 (63) 27 (73)More than 48 hour (n%) 6 (18) 6 (16)

Surgical durationMedian (min–max) minutes 160 (60–360) 160 (60–420)

Corticotomy DiameterMedian (min–max) mm 10 (5–30) 15 (3–30)

were equal. Risk factors for poor outcome such as elderly age (age over 60 years);14 female,15 history of hypertension and diabetes, and abnormal mean arterial pressure (MAP)17 were found greater in the tigecycline group. On the contrary, mid line shifting (MLS) of ≥10 mm, which is a sign of brain herniation and associated with poor outcome,18 was greater in the fosfomycine group.

MAP= mean arterial pressure; CGS= Glasgow coma scale; MMP-9= matrix metalloproteinase-9

Table 1. Subject characteristics



MMP-9 plasma levelsChanges of the MMP-9 plasma levels were analyzed based on mean value and proportion of subject with decreased level. Changes of plasma levels of MMP-9 are shown in table 2.

Post-operative day 1 Either the median of MMP-9 plasma level or proportion of subjects with decrease MMP-9 plasma level was statistically non significant different (table 2). Instead of lowering the plasma levels of MMP-9, tigecycline slightly increase the plasma MMP-9 levels. The chance for tigecycline to reduce the plasma levels of MMP-9 was 0.9 time compared to control group.

Post-operative day 7Similar to the result on the day 1; on the day 7, either median MMP-9 plasma level or proportion of subjects with decrease MMP-9 plasma level were also statistically non significant. However, tigecycline group showed significant increase of the median MMP-9 plasma level; increased from 216.2 before surgery to 305.0 ng/mL on day 7 after surgery (increased 40%) compared to control group ie increased from 250.8 ng/mL before surgery to 256.7 ng/mL (increased 2%). The chances of patients in tigecycline group to decreased plasma levels of MMP-9 was 0.5 time compared to patients in the control group. Although statistically unsignificant, the increasing median level of plasma level of MMP-9, as well as a

Experimental groupp RR OR

Tigecycline ControlDay 1MMP-9

Median (min–max) ng/mL 220.6 (104.6–545.7) 253.1 (101.5–542.9) 0.395Normal Level (n,%) 21 (68) 16 (55) 0.317 0.72 0.6Decreased Level (n,%) 15 (48) 14 (50) 0.902 1.03 1.1

Day 7MMP-9

Median (min – max) ng/mL 305.0 (119.0–511.2) 256.7 (96.4–915.0) 0.720Normal Level (n,%) 10 (40) 15 (58) 0.157 1.43 2.1Decreased Level (n,%) 8 (33) 12 (48) 0.296 1.29 1.9

Brain edema degreeDecreased (n,%) 24 (86) 20 (80) 0.58 0.7 0.68

Table 2. Changes of plasma level of MMP-9 and brain edema

MMP-9= matrix metalloproteinase-9; RR= relative risk; OR= odds ratio

less proportion of patients experienced of reduce plasma level of MMP-9 in the tigecycline group may to be an early sign that tigecycline trigger an increase MMM-9 plasma level.

Brain edemaThe CT scan on the day seven could be done in 74% subjects ie 89% subjects in tigecycline group and 65% subjects in control group. The mean reason for not to do CT scan were patients already death or unstable conditions. The chance of patients in tigecycline to reduce the brain edema levels is 1.5 times compared to patients in the control group.

Length of hospital stay (LOS)The chance of patients recieved tigecycline as prophyactic antibiotic experience LOS less than 15 days was 0.5 time compared to patients in the control group. However, control group showed more subjects experience an early death. Inhospital mortality before seven days after surgery in this study were 13 subjects; one subjects from tigecycline group and twelve subjects from control group (Table 3).

DISCUSSION

Phenolic β-diketone compound in the chemical structure of tetracycline has been known as metal chelator.19 Tetracycline derivates; doxycycline and minocycline has been known to have iron chelator

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Experimental groupp RR OR NNT

Tigecycline ControlInhospital mortality (n,%) 6 (17) 13 (35) 0.083 0.49 0.38 5LOS

Median (min-max) day 12 (4–46) 13 (2–30) 0.264LOS ≥15 days 14 (40) 10 (27) 0.243 1.48 1.81 8

Table 3. Inhospital mortality and LOS

LOS= length of stay; RR= relative risk; OR= odds ratio; NNT= number needed to treat

activity.20 Matrix metalloproteinases are zinc-dependent endopeptidase, therefore, tetracycline and its derivative are believed to have an anti-matrix metalloproteinases.21 Tigecycline is a new tetracycline derivate and its chemical structure also contains phenolic β-diketone.22 Study in animals showed that tigecycline reduced the MMP-9 levels;10 however, result of the present study is not inline. Neither at the first nor at the seventh day after administration showed significant differences between the two groups. Inability of tigecycline to reduce the MMP-9 plasma levels in this study expected to be due to many factors such as different subjects, i.e. humans vs rats; different pathology, i.e. SICH vs staphylococcus-infected burn wound; terms or amount of tigecycline administered, i.e. single dose vs 21 times; MMP-9 measurement time, i.e. day one or day seven after single dose administration vs day one after 21 times or doses administration (at day twenty-second).9 MMP-9 also has twice-peak levels; the first peak is on the first three days after SICH attack and the second peak is on the seventh day.23 It is believed that the distinction between study in animals and study in humans stems from the aforementioned conditions.

MMP-9 is associated with disruption of BBB that promote brain edema.24 This study showed the changes of brain edema between tigecycline group and control group was not statistically significantly diffeerent. This result was in accordance with the non statistically significantly different in either median MMP-9 level or the proportion of subjects with decrease MMP-9 plasma level. However, there was slightly higher on the proportion of subjects with decrease brain edema (table 2). Considering that the proportion of subjects with decrease MMP-9 plasma level was lower in tigecycline group; as well as the median MMP-9 level was higher in tigecycline

group; the comparability of severity of trauma related surgical procedure (surgical duration and the diameter of corticotomy; table 1); as well as comparability of hematoma volume before surgery, the amount of hematoma that evacuated was expected to be a main factor of the reduction of brain edema. The remaining hematoma volume after surgery ≥30 mL on CT scan control were 16% in control group compared to 4 % in tigecycline group. Therefore, the effects of tigecycline on reduction of brain edema in SSICH after surgical evacuation of hematoma could not be concluded.

Study on traumatic brain injury patients showed MMP-9 plasma level in the first 48 hours after onset was associated with endpoint intensive care unit (ICU) LOS.7 Meanwhile, in-hospital morbidity and complications were the main factors affecting the LOS of ICH patients.25,26

Considering that a longer LOS will increase the hospital cost, LOS could be an indicator of effectiveness and efficiency of a treatment choice as well as a hospital management.27 The present study showed the differences between the two groups; either the median of LOS or LOS more than 15 days, were statistically not significantly different. Previous study showed that early mortality was correlated with shorten LOS.28 This study showed 92% subjects who died prior to day seven after surgery was subjects from control group. It may be that the more higher proportion of early mortality in the control group was be a factor of more higher proportion of subjects in the tigecycline group that having LOS ≥15 days. Overall, this study could not conclude the effects of tigecycline on LOS of SSICH patients after surgical evacuation of hematoma, whether shorten or extend the LOS.

Contrary to what has been reported in animal study, instead of reducing the plasma levels of



MMP-9, tigecycline increased MMP-9 plasma levels on day seven, with the median of 40% in tigecyline group, and 2 % on control group. The proportion of subjects with normal plasma level of MMP-9 on tigecycline group was decrease by 29%. In contrary, the control group showed increase proportion of subjects with normal level of MMP-9 by 57%. The proportion of subjects with increase plasma levels of MMP-9 in tigecycline group was higher than control group (67% vs 52% respectively). This trend was in accordance with study in human retinal pigment epithelial cells culture that showed minocycline; the prototype of tigecycline, promote the increase of MMP-9 levels.29 Beside having dual peak levels, MMP-9 also has dual roles. On the first three days or the first peaks, MMP-9 play a role in pathologic process, ie disruption of BBB. Meanwhile, on the second peak, MMP-9 plays role in neuronal recovery.23 The increase levels of MMP-9 is needed for healing process as well as neurogenesis by promoting migration of activated microglia30 and endogen stem cells.31,32 Endogenous neurogenesis was believe to be a powerful tool to repair the brain after SICH.33 However, this study could not conclude the effects of tigecycline on short-term clinical outcome, mainly on patient’s LOS.

There were some limitations in this study. This RCT involving 11 hospitas in Jakarta, This study showed the median of LOS was comparable with the studies in US (14.9 days)34 and Germany (15 days).26 The median of LOS in the present study was 12 days in tigecycline group and 13 days in control group. According to the formula for sample size, this study needs 22 subjects each group. During two years peiode of study, 72 subjects was colected. Compared to the study in US that involves 45,159 subjects and study in Germany that involves 1,405 subjects, subjects involved on this study was only slightly. However, to the best of our knowledge, this is the first RCT of effetcs of tigecycline on MMP-9 levels as well as LOS of SSICH patients that surgically treated in Indonesia. Considering that tigecycline clinically effective to reduce in-hospital mortality,11 further study is needed to determine the effects of multiple adminitrations of tigecycline on clinical outcomes in meddle to long-term clinical outcomes.

In conclusion, the administration of single dose of 100 mg tigecycline for SSICH patients that surgically treated to evacuate the hematoma

has not been able to significantly change MMP-9 plasma levels, brain edema, and the LOS. However, the increase of MMP-9 plasma levels on the day seven in tigecycline group need further studies to explore the effects of tigecycline for better recovery after SICH.

Acknowledgment The author would like to thank Prof. Sarwono Waspadji, M.D., Ph.D. and all medical staff members of the Department of Neurosurgery, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, as well as all hospitals that have participated in this study.


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Indonesian local fetal-weight standard: a better predictive ability for low Apgar score of small-for-gestational age neonates

Keywords: Apgar, small-for-gestational-age, standard, weight

pISSN: 0853-1773 • eISSN: 2252-8083 • http://dx.doi.org/10.13181/mji.v25i4.1301 • Med J Indones. 2016;25:228–33• Received 30 Oct 2015 • Accepted 04 Sep 2016

Corresponding author: Adly N.A. Fattah, [email protected]


Adly N.A. Fattah,1 Karina N. Pratiwi,1 Sulaeman A. Susilo,1 Jimmy S.N. Berguna,1 Rima Irwinda,1 Noroyono Wibowo,1 Budi I. Santoso,1 Jun Zhang2

1 Department of Obstetrics and Gynecology, Faculty of Medicine Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesa

2 Ministry of Education-Shanghai Key Laboratory of Children’s Environmental Health, Xinhua Hospital, Shanghai, China

Cl inical Research


ABSTRAK

Latar belakang: Keakuratan penilaian pertumbuhan janin adalah salah satu komponen penting dalam asuhan antenatal. Rujukan umum berat janin dan berat lahir yang dapat diadaptasi pada pasien lokal telah dikembangkan oleh Mikolajczyk dan rekan. Penelitian ini bertujuan untuk memvalidasi standar presentil lokal dengan mengevaluasi odds ratio (OR) dari skor Apgar <7 pada menit ke-1 dan ke-5 pada small-for-gestational age (SGA) dibandingkan bayi yang tidak SGA.

Metode: Kami menggunakan rujukan umum persentil berat janin dan berat lahir yang dikembangkan Mikolajczyk dan rekan untuk menghasilkan standar local Indonesia kemudian merumuskan neonatus dengan SGA. Data divalidasi menggunakan basis data bayi lahir tunggal hidup (2.139 kelahiran) dari 1 Januari hingga 31 Desember 2013 di Rumah Sakit Cipto Mangunkusumo, Jakarta, Indonesia. Kami membandingkan rujukan kami dengan rujukan dari Hadlock dan rekan. Dari setiap rujukan, OR Apgar <7 pada menit 1 dan 5 pada bayi SGA dibandingkan dengan tidak SGA diukur menggunakan analisis bivariat dan multivariat.

Hasil: SGA didapatkan pada 35% (748/2.139) dan 13% (278/2.139) dari neonatus sesuai definisi standar Indonesia dan Hadlock. OR dari skor Apgar <7 pada menit 1 dan 5 adalah 3,45 (95% IK=2,56–4,65) dan 3,05 (95% IK=1,92–4,83) untuk berat janin standar lokal Indonesia dibandingkan 2,14 (95% IK=1,65–2,76) dan 1,83 (95% IK=1,21–2,77) sesuai rujukan Hadlock dan rekan.

Kesimpulan: Berat janin Indonesia terstandar memiliki kemampuan lebih baik dalam memprediksi skor Apgar <7 pada menit ke-1 dan ke-5 pada neonatal dengan SGA dibandingkan rujukan dari Hadlock dan rekan.

ABSTRACT

Background: Accurate assessment of fetal growth is one of crucial components of antenatal care. A generic reference for fetal-weight and birthweight percentiles that can be easily adapted to local populations have been developed by Mikolajczyk and colleagues. This study aimed to validate our own local percentile standard by evaluating the odds ratio (OR) of low 1st and 5th minute Apgar score for small-for-gestational age (SGA) versus those not SGA.

Methods: We used the generic reference tools for fetal-weight and birthweight percentiles developed by Mikolajczyk and colleagues to create our own local standard and then defined the SGA neonates. For validation, we used the database of singleton live deliveries (2,139 birth) during January 1st to December 31st 2013 in Cipto Mangunkusumo Hospital, Jakarta, Indonesia. We compared our reference with that of Hadlock and colleagues. For every reference, the OR of Apgar score <7 at 1st and 5th minutes for infants who were SGA versus those not estimated with bivariate and multivariate analyses.

Results: SGA found in 35% (748/2,139) and 13% (278/2,139) of neonates using the definition derived from Indonesian standard and Hadlock’s. OR of Apgar score <7 at 1st and 5th minutes were 3.45 (95% CI=2.56–4.65) and 3.05 (95% CI=1.92–4.83) for the Indonesian local fetal-weight standard compared with respectively 2.14 (95% CI=1.65–2.76) and 1.83 (95% CI=1.21–2.77) for Hadlock and collegues’ reference.

Conclusion: Indonesian local fetal-weight standard has a better ability to predict low 1st and 5th minutes Apgar scores of SGA neonates than has the Hadlock and collegues’ reference.


Fattah, et al.Indonesian local fetal-weight standard

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Accurate assessment of fetal growth is one of crucial components of antenatal care,1 because intrauterine growth restriction (IUGR) could lead to the increased rate of perinatal mortality and morbidity.2 However, defining normal or abnormal fetal growth has become a great challenge in clinical research.3 Many studies have been conducted to find the best method to assess fetal growth.4 They are widely ranging methods from a simple-inexpensive fundal height measurement5 to the Doppler velocimetry and biomarkers work-up.3

In 2014, the International fetal growth standards for the clinical interpretation was issued. It was derived from women who were at low risk of IUGR in eight countries (Brazil, Italy, Oman, United Kingdom, United States, China, India, and Kenya).6 However, there was contrary statement that the use of customized or individualized fetal weight charts, with adjustment for maternal and fetal factors, have a better accuracy in predicting fetal weight compared to non-customized fetal-weight reference.7,8 Dutch already constructed their own reference standard for birthweight by parity, sex, and ethnic background, seperately.9 United States also have their own customized standard to assess the fetal growth and birthweight among their population by adjusting physiological coefficients (maternal height, weight, parity, ethnic origin, and sex of the baby), positive (diabetic mothers) and negative effects (smoking, history of preterm delivery, and hypertensive diseases) on birthweight.10 These standards were created as the consequences of statement that optimal neonatal outcome is achieved at different birth weights in different populations.4

It was internationally accepted that customized standards for fetal growth and birth weight improve the accuracy to detect IUGR by better distinction between physiological and pathological smallness.2,7 Ideal diagnosis of IUGR is complex because it should involve the growth potential of the fetus, current fetal size, fetal and placental health, and, if available, fetal growth velocity.3 Miller et al11 stated that normal fetal growth is affected by the genetically predetermined growth potential and later controlled by maternal, fetal, placental, and external factors11.

Mikolajczyk et al12 created generic reference for fetal-weight and birthweight percentiles that can be easily adapted to local populations. It has a better

ability to predict adverse perinatal outcomes than the non-customized fetal-weight reference, and is simpler to use than the individualized reference without loss of predictive ability. They created a weight percentiles calculator that allow us to obtain weight percentile standards for our own local population by inserting the mean birthweight at 40 weeks of our population into the formula.12

In this present study, we aimed to develop our own local percentile standard of fetal birthweight using the generic reference tool developed by Mikolajczyk et al.12 We applied the correlation between small-for-gestational age and low 5th minute Apgar score to test the predictive ability of our fetal weight standard. It will be compared with the previous birthweight percentile developed by Hadlock et al13 that commonly used in our practical setting.

METHODS

The generic reference tool developed by Mikolajczyk et al12 was first downloaded in a form of excel (.xls) file. The original article was downloaded in order to analyze the method used in the development of the generic reference of the fetal weight chart. They made the weight reference easily adjustable according to the mean birthweight at 40 weeks of gestation for any local population after considering the fetal-weight reference developed by Hadlock et al13 and the notion of proportionality proposed by Gardosi et al.14 Optimum growth equation proposed by Hadlock et al13 was used to create a generic global reference.

Fetal weight (g) = exp(0.578 + 0.332 × GA – 0.00354 × GA2)

Hadlock et al13 performed ultrasound examination between 10 and 41 weeks of gestation of 392 pregnant women of the European Continental Ancestry Group in the USA. The statistical variation of fetal weight in a given gestational week was also provided and resulted in a constant fraction of mean. Therefore, the fetal-weight percentiles of each gestational week were developed13.

Gardosi et al5 created the individualized reference by using the Hadlock et al13 formula. They transformed the formula into a proportionality


function. Therefore, a percentage of the expected weight based on Hadlock’s formula could be derived from a given individual birth weight. They also adjusted it for ethnic group, parity, sex of the infant, and maternal height and weight.14 To determine the mean birthweight, we use at least 100 deliveries at 40 weeks (40+0 to 40+6) and with no risk factors for having small-for-gestational age (SGA) infants. Therefore for this purpose, pregnancy complicated with preeclampsia, intrauterine growth restriction, known congenital anomalies, maternal systemic disease i.e heart failure, diabetes mellitus were excluded from the initial database.

For application and validation, we used data from our delivery database at Cipto Mangunkusumo Hospital, Jakarta, Indonesia. This database contains clinical information on all women who delivered their babies during 1st January to 31st December 2013. Gestational age was determined based on first trimester ultrasound examination, and if not available, last menstrual period. SGA was defined as below 10th percentile for completed week of gestational age based on the local percentile standards. The percentile of each neonates’ birthweight was obtained from Fetal/Birth Weight Percentiles Calculator developed by Mikolajczyk et al.12 Apgar scores at 1st and 5th minutes below 7 established by competent perinatology residents was considered as low.

Bivariate analysis was used to find the association between the low Apgar score with SGA defined by both of Indonesian local fetal weight standard and Hadlock’s Fetal weight standard. P<0.05 was considered as significant association. Statistical analyses were conducted using software IBM® SPSS® Statistics version 20 (Armonk, NY: IBM Corp).

RESULTS

Mean birthweight at 40 completed weeks of gestation were respectively 3,705 grams and 3,217 grams by Hadlock et al13 and by our local standard. Hadlock et al13 performed ultrasound measurements between 10 and 41 weeks of gestation among 392 pregnant women of the European Continental Ancestry Group living in the USA in order to create the optimum growth equation. We calculated the mean birthweight of 285 neonates born in during 2013 who were not at risk of having SGA. Therefore, preeclamptic deliveries, IUGR fetus, fetal with known congenital anomaly were excluded prior to the calculation.

We developed two growth percentile standards (Figure 1) using the generic reference tool and then defined the SGA from both standards (Table 1).

Figure 1. Indonesian and Hadlock’s13 Fetal Growth Chart

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The 10th percentile value of fetal weight at 28 wga were respectively 1,074 g and 957 g defined by Hadlock’s standard and by Indonesian fetal growth standard. At 40 wga, the 10th percentile value was respectively 3,077 g and 2,744 g defined by Hadlock’s standard13 and by Indonesian fetal growth standard (Table 2).

We validated the definition of SGA using the delivery database of 2,139 deliveries during 1st January to 31st December 2013 in Cipto Mangunkusumo Hospital Jakarta, Indonesia. Table 2 shows the characteristics of women underwent delivery registered in our database. The median (range) of maternal age was 29 years (13–48). More than half of the population was multigravida (53.6%). The median (range) of birthwight was 2,800 (500–4,935) grams. SGA was diagnosed in 35% (748/2,139) of the population using the definition derived from Hadlock’s standard13. However it found in 13% (278/2,139) of neonates when defined by Indonesian fetal growth standard definition. Low Apgar scores were found respectively in 12.9% (275/2,149) and 4.4% (94/2,139) of neonates (Table 3).

Low Apgar scores at both of 1st and 5th minutes were significantly associated with the SGA neonates defined by both standards (p>0.005). OR of Apgar scores lower than 7 at 1st and 5th minutes were respectively 3.45 (95% CI=2.56–4.65) and 3.05 (95% CI=1.92–4.83) for the Indonesian local fetal-weight standard compared with respectively 2.14 (95% CI=1.65–2.76) and 1.83 (95% CI=1.21–2.77) for Hadlock et al13 standard. After adjusted with maternal age, parity, and preterm delivery, the OR of low 1st minute AS was improved from 3.45 to 3.53 using the definition of SGA derived from Indonesian standard. However, using the Hadlock’s et al13

Variable

Birthweight

Hadlock’s Fetal Growth(n=392)

Indonesian Fetal Growth

(n=285)

Mean (g) 3,705 3,21790th percentile at 28 wga (g) 1,512 1,28890th percentile at 40 wga (g) 4,333 3,69110th percentile at 28th wga (g) 1,074 95710th percentile at 40th wga (g) 3,077 2,744

Table 1. Birthweight mean and percentiles on Hadlock’s standard13 and Indonesian fetal growth standard

Wga= weeks of gestational age

Variable n=2,139Maternal age (year) n%

<35 1,678 (78.4%)≥35 461 (21.6%)

Parity, n %Primigravid 990 (46.3%)Multigravid 1,147 (53.6%)

Preterm birth n% 752 (35.2%)Gestational age at deliveries (weeks) * 38 (23–43)Birthweight (g) * 2,800 (500–4,935)SGA Hadlock n% 748 (35%)SGA Indonesian Fetal growth n% 278 (13%)Apgar Score <7 at 1st min n% 275 (12.9%)Apgar Score <7 at 5th min n% 94 (4.4%)

Table 2. Delivery characteristics of database used for valida-tion of Hadlock’s and Indonesian fetal growth chart

SGA: small-for-gestational-age; *: variables expressed as me-dian (range).

SGA (Indonesian Local Fetal Weight Standard) SGA (Hadlock’s Fetal Weight Standard)n % OR(95%CI) Adj OR(95%CI)* n % OR(95%CI)* Adj OR(95%CI)

AS 1st min < 7 80/278 28.8% 3.45 (2.56–4.65) 3.53 (2.56–4.89) 140/748 18.7% 2.14 (1.65–2.76) 1.57 (1.20–2.07)AS 5th min < 7 28/278 10.1% 3.05 (1.92–4.83) 2.80 (1.73–4.51) 46/748 6.1% 1.83 (1.21–2.77) 1.21* (0.79–1.87)

Table 3. Odds ratio of Apgar score < 7 at 1st and 5th minutes for infants who were SGA versus those not estimated with bivariate and multivariate analysis

SGA: small-for-gestational-age; AS: Apgar Score; OR: odds ratio; Adj OR: adjusted odds ratio; CI: confident interval; *: non-signifi-cant result (p>0.05). Multivariate analysis was performed by adjusting maternal age, parity, and preterm delivery

definition, the SGA was no longer associated with low 5th minute AS (p>0.05) after adjusting other variables (Table 3).


DISCUSSION

We showed that Indonesian local fetal-weight standard has a better ability to predict low 1st and 5th minutes Apgar scores of SGA neonates than has the Hadlock13 and collegues13 reference. The evidences that SGA neonates have an increased risk of low 1st and 5th minutes Apgar scores were irrespective of the standard used for definition. In our study, however, most of low Apgar scores were found in infants without SGA. Consequently, the predictive value of status of SGA for low Apgar score is very low.12 Low Apgar score was closely related to most cases of early neonatal mortality as well as low birth weight and prematurity.15 It also has a significant association with SGA neonates.12

Mikolajczyk et al12 have published a reliable generic reference for fetal-weight and birthweight percentiles that can be easily adapted to local populations. They compared the prediction ability for adverse perinatal outcomes of their non-customized fetal-weight reference with the fully individualized reference developed by Gardosi et al.14 Adverse perinatal events include any of stillbirth, dead within or after 24 hours of birth, alive on 7th day postpartum but referred to a higher level or special care unit, or Apgar score lower than seven at five minutes. We studied Apgar scores <7 at both of 1st and 5th minutes as the adverse perinatal event because the data are available in every deliveries record therefore the probability of having missing data would be reduced.

Odds ratio of Apgar score <7 at 5th minute was 3.05 (95% CI=1.92–4.83) for the Indonesian local fetal-weight standard. It was similar with OR of adverse outcomes (stillbirth, dead within or after 24 hours of birth, alive on 7th day postpartum but referred to a higher level or special care unit, or Apgar score <7 at five minutes) for infants SGA versus those SGA age determined by the country-specific reference developed by Mikolajczyk and colleagues 2.87 (95% CI=2.73–3.01).12 In addition, our findings supported the evidence that the local standard derived from generic tool12 has a better ability to predict adverse perinatal outcomes than has the non-customized fetal-weight reference.

We used the generic reference tool due to its proven-applicability and flexibility. It was validated in a large multinational study that included various ethnic groups. We considered that such tool is very useful in low-resource settings, especially where longitudinal ultrasound studies needed to examine variation in fetal growth were difficult to be conducted. To our knowledge, this is the first study that validates the generic reference tool developed by Mikolajczyk et al12 in Indonesia. Also, deliveries occurred over a year period in a tertiary medical center, is unlikely to affect to the Apgar score. However it has several limitations. First, by using this generic reference tool which is developed using the method proposed by Gardosi et al,14 a similar fetal growth pattern was assumed for all ethnic groups. However, this assumption might not always be accurate. Our hospital is a tertiary national referral hospital that allows multi-ethnic patients to be managed. In addition, we considered that defining normal and abnormal fetal growth using the reference or standard is found to be problematic in both of research fields and clinical practice.3 Second, we found similarly high prevalence of SGA in both standards (35% and 13% respectively for Indonesian standards and Hadlock’s standard13) using the cut off of 10th percentile. In undernourished population the prevalence would probably different. Therefore, this study result should only be generalized to pregnant women who have adequate nutrition.

Finally, utilization of a generic reference for fetal weight and birthweight can be completed with the approach developed by Mikolajczyk et al.12 A definition of SGA was largely dependent on the study population, i.e ethnic origin and maternal nutritional status. Indonesian local fetal-weight standard produced lower prevalence of SGA than Hadlock’s.13 It also has a better ability to predict low 1st and 5th minutes Apgar scores of SGA neonates than has the Hadlock et al13 reference.

AcknowledgmentThis paper is based upon previous research work of Jun Zhang from MOE and Shanghai Key Laboratory of Children’s Environmental Health, Xinhua Hospital, Shanghai, China.

Conflicts of interestThe authors affirm no conflict of interest in this study.

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REFERENCES

1. Bloemenkamp KWM. Fetal growth. Int Congr Series. 2005;1279:295–301.

2. Gardosi J. Fetal growth standards: individual and global perspectives. Lancet. 2011;377(9780):1812–4.

3. Zhang J, Merialdi M, Platt LD, Kramer MS. Defining normal and abnormal fetal growth: promises and challenges. Am J Obstet Gynecol. 2010;202(6):522–8.

4. Reeves S, Bernstein IM. Optimal growth modeling. Semin Perinatol. 2008;32(3):148–53.

5. Morse K, Williams A, Gardosi J. Fetal growth screening by fundal height measurement. Best Pract Res Clin Obstet Gynaecol. 2009;23(6):809–18.

6. Papageorghiou AT, Ohuma EO, Altman DG, Todros T, Cheikh Ismail L, Lambert A, et al. International standards for fetal growth based on serial ultrasound measurements: the Fetal Growth Longitudinal Study of the INTERGROWTH-21st Project. Lancet. 2014;384(9946):869–79.

7. Gelbaya TA, Nardo LG. Customised fetal growth chart: a systematic review. J Obstet Gynaecol. 2005;25(5):445–50.

8. Kiserud T, Johnsen SL. Biometric assessment. Best Pract Res Clin Obstet Gynaecol. 2009;23(6):819–31.

9. Visser GH, Eilers PH, Elferink-Stinkens PM, Merkus HM, Wit JM. New Dutch reference curves for birthweight by gestational age. Early Hum Dev. 2009;85(12):737–44.

10. Gardosi J, Francis A. A customized standard to assess fetal growth in a US population. Am J Obstet Gynecol. 2009;201(1):25e1–7.

11. Miller J, Turan S, Baschat AA. Fetal growth restriction. Semin Perinatol. 2008;32:274–80.

12. Mikolajczyk RT, Zhang J, Betran AP, Souza JP, Mori R, Gülmezoglu AM, et al. A global reference for fetal-weight and birthweight percentiles. Lancet. 2011;377(9780):1855–61.

13. Hadlock FP, Harrist RB, Martinez-Poyer J. In utero analysis of fetal growth: a sonographic weight standard. Radiology. 1991;181(1):129–33.

14. Gardosi J, Mongelli M, Wilcox M, Chang A. An adjustable fetal weight standard. Ultrasound Obstet Gynecol. 1995;6(3):168–74.

15. Ersdal HL, Mduma E, Svensen E, Perlman J. Birth asphyxia: a major cause of early neonatal mortality in a Tanzanian rural hospital. Pediatrics. 2012;129(5):e1238–43.



The effects of intra-articular tranexamic acid given intraoperatively and intravenous tranexamic acid given preoperatively on post surgical bleeding and transfusion rate post total knee arthroplasty

Keywords: blood transfusion, intravenous, intra-articular, postoperative bleeding, total drain, total knee replacement, tranexamic acid

pISSN: 0853-1773 • eISSN: 2252-8083 • http://dx.doi.org/10.13181/mji.v25i4.1502 • Med J Indones. 2016;25:234–9• Received 18 Jul 2016 • Accepted 13 Nov 2016

Corresponding author: Aryo N. Triyudanto, [email protected]


Aryo N. Triyudanto, Andri M.T. LubisDepartment of Orthopaedy and Traumatology, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia

Clinical Research


ABSTRAK

Latar belakang: Terlepas dari kemajuan dalam berbagai bidang total knee replacement (TKR), besarnya jumlah perdarahan hingga kini masih merupakan masalah yang sulit diatasi. Penelitian ini bertujuan untuk menilai efektivitas pemanfaatan asam traneksamat pada TKR melalui berbagai metode administrasi.

Metode: Penelitian ini merupakan uji klinik berpembanding yang dilakukan pada 22 pasien di RS Cipto Mangunkusumo, Jakarta, pada Agustus 2014 hingga Februari 2016. Pasien menjalani TKR dan dibagi menjadi tiga kelompok yaitu kelompok kontrol, asam traneksamat intra-artikular intraoperatif, dan intravena preoperatif. Perdarahan intraoperatif, kadar hemoglobin (Hb), total produksi drain, jumlah transfusi total, dan hari pencabutan drain dicatat dan dibandingkan. Uji perbandingan numerik parametrik dan non-parametrik digunakan untuk perbandingan antargrup dengan sebelumnya dilakukan pengujian normalitas data.

Hasil: Jumlah transfusi pada kelompok intra-artikular (200±SB 100 ml) dan intravena (238±SB 53 mL) secara signifikan berbeda dengan kelompok kontrol (1.016±SB 308,2 mL) (p=0.001). Total produksi drain pada kelompok intra-artikuler (328±SB 193 mL) maupun intravena (391±SB185 mL) berbeda bermakna dengan kelompok kontrol (652±SB 150 mL) (p=0,003). Tidak terdapat perbedaan yang bermakna antara jumlah transfusi antara grup intravena dengan grup intra-artikuler. Tidak terdapat perbedaan yang bermakna antara kadar Hb baik preoperasi maupun pascaoperasi, jumlah perdarahan intraoperatif, maupun hari drain dicabut pada setiap kelompok.

Kesimpulan: Pemberian asam traneksamat baik intravena maupun intra-artikuler dapat mengurangi jumlah transfusi dan total produksi drain secara efektif pada pasien yang menjalani prosedur TKR.

ABSTRACT

Background: Despite the advances in the design and fixation of implants in total knee replacement (TKR). the amount of postoperative bleeding is still an important issue that has not been resolved. This study aimed to measure the effectiveness of various tranexamic acid administration.

Methods: This was a randomized controlled trial study, held from August 2014 to February 2016 at Cipto Mangunkusumo Hospital, Jakarta. Twenty two patients having TKR were divided into three groups: the control group, the tranexamic acid intra-articular-intraoperative group, and the intravenous preoperative group. Intraoperative bleeding, haemoglobin (Hb) level on preoperative to five-day-post-surgery, total drain production, total blood tranfusion needed and the drain removal timing were recorded and compared. Numerical data were analyzed by using parametric and non-parametric test, depended on the normality of the data.

Results: The amount of blood transfusion needed in both the intra-articular group (200±SD 100 mL) and the intravenous group (238±SD 53 mL) were significantly different compared to those in the control group (1,016±SD 308.2 mL) (p=0.001). Meanwhile, there was no significant difference between the amount of blood transfusion needed in the intra-articular group and the intravenous group. Total drain production in the intra-articular group (328±SD 193 mL) and intravenous group (391±SD 185 mL) was significantly different compared to the control group (652±SD 150 mL) (p=0.003). No significant difference between the levels of both preoperative and postoperative haemoglobin, the amount of intraoperative bleeding, and the duration of drain usage.

Conclusion: Intravenous and intra-articular tranexamic acid effectively decreased transfusion volume and drain production in patients undergoing TKR.


Triyudanto and LubisTranexamic acid in total knee arthroplasty

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Osteoarthritis (OA) is a degenerative joint disease that leads to disability with prevalence increasing with ages.1 Despite mechanical factor, systemic process within the body and abnormalities in biomechanic load make joints more vulnerable. About 15% of the world population has OA, and the number is predicted to increase twice in 2020.2

Severe pain caused by progressive destruction of cartilago, intermitten inflamation, and other pathological process will limit the joint movement. Some of non-operative therapy such as non-steroidal anti inflamatory drugs, glucosamine, condhroitin sulphate become non-invasive solution to relieve pain and modified the pathological process with various rate of success.

Total knee arthroplasty (TKA) is thought to be the choice of treatment in OA with Kelgren Lawrence III and IV classification. TKA is aimed to restore the joint function nearly to normal. Yet, this prcedure has blood lost risk estimated from 800–1,700 cc; thus it is estimated that 50% of post TKA patients need blood transfusion. However, blood transfusion is a procedure with diverse risk and adverse effects. Hepatitis B has a transmission risk one in every 350,000 blood unit. Occasionally, blood transfusions lead to transfusion reactions. The reactions may vary from mild to severe. Transfusion related acute lung injury (TRALI) is a serious reaction that occurs one in every 5,000 transfusion. Several attempts have been done to avoid complications post-transfusion. Moreover, some studies showed that fibrin topical and intravenous injection of tranexamic acid (TXA) could decrease the bleeding in TKA.3

Many studies have been done to reveal the effects of tranexamic acid application in bleeding post-TKA. Nevertheless, specific research that compares the effects of intraoperative intra-articular tranexamic acid administration and pre-operative intravenous administration of rivaroxaban anti coagulant as well as modifies Robert Jones bandage has never been done in Indonesia. Therefore, we did a research to compare the effects of intravenous administration and intra-articular intraoperative injection of TXA on patients that underwent TKR in terms of total bleeding and transfusion rate post-TKA.

METHODS

This is a randomized control trial study that was held from August 2014 to February 2016. Twenty two patients (of total 28 patients available) were included according to the inclusion criteria: aged 50–80 years old, visiting outpatient clinic Cipto Mangunkusumo Hospital, Jakarta, and had severe OA end-stage of Grade III and Grade IV Kelgren Lawrence criteria that underwent total knee arthroplasty; while the exclusion criteria were those who consumed anticoagulant and anti-thrombocyte aggregation, had preoperative Hb ≤10.5 g/dl for man and woman, had intraoperative blood loss ≥500 cc, with mental illness, had uncontrolled diabetes mellitus (DM), rheumatoid arthritis, malignancy, and immunosuppression, had infected knee, had abnormal prothrombin time (PT) and activated partial thromboplastin test (APTT). The patients were later divided into three groups of treatment: the control group (Group I), the intra-articular intra operative TXA (Group II), and the intravenous perioperative TXA (Group III). Each sample was allocated randomly to each group of treatment. For intravenous group, tranexamic acid was adminstered by injecting 500 mg of tranexamic acid 10 minutes before tourniquet was extended, while for the intra-articular group, tranexamic acid was given moments after prosthesis was implanted.

For each of the treatment group, a drain was installed into the articular cavity, ensuring routine measurement of drain production. Drain was clamped for six hours after surgery. In addition, a modified-Robert-Jones wrapping and daily rivaroxaban administration were conducted for all of the patients. Bleeding postoperative was considered as the main outcome that was measured by total drain production, haemoglobin rate, and number of transfusion that was needed for each group.

Analysis of data utilized Statistical Package for Social Science (SPSS) v.15 (Chicago, Illinois). Demographic analysis, normality of data, and bi-variate analysis were done on all of the data. The protocol of this study has been approved by Medical Ethics Committee of Faculty of Medicine, Universitas Indonesia (No. 34/UN2.F1/ETIK/2014) and patients were given verbal and



written explanation and asked for consent for each of the groups.

RESULTS

A total of 22 patients were included in the trial. Mean age of the subjects were 65.1 years old (SD±6.82). Most of the samples were female (n=17, 77.3%), and the right knee was the most operated location (n=12, 54.5%). For each of the treatment group, bivariate analysis was done in order to assess homogenicity with a result showing that there was no difference for each of the demographic characteristics.

n=22Age, year –mean (SD) 65.1 (6.82)Sex

Male 5 (22.7)Female 17 (77.3)

Affected sideRight 12 (54.5)Left 10 (45.5)

Pre-operation Hb, g/dL –mean (SD) 12.1 (1.13)Intraoperative bleeding, ml, –median (IQR)

200.0 (187.5–325.0)

Table 1. Demographic and clinical characteristics of patients with OA Grade III and IV who underwent Total Knee Arthro-plasty

Result is presented in mean (SD) for numeric and normal distribution or median (interquatile range) for numeric ab-normal distribution, and frequency (percentage) for propor-tional data

Control n=9

Group 2 n=7

Group 3 n=6 p

Preoperative Hb rate 12.0 (1.31) 12.3 (0.91) 11.9 (1.23) 0.805postoperative serial Hb rate (g/dL)

Day -1 11.3 (1.77) 10.9 (0.50) 10.6 (0.87) 0.597Day -2 9.5 (0.77) 9.7 (0.76) 9.4 (1.03) 0.711Day -3 9.7 (0.86) 10.6 (1.26) 9.1 (1.18) 0.075Day -4 9.7 (1.11) 10.2 (0.79) 10.3 (1.25) 0.497Day -5 10.6 (0.37) 10.7 (0.45) 11.0 (0.72) 0.313

Table 2. Rate of serial haemoglobin Postoperative based on groups, tranexamic acid intra-articular, intravenous and control

Hb rate is presented in mean (SD). *p value is calculated using parametric analitical varians (ANOVA) for mean comparison in three groups

Mean preoperative haemoglobin was 12.1 g/dl (SD±1.13) with median blood loss of 200 ml (range 187–325ml) (Table 1). Haemoglobin (Hb) rate was measured serially before and five days after TKA operation was done. Generally, in all groups, Hb rates tended to decrease in the first and the second day after operation. Hb rate then gradually increased from day three to day five as can be seen in Table 2. There was no signifcant difference (p=0.305) between the groups in Hb rate changes from time to time.

For drain production, the volume decreased for all groups every day during the three-day-follow up. There was a significant difference between each group from day to day and the total drain production. The highest drain volume was measured from the control group with the median of 620 (range: 585–775), while the least volume was measured from the second group with the median of 230 (range: 150–550) (Table 3).

The median of blood transfusion volume in all patients was 200 (300–1,000) ml. There was a significant difference (p=0,001) between the groups, with control group held the highest transfusion number. However, there was no significant difference between the amount of blood transfusion needed in the second group and the amount needed in the third group (Table 4).

Further analysis was done in between the main treatment groups in order to know the difference of outcome of the groups. Drain volume was compared between the intra-articular and

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Control n=9

Group 2 n=7

Group 3 n=6 p*

Postoperative drain volume (mL)Day 1 350 (310–350) 150 (150–300) 260 (120–300) 0.013Day 2 200 (150–250) 50 (30–100) 150 (112.5–200) 0.017Day 3 100 (75–100) 50 (0–50) 40 (0–50) 0.004

Total drain volume (mL) 620 (585–775) 230(150–550) 435 (307.5–512.5) 0.006

Table 3. Outcome comparison for each group treated with intra-articular and intravenous TXA, and the control group

*ANOVA was used for normally distributed data, otherwise, Kruskall-wallis was used. TXA= tranexamic acid

Control n=9

Group 2 n=7

Group 3 n=6 p

Blood transfusion volume (mL) 775 (1,000–1,250) 200 (200–300) 195 (225–300) 0.001

Table 4. Volume of blood transfusion based on interventions, intra-articular tranexamic acid, intravenous tranexamic acid and control

Presented in median (inter-quartile range) for numerical with abnormal distribution. *p value is measured with non-parametric Kruskall- Wallis for mean groups comparison.

intravenous group, with no significance result (p>0.05) as the difference. Transfusion volume was also compared between the intravenous and intra-articular group, with no significance result (p>0.05) as the difference.

DISCUSSION

OA is a disease which incidentally increased as people get old. A study revealed that 8.9% of adult population are having OA. It implies that the probability of someone having OA is increased as ages.4 This result is concomitant with this study in which the median of subjects’ age was 65.1 years old. Yet, from all the groups above, there was no significant difference in ages.

Another characteristic is female with OA are more in number compared to male with OA. Sharon et al5 showed that anatomic, genetic, and hormonal were assumed to affect this difference in gender.5 Linear to Sharon et al,5 Heidari6 stated that female had a bigger OA risk than males. Yet, in this study the p=0.304 which implied that there was no significant difference between sexes in the groups.

Tranexamic acid is one of the most common anti-bleeding used in clinical practice. However, in orthopedic field, the usage of the tranexamic acid is still debatable due to its benefit and

risk balance.7,8 Some research have suggested a beneficial usage for this medicine. Research that was done by Chen et al9 implied that intra-articular tranexamic acid could lessen blood loss in TKR without increasing the surgery duration significantly. This study was conducted to 50 patients who underwent TKR and were given 1,500 mg tranexamic acid. Intravenous tranexamic acid was proven to decrease drain after operation.9

We conducted three main outcome measurements for this study. Drain volume was measured serially from the first day to the third day after operation was done. Blood lost decreased gradually from day one to day four post-operation. There was a significant difference in day one drain volume (p=0.013), day two (p=0.017), and day three (p=0.004) post-operation. The control group had the biggest number of blood in drain volume. Drain volume was significantly different (p=0.006) between groups. Generally, bleeding volume is found in the control group. A study conducted by Zhu10 showed that patients with bilateral TKR significantly bled more than those who was given tranexamic acid intra-articular or intavenous. Moreover, Zhu10 also showed that the less drain volume existed, the less Hb decreased, and the less the need of transfusion in group with tranexamic acid compared to the control group.7

This result clearly implied the rule of tranexamic



acid as anti-bleeding. Fibrinolisis was avoided because tranexamic acid competitively blocked the lisin receptor in plasminogen.8

Haemoglobin was measured serially before and until five days after operation. According to Zhu10 Hb measurement was the best way to predict intraoperative blood loss, especially for blood loss that could not be measured by drain. Formby et al11 showed that Hb rate in group which was given tranexamic acid tended to be higher compared to group without tranexamic acid. In all group, there was a trend of Hb decrease up to two days postoperatively. Meanwhile in intravenous tranexamic acid group, it kept on decreasing until the third day. This phenomenon is linear with the study held by Sabatini et al12 where Hb decreased in the first three days after operation. Delta Hb per day was calculated by distracting the pre-operative Hb with the postoperative Hb. From this study, we know that the biggest delta Hb was in the intravenous tranexamic group in day two and day three and the control group in day two. Besides, there was no significant difference in delta Hb in each group. The importance of knowing preoperative Hb was explained by Basora et al.13 They concluded that low Hb preoperative was one of the predictor of red blood cell transfusion. Moreover, every 1 g/dL decreasing in Hb would increase the risk of transfusion 3.7 times.10

A good preoperative haemoglobin could predict the need for transfusion in operated patients generally. It was proposed that a low number of preoperative haemoglobin might increase the need for transfusion postoperative, necessitating a good preoperative blood preparation for patients in general. A lower preoperative haemoglobin level might be one of the reasons why the haemoglobin level for the intravenous group kept decreasing after the third day.10,14

Transfusion is the most important aspect in assesing preoperative bleeding. Administration of tranexamic acid intra-articular or intravenous had no significant difference in blood loss or transfusion rate.15-18 However, there was a significant difference between the group that was given tranexamic acid with the control group that was not given tranexamic acid in term of transfusion need. This result is linear with Sabatini et al12 whom stated that tranexamic acid had protective effect in the need of transfusion.

In conclusion, intra-articular and intravenous tranexamic acid group had better result in drain volume and transfusion volume needed in patient with grade III and IV OA who underwent TKA compared to the control group. Intra-articular or intravenous gave the same effects to patients with OA grade III and IV post-TKA. The control group had the biggest drain volume compared to other group and this result established the role of tranexamic acid in blocking bleeding in patients.

AcknowledgmentAuthors would like to give their best regards to the Department of Orthopaedy and Traumatology, Faculty of Medicine, Universitas Indonesia, and Cipto Mangunkusumo Hospital, for the support given in completion of this study.

Conflict of interestThe authors affirm no conflict of interests in this study

REFERENCES

1. Civinini R, Nistri L, Martini C, Redl B, Ristori G, Innocenti M. Growth factors in the treatment of early osteoarthritis. Clin Cases Miner Bone Metab. 2013;10(1):26–9.

2. Flanigan DC, Harris JD, Trinh TQ, Siston RA, Brophy RH. Prevalence of chondral defects in athletes’ knees: a systematic review. Med Sci Sports Exerc. 2010;42(10):1795–801.

3. Molloy DO, Archbold HA, Ogonda L, McConway J, Wilson RK, Beverland BE. Comparison of topical fibrin spray and tranexamic acid on blood loss after total knee replacement: a prospective randomized control trial. J Bone Joint Surg Br. 2007;89(3):306–9.

4. Turpie AG, Lassen MR, Davidson BL, Bauer KA, Gent M, Kwong LM, et al. Rivaroxaban versus enoxaparin for thrombophylaxis after total knee arthroplasty (RECORD4): a randomised trial. Lancet. 2009;373(9676):1673–80.

5. Hame SL, Alexander RA. Knee osteoarthritis in women. Curr Rev Musculoskelet Med. 2013;6(2):182–7

6. Heidari B. Knee osteoarthritis prevalence, risk factors, pathogenesis and features: Part I. Casp J Intern Med. 2011;2(2):205–12.

7. Karaaslan F, Karaoğlu S, Mermerkaya MU, Baktir A. Reducing blood loss in simultaneous bilateral total knee arthroplasty: combined intravenous-intra-articular tranexamic acid administration. A prospective randomized controlled trial. Knee. 2015;22(2):131–5.

8. Iwai T, Tsuji S, Tomita T, Sugamoto K, Hideki Y, Hamada M. Repeat-dose intravenous tranexamic acid further decreases blood loss in total knee arthroplasty. Int Orthop. 2013;37(3):441–5.

9. Chen JY, Rikhraj IS, Zhou Z, Tay DK, Chin PL, Chia SL, et al. Can tranexamic acid and hydrogen peroxide reduce

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blood loss in cemented total knee arthroplasty? Arch Orthop Trauma Surg. 2014;134(7):997–1002.

10. Zhu Y YM, Meng HY, Way AY, Guo QY, Wang Y, Peng J. Basic science and clinical application of platelet-rich plasma for cartilage defects and osteoarthritis: a review. Osteoarthritis and Cartilage. 2013:1–11

11. Formby P, Pickett AM, Van Blarcum GS, Mack AW, Newman MT. The use of intravenous tranexamic acid in patients undergoing total hip or knee arthroplasty: a retrospective analysis at a single military institution. Mil Med. 2015;180(10):1087–90.

12. Sabatini L, Atzori F, Revello S, Scotti L, Debiasi F, Massè A. Intravenous use of tranexamic acid reduces postoperative blood loss in total knee arthroplasty. Arch Orthop Trauma Surg. 2014;134(11):1609–14.

13. Basora M, Tió M, Martin M, Lozano L, Salazar F, Sánchez-Etayo G, et al. Should all patients be optimized to the same preoperative haemoglobin level to avoid transfusion in primary knee arthroplasty? Vox San. 2014;107(2):140–52.

14. Wong J, Abrishami A, El Beheiry H, Mahomed NN, Roderick Davey J, Gandhi R, et al. Topical application

of tranexamic acid reduces postoperative blood loss in total knee arthroplasty: a randomized, controlled trial. J Bone Joint Surg Am. 2010;92(15):2503–14.

15. Aguilera X, Martinez-Zapata MJ, Bosch A, Urrútia G, González JC, Jordan M, et al. Efficacy and safety of fibrin glue and tranexamic acid to prevent postoperative blood loss in total knee arthroplasty: a randomized controlled clinical trial. J Bone Joint Surg Am. 2013;95(22):2001–7.

16. Panteli M, Papakostidis C, Dahabreh Z, Giannoudis PV. Topical tranexamic acid in total knee replacement: a systematic review and meta-analysis. Knee. 2013;20(5):300–9.

17. Iwai T, Tsuji S, Tomita T, Sugamoto K, Hideki Y, Hamada M. Repeat-dose intravenous tranexamic acid further decreases blood loss in total knee arthroplasty. Int Orthop. 2013;37(3):441–5.

18. Wang H, Shen B, Zeng Y. Comparison of topical versus intravenous tranexamic acid in primary total knee arthroplasty: a meta-analysis of randomized controlled and prospective cohort trials. Knee. 2014;21(6):987–93.



Endoscopic incision of protruding right ureterocele in a single collecting system: a case report

Keywords: endoscopic incision, protruding ureterocele

pISSN: 0853-1773 • eISSN: 2252-8083 • http://dx.doi.org/10.13181/mji.v25i4.1352 • Med J Indones. 2016;25:240–4• Received 12 Jan 2016 • Accepted 01 Nov 2016

Corresponding author: Harrina E. Rahardjo, [email protected]


Rinto Hariwibowo, Harrina E. RahardjoDepartment of Urology, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia

Case Report


ABSTRAK

Ureterokel yang menonjol keluar merupakan kasus yang jarang ditemukan pada literatur. Kami melaporkan perempuan 21 tahun dengan keluhan massa yang menonjol keluar dari uretra, disertai disuria, hematuria, dan infeksi saluran kemih berulang. Pada inspeksi genitalia eksterna ditemukan massa menonjol keluar dari uretra yang dapat direduksi secara manual. Urografi ekskretorik menunjukkan single collecting system bilateral, hidronefrosis derajat II pada ginjal kanan, dan gambaran cobra head appearance pada pelvis sisi kanan bawah. Pasien didiagnosis dengan ureterokel kanan pada single collecting system, dan insisi ureterokel dilakukan secara endoskopik. Ultrasonografi yang dilakukan tiga minggu pasca prosedur tidak menunjukkan adanya hidronefrosis atau ureterokel residual. Voiding cystourethrography (VCUG) tiga bulan pasca-prosedur menunjukkan vesico-uretereal reflux (VUR) derajat 5 pada sisi kanan. Pasien lalu direncanakan untuk menjalani operasi reimplantasi ureter. Dapat disimpulkan bahwa insisi endoskopik untuk ureterokel cukup aman dan dapat memberikan hasil yang baik, namun harus diperhitungkan kemungkinan diperlukannya operasi sekunder pada beberapa kondisi.

ABSTRACT

Protruding ureterocele is a very rare case found in the literature. We are reporting a 21 year-old female with an intermittent protruding mass from urethra, accompanied by dysuria, hematuria, and recurrent urinary tract infection. Inspection of the external genitalia revealed a protruding mass from the urethra which could be reduced manually. Excretory urography showed bilateral single collecting systems, grade II hydronephrosis of the right kidney, and a cobra head appearance of the lower right pelvis. The patient was diagnosed with a protruding right ureterocele in a single collecting system, and thus, endoscopic incision of a ureterocele was performed. Ultrasonography which was carried out three weeks after the procedure confirmed no residual hydronephrosis or ureterocele in the bladder. Voiding cystourethrography (VCUG) underwent at a three-month-follow up revealed a grade 5 vesico-ureteral reflux (VUR) on the right side. Surgical reimplantation was then considered. In conclusion, endoscopic incision was safe and yielded good result for protruding ureteroceles, but the need for secondary surgery in several conditions should be considered.


Hariwibowo and Rahardjo.Protruding ureterocele in a single collecting system

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A ureterocele is a cystic dilatation of the distal ureter.1 It has been proposed that incomplete dissolution of the Chwelle’s membrane is the embryological basis for ureterocele formation.2,3

Ureteroceles can be further classified as intravesical or extravesical. Intravesical ureteroceles (orthotopic) are entirely located within the bladder and above the bladder neck, while extravesical ureteroceles (ectopic) are partially located at the bladder neck or urethra.1,2 Ureteroceles can also be presented with a double collecting system (duplex system ureterocele) or a single collecting system (single system ureterocele).1,3,4

The incidence of ureteroceles is around 1:4,000 children and is 4–7 times more frequently found in female.2,3 At the moment, there is no epidemiological data for ureteroceles in South East Asia or in adult population. Ureteroceles are mostly associated with upper pole moiety in double collecting systems (~80%), but a small percentage of ureteroceles (~20%) can be found in single systems.1,3,4

Ureteroceles may exhibit varying symptoms from urinary tract infection, obstruction to incontinence, and it may even protrude out of the urethra. Protruding ureteroceles have been reported, but are rarely found in the literature. Ureteroceles can be managed with several modalities, including a minimal invasive endoscopic incision. We are reporting a protruding orthotopic ureterocele in an adult female with a single collecting system, which was successfully treated with an endoscopic incision.

CASE REPORT

A 21 year-old female came to our hospital with a protruding mass from the urethra. She has experienced this condition since she was 12 years old. The patient also complained of dysuria, hematuria, and recurrent urinary tract infection. There was no history of trauma, flank pain, vaginal birth, or previous gynecological/urological surgery. On the physical examination, inspection of the external genitalia revealed a protruding mass from the external urethral orifice with a round shape, bright brown-red color, cystic consistency, smooth surface, and a size of 3x2x2 cm. It could be reduced manually back through

the urethra. Ultrasonography of the upper urinary tract showed a mild hydronephrosis of the right kidney. Excretory urography revealed bilateral single collecting systems, grade II hydronephrosis of the right kidney, and a cobra head appearance of the lower right pelvis. A urinary tract infection was evident from urine analysis. Other laboratory results were unremarkable.

The patient was then diagnosed with a protruding right ureterocele in a single collecting system. Therefore, an endoscopic incision of the ureterocele was performed. The patient was discharged one day after the procedure with continued oral antibiotic and analgesia. At a three-week-follow up, urinalysis of the patient who had dysuria revealed urinary tract infection which was then resolved by antibiotic. There was no residual hydronephrosis of the right kidney or ureterocele in the bladder on ultrasound examination performed three weeks after the procedure. Voiding cystourethrography (VCUG) was done at the third month and a grade 5 vesicoureteral reflux (VUR) on the right side was observed. The patient was scheduled for ureteral reimplantation. However, she decided to postpone the surgery due to the lack of complaints.

DISCUSSION

Patients with ureterocele may have varying presenting symptoms such as urinary tract infection, obstruction, or incontinence.2,6 There are several reported cases in the literature on protruding ureteroceles through the urethra, but they are very rare, as first reported by Sen et al.12 In children, the exact incidence of protruding ureteroceles is unknown, but it has been reported to be less than five percent.7 In adults, the exact number of incidence is also unknown, with no more than five cases reported in the last ten years. Such urethral prolapse may be caused by weakness of the urethral wall distal to the ureterocele.8 Our patient experienced a protruding mass from the urethra with a history of intermittent dysuria, hematuria, and recurrent urinary tract infection. The mass had a smooth round shape with bright brown-red color, and it could still be reduced back through the urethra, suggesting a diagnosis of a protruding ureterocele.9 The abscence of history of flank pain might indicate there was no severe urinary tract obstruction or upper urinary tract infection.



A diagnosis of a protruding ureterocele can further be confirmed by the presence of a ureterocele in bladder through radiologic imaging. Excretory urography and ultrasonography have been widely used to reveal ureteroceles.2,4 Excretory urography can identify the duplication of urinary tract, urinary tract obstruction, and ureterocele itself (as a cystic dilatation in the bladder).4 Voiding cystourethrography and renal scanning can also be performed to evaluate reflux and split renal function.2

Our patient’s excretory urogram showed a bilateral single collecting system and a cobra head appearance suggestive of a ureterocele on the right side (Figure 1). A hydronephrosis of the right kidney was also observed using ultrasonography, suggestive of an obstruction caused by the ureterocele. Based on physical

Figure 1. Excretory urogram revealing hydronephrosis of the right kidney and a “cobra head” appearance of the right vesical junction area

Figure 2. Endoscopic incisionn of the ureterocele

and radiologic examination, we confirmed the patient’s diagnosis of a protruding right ureterocele in a single collecting system. Single system ureteroceles in adults are very rare and usually associated with preserved ipsilateral renal function and minimal hydronephrosis. It may occasionally be complicated with calculus formation, which was not found in our patient.4,5

Management of ureteroceles should be oriented toward symptoms improvement and preservation of renal function.3,10,11 Several modalities include conservative approach, endoscopic decompression, ureteral reimplantation, partial nephroureterectomy, or complete primary reconstruction. The choice of management should depend on patient’s clinical status, age, renal function, presence of reflux or obstruction, presence of double or single collecting system, ectopic or orthotopic ureterocele, and the preferences of the patient and the surgeon.3,11

In our patient, endoscopic incision of the ureterocele was performed to decompress the ureterocele and to release the obstruction (Figure 2). In several studies, endoscopic incisions have been reported to yield good result in terms of safety and symptoms or ureterocele resolution.10,11 Three cases reported by Scovell et al,5 Sen et al,12 and Westesson13 also showed the success of endoscopic management of ureteroceles. In our case, at a three- week-follow up, there was no residual hydronephrosis or ureterocele found from ultrasonography, indicating resolution of the disease (Figure 3).

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Figure 4. Voiding cystourethrogram at a three-month-follow up revealed reflux of the right ureter

Endoscopic incision of ureteroceles might result in VUR in some patients, with varying reports of incidence (0–32%). Therefore, VCUG was performed three months after the endoscopic incision.10 Secondary surgery is necessary when the decompression is not effective, significant reflux is found, or when there is significant obstruction.3,11 In our patient, significant vesicoureteral reflux (grade 5) was found on the right side (Figure 4), indicating a need for a secondary surgery. Reimplantation of the ureter was then scheduled. We would expect a good prognosis for our patient because the procedure is generally safe and provides excellent success rates (92–98%) in such case.3

In conclusion, ureteroceles may exhibit varying symptoms and may even protrude out of the urethra. While several modalities up to open surgery can be selected, endoscopic approach can be used and has been reported to be safe and yield good results in the management of ureteroceles.

Conflict of interestThe authors confirm no conflict of interest in this study.

REFERENCES

1. Peters CA, Schlussel RN, Mendelsohn C. Ectopic ureter, ureterocele, and ureteral anomalies. In: Kavoussi LR, Novick AC, Partin AW, Peters CA, editors. Campbell-Walsh urology. 10th ed. Philadelphia: Elsevier Saunders; 2012. p. 3236–60.

2. Kogan BA. Disorders of the ureter & ureteropelvic junction. In: Tanagho EA, McAninch JW, editors. Smith’s general urology. 17th ed. New York: McGraw-Hill; 2008. p. 560–2.

3. Tekgül S, Dogan HS, Erdem E, Hoebeke P, Kočvara R, Nijman JM, et al. Guidelines on paediatric urology. Eur Assoc Urol. 2014. p.90–4.

4. Zerin JM, Baker DR, Casale JA. Single-system ureteroceles in infants and children: imaging features. Pediatr Radiol. 2000;30(3):139–46.

5. Scovell JM, Chan RC, Khavari R. Prolapse of a single system large ureterocele containing multiple stones in a pregnant woman. Urology. 2014;83(3):e3–4.

6. Muhammed A, Hussaini MY, Ahmad B, Hyacinth MN, Garba KD. Ureteroceles in adults: management of patients in Zaria, Nigeria. Arch Int Surg. 2012;2(1):24–8.

7. Ilica AT, Kocaoğlu M, Bulakbaşi N, Sürer I, Tayfun C. Prolapsing ectopic ureterocele presenting as a vulval mass in a newborn girl. Diagn Interv Radiol. 2008;14(1):33–4.

8. Konami T, Wakabayashi Y, Takeuchi H, Tomoyoshi T. Female wide urethra masquerading as a urethral diverticulum in association with ectopic ureterocele. Hinyokika Kiyo. 1988;34(8):1437–41.

9. Waihenya CG. Prolapsed single system ureterocele in a female adult: case report. East Afr Med J. 2008;85(4):197–200.

10. Vijay MK, Vijay P, Dutta A, Gupta A, Tiwari P, Kumar S, et al. The safety and efficacy of endoscopic incision

Figure 3. Ultrasonography at a three-week-follow up



of orthotopic ureterocele in adult. Saudi J Kidney Dis Transpl. 2011;22(6):1169–74.

11. Timberlake MD, Corbett ST. Minimally invasive techniques for management of the ureterocele and ectopic ureter: upper tract versus lower tract approach. Urol Clin N Am. 2015;42(1):61–76.

12. Sen I, Onaran M, Tokgoz H, Tan MO, Biri H, Bozkirli I. Prolapse of a simple ureterocele presenting as a vulval mass in a woman. Int J Urol. 2006;13(4):447–8.

13. Westesson KE, Goldman HB. Prolapse of a single-system ureterocele causing urinary retention in an adult woman. Int Urogynecol J. 2013;24(10):1761–3.

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Wibisono and Rahardjo.Percutaneous tibial nerve stimulation for OAB

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Management of overactive bladder review: the role of percutaneous tibial nerve stimulation

Keywords: electrical stimulation, overactive bladder, percutaneous tibial nerve stimulation

pISSN: 0853-1773 • eISSN: 2252-8083 • http://dx.doi.org/10.13181/mji.v25i4.1385 • Med J Indones. 2016;25:245–54• Received 14 Feb 2016 • Accepted 24 Sep 2016

Corresponding author: Harrina E. Rahardjo, [email protected]


Elita Wibisono, Harrina E. RahardjoDepartment of Urology, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia

Review Article


ABSTRAK

Overactive bladder (OAB) merupakan suatu kondisi yang sering terjadi dan diperkirakan sekitar 455 juta orang (11% penduduk dunia) pernah mengalami gejala tersebut. Kondisi ini dapat memberikan dampak yang signifikan bagi kualitas hidup pasien. Tatalaksana lini pertama OAB adalah terapi konservatif dan medikamentosa dengan obat antimuskarinik. Bagi pasien yang resisten terhadap pilihan terapi tersebut, terdapat beberapa alternatif tatalaksana, antara lain operasi, stimulasi elektrik, dan injeksi toksin botulinum. Dari antara pilihan tersebut, percutaneous tibial nerve stimulation (PTNS) merupakan pilihan yang invasif minimal. PTNS bekerja dengan menstimulasi pleksus saraf sakral, sekelompok saraf yang berperan dalam regulasi fungsi kandung kemih. Setelah mendapat sertifikasi food and drug administration (FDA) pada tahun 2007, PTNS semakin banyak digunakan dengan hasil menjanjikan. Pada tinjauan pustaka ini disajikan berbagai studi nonkomparatif dan komparatif yang membandingkan PTNS dengan prosedur sham, terapi antimuskarinik, dan terapi kombinasi yang menggabungkan PTNS dan antimuskarinik dengan data yang mendukung penggunaan PTNS pada OAB.

ABSTRACT

Overactive bladder (OAB) is a common condition that is experienced by around 455 million people (11% of the world population) and associated with significant impact in patients’ quality of life. The first line treatments of OAB are conservative treatment and anti-muscarinic medication. For the refractory OAB patients, the treatment options available are surgical therapy, electrical stimulation, and botulinum toxin injection. Among them, percutaneous tibial nerve stimulation (PTNS) is a minimally invasive option that aims to stimulate sacral nerve plexus, a group of nerve that is responsible for regulation of bladder function. After its approval by food and drug administration (FDA) in 2007, PTNS revealed considerable promise in OAB management. In this review, several non-comparative and comparative studies comparing PTNS with sham procedure, anti-muscarinic therapy, and multimodal therapy combining PTNS and anti-muscarinic had supportive data to this consideration.



Overactive bladder (OAB) is a common disorder and pervasive problem throughout all aspects of life that causes considerable effects on patients’ quality of life.1 This term specifically refers to a type of voiding dysfunction that consists of urgency, with or without urgency incontinence, without other pathology. Usually, it is accompanied with frequency and nocturia.2

It is estimated that there are around 455 million people (11% of the world population) having experienced OAB symptoms during their life. The reported OAB prevalence is dominated by females varying from 7.7% to 31.3% compared with males from 10.2% to 17.4%.2–4 In Indonesia, the prevalence of OAB is 4.1% and 1.6% for wet and dry OAB, respectively. In pediatric population, the majority urinary incontinence (UI) is wet OAB (2.1%), similar to in adult and geriatric population (4.6%).5

As previously stated, OAB can give significant impact to quality of life, involving physical, sexual function, psychological and social relation.1,6 Sumardi et al5 found that UI, including OAB, impaired family life (25.3%), school/job performance (23.7%), and sexual relationship (13.6%). Therefore, the proper choice of treatment is necessary to overcome OAB problem. First line treatments of OAB are conservative treatment combined with anti-muscarinic medication. For those resistant to the first line option, the treatment options available are surgical therapy, electrical stimulation and botulinum toxin injection.7

EtiologyGenerally, the etiology of OAB is unknown, but it can be related to neurologic problems, as they may affect bladder innervation. The most common neurologic diseases mentioned are multiple sclerosis and Parkinson’s disease. Increased lower urinary tract symptoms (LUTS) is also related to aging with decrease on brain function and inhibitory innervation. There is an age-related decrease of neuronal acetylcholine release and increase of stretch related acetylcholine from urothelium non-neuronal cells that may cause detrusor contraction. Another factor that is important to be considered in OAB is bladder outlet obstruction (BOO) with its impact in detrusor overactivity. The mechanism is hypersensitivity to acetylcholine and denervation of cholinergic

detrusor. Moreover, there are several effects caused by prolonged BOO such as nerve growth factor production increase, neuronal enlargement, and spinal micturition reflex enhancement.7,8 A study by Steers9 showed that there were increases of bladder’s nerve growth factor and alteration of neuron excitability related to increased nerve growth factor access, in patients with OAB and benign prostatic hyperplasia.

Neurophysiology of bladder functionThe regulation of bladder is controlled by sacral nerve plexus, located at the base of the spine.6 In order to maintain continence, the normal detrusor function is regulated in sacral balance under the control of sympathetic and parasympathetic nerves. Usually, the sympathetic tone plays a role in storage of urine, while the parasympathetic nerve controls the emptying of the bladder.10 The innervation of the bladder can be seen in Figure 1.

The detrusor muscle contraction in sacral micturition reflex pathway is started by excitation of bladder afferent and followed by bladder efferent. The balance of voiding control is influenced by pontine micturition center. Therefore, involuntary voiding can occur as a result of any signaling problem in central inhibition or sensitization of bladder afferent.7,10

In bladder storage process, there are two reflexes: guarding reflex and bladder afferent loop reflex. The guarding reflex prevents urine loss from any stress that turned off by supra-pontine input to control bladder emptying. The bladder afferent reflex, responsible for promoting continence during

Figure 1. Nerve pathway of bladder function

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bladder filling, is mediated by pudendal nerve efferent pathways, activated by sacral interneuron, that directly control urethral sphincter.

In bladder emptying process, stimulation of bladder afferent nerve fibers (Aδ or C fibers) comes from any stimulus, like pain, pressure, stretch, and fullness. Then, these fibers connect with parasympathetic efferent regulating bladder-bladder reflex and parasympathetic efferent regulating bladder-urethral reflex. As a result, bladder-urethral reflex is inhibited and bladder-bladder reflex is permitted leading to voiding.10

It is presumed that in the spinal cord, there are reflexes induced by nerve stimulation. Based on the theory, pelvic muscle contractions or detrusor contractions can be influenced by neurostimulation. Sacral nerve stimulation (SNS) is a method using implantable nerve stimulators which is considered invasive. Recently, there are many studies referring to percutaneous tibial nerve stimulation (PTNS) with its less invasive approach. It will be offered when other conservative measures fail.6

Pathophysiology of OAB In micturition, there are involvement of the central nervous system, the peripheral somatic, autonomic, sensory afferent innervation, and anatomical components of bladder and urethra. Therefore, any malfunction of these components can induce OAB symptoms.8 The typical objective

Figure 2. The guarding reflex promotes continence and al-lows the outlet to contract the urinary sphincter during peri-ods of stress. The brain can turn this reflex off during voiding

urodynamic finding in OAB is detrusor over-activity (DO). During filling phase, common signs of this unstable bladders are sudden increase of pressure in bladder at low volume and myogenic activity, as well as changes of smooth muscle structure and stimuli responsiveness.7,11

Basically, there are three hypotheses that can explain the above findings; neurogenic, myogenic and afferent hypothesis. In the neurogenic hypothesis, the nerve-mediated detrusor muscle excitation induces DO. This excitation may be due to any disturbance in cerebral inhibitory centers, cerebral infarction inducing alteration in several neurotransmitters that are important in micturition, and activation of spinal cord capsaicin-sensitive c-fiber-mediated micturition reflex in spinal cord damage above sacral level. In myogenic hypothesis, DO results from a combination of changes in structure and function of bladder detrusor smooth muscle. Therefore, there will be an increase of muscle excitability and enhancement of muscle connectivity generating uninhibited contraction. In afferent mechanism, there are numerous abnormal sensitized afferent nerve endings leading to inappropriately high sensory activity for any distention level of the bladder. In conclusion, the key points in OAB are the degree of excitation and generation in myovesical plexus structures in the bladder wall.7–9

ManagementAccording to The Fourth International Consultation of Incontinence (ICI) recommendations, first line treatment of OAB should be a combination of conservative treatment and pharmacological therapy. In case this method is not successful, it is necessary to perform a more specialized treatment.12

There is a wide range of OAB treatments. To simplify, the management of OAB can be divided into four classes: conservative treatment, pharmacotherapy, surgical therapy, and additional therapy for intractable OAB.7 First option of OAB treatment is conservative or standard treatment including lifestyle modification, pelvic floor muscle exercise and bladder training.6 For lifestyle interventions, patients should pay attention to their dietary irritants (caffeine, alcohol), fluid intake and smoking habit. Exercise of pelvic floor muscle and bladder training will assist patient to resist and terminate overactivity, as well as improving



inhibitory mechanism on lower urinary tract.7 The second class treatment is pharmacotherapy. The primary mechanism of action of anti-muscarinic is to decrease urgency and improve bladder capacity. Systematic review and meta-analysis by Chapple et al13 has shown that these medications were better than placebo significantly in incontinence and voiding symptoms. Unfortunately, despite its efficacy and effectiveness, approximately 80% of OAB patients stop antimuscarinic after a year, 17% of them is related to adverse side effects.6,14 The most common adverse events reported are dry mouth and constipation, followed by drowsiness, blurred vision, and declined cognitive function. Patients with such condition have the third line treatment options such as bladder augmentation, detrusor myomectomy, or less invasive methods of treatment like botulinum toxin injection to detrusor muscle and electrical stimulation (neuromodulation and neurostimulation).6,15,16 For intractable OAB, there is a place for appliances, catheters, urethral closure, and urinary diversion.7

Most OAB patients can take advantages from multimodal therapy consisting of medical therapy, bladder training and pelvic floor muscle training (PFMT). When symptoms do not disappear at the maximal dosage of pharmacotherapy, called refractory OAB, the remaining options are neuromodulation or surgery.7

Electrical stimulationIn 1878, the early finding of direct bladder stimulation via a metal transurethral catheter in urinary retention patients increased development of bladder direct stimulation through detrusor and transurethral routes. On the other hand, the direct

stimulation of pelvic nerve in 1965 seemed to be unsuitable for treatment of bladder dysfunction. It was due to increase of outlet resistance through stimulation of hypogastric nerve. Therefore, stimulation methods were more focused on the spinal cord, sacral roots, or pelvic floor muscles.10

In 1990, the studies showed that stimulation of sacral anterior root accompanied with posterior sacral 2 (S2) to sacral 4 (S4) rhizotomies was necessary for optimal bladder emptying as it would increase bladder compliance and decrease detrusor activity.

Then, the concept of neuromodulation was defined as generation of external sphincter function by sacral roots activation that decrease normal inhibitory reflex of detrusor. These findings made the basic knowledge of neurostimulation and neuromodulation.10

Neurostimulation is the electrical stimulation applied on nerves and muscles to get rapid clinical changes in neurogenic conditions, while neuromodulation is the electrical stimulation of nerves to make neurotransmission process alteration, both in neurogenic and non-neurogenic condition.10

In order to find methods of restoring complete voluntary control over storage and emptying function of the bladder, many researches have been performed. There are several ways of electrical stimulation which are determined by the purpose or selection of the desired bladder function and sites of stimulation with the related mechanism (Table 1).17

Purpose Sites of stimulation MechanismFacilitate filling-storageInhibit detrusor contractilityIncrease bladder capacityDecrease urgency and frequency

vagal, anal, suprapubic, posterior tibial, common peroneal, sacral roots, intravesical

Neuromodulation

Decrease nociception vaginal, anal, suprapubic, sacral roots NeuromodulationIncrease outlet resistance vaginal, anal, sacral roots Direct stimulation (efferent nerves or roots)Facilitate emptyingStimulate detrusor contraction (spinal cord-injured patient)

sacral anterior (ventral) roots Direct stimulation (efferent nerves or roots)

Restore micturition reflex (idiopathic retention)

sacral roots, intravesical Neuromodulation

Table 1. Choices of electrical stimulation in voiding dysfunction treatment10

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In patients with detrusor hyperreflexia, neurostimulation of sacral roots showed effective hyperactivity suppression. This suppression of detrusor contractility is a result of the sacral plexus somatic component stimulation by reflex response of detrusor muscle.10

Sacral nerve stimulation (SNS) has been proved to be effective in the treatment OAB but is considered invasive and related to complications such as infection, pain, and need of surgical revision because of lead migration in 33% of cases. In SNS procedure, implanted device is used to stimulate S3 and nearby nerve roots.18 Other stimulation called transcutaneous electrical nerve stimulation (TENS) has been explained using electrodes in supra-pubic or sacral attachment. In spite of its high continence rates (54%), the long therapy is associated with skin irritation.19 Gungor Ugurlucan et al20 assessed the comparison of percutaneous tibial nerve stimulation (PTNS) and transvaginal electrical stimulation (ES) in OAB treatment. The result showed that both ES and PTNS were effective in OAB treatment with significant improvement in subjective and objective parameters, but without significant difference between the two groups.20 Thus, PTNS is considered more effective than TENS and suggested as a cheaper, safer and less invasive alternative to SNS and ES.21

Percutaneous tibial nerve stimulation (PTNS)DefinitionPTNS is the least invasive form of neuromodulation that percutaneously stimulate posterior tibial nerve and deliver electrical stimulation to sacral nerve plexus. This plexus consists of sensory and motor nerve fibers originating from lumbal 4 (L4) through sacral 3 (S3) which control the function of bladder, urinary sphincter, and pelvic floor muscles, both through the somatic and autonomic nerves. The system may have an effect both on the detrusor and micturition centers in the brain.10,22 PTNS pathway to sacral plexus is represented in Figure 3.

PTNS (Urgent PC, Anoka, Mn, CystoMedix) is approved by U.S. FDA. It was first improved and promoted by Stoller.1 The use of PTNS for refractory OAB is recommended by European Association of Urology (EAU), American Urological Association (AUA), and National Institute for Health and Clinical Excellence (NICE).23–25

Figure 3. PTNS pathway to sacral plexus

ProcedureThere is no special preparation for doing PTNS. The patients are asked to sit comfortably in a chair with a leg elevated. A small, slim needle, specifically 34-gauge needle, is inserted percutaneously near the ankle, just posterior to tibia margin and 5 cm cephalad from the medial malleolus. On the medial surface of the calcaneus, the grounding electrode pad is placed. An adhesive pad will be applied to the foot to complete the circuit. It is important to insert the needle at the right location. These settings are illustrated in Figures 4 and 5.

The component of PTNS is connected to a battery-powered stimulator and then stimulator is turned on. It is necessary to adjust the strength of stimulation. First, the sensation will emerge in the ankle, foot, toes and spread out. The patient’s



response should be observed to get the ideal strength of impulse needed. Then, stimulator’s impulses are delivered to tibial nerve, transferred to sacral nerve plexus and thereby stimulate the inhibition of detrusor contractility.10

Commonly, PTNS cycles consist of 12-weekly treatment of 30 minutes with parameters evaluated are nocturia and urge incontinence after four to six treatment. At least six treatments of PTNS are needed for the patients to get a change of symptoms. Patients who successfully respond to PTNS treatment need retreatment to get sustainable results.10,24,26

ContraindicationThe contraindications of PTNS are patients with pacemaker, nerve damage, coagulopathy that favors bleeding, and pregnancy.

EfficacyThe early multicenter trial by Govier et al21 found that from 53 patients with a various lower urinary tract diseases, there were 71% response after 12 weekly PTNS treatments. The responders with positive response of PTNS were also shown in several studies from 2005 to 2006, ranging from 37.3% to 81.8%.27–31 The terms of responders were more than 50% reduction in symptoms including urgency, frequency, incontinence episodes,27,29,30 complete recovery after treatment,28 overall treatment response rate31 and subjective feeling of improvement.27 In an RCT by Finazzi Agrò et al32 comparing PTNS and sham procedure, there were 75% and 0% patients with 50% reduction

Figure 4. Setting of PTNS procedure

Figure 5. Needle electrode placement

in urinary incontinence episodes in PTNS and sham group, respectively.

After its approval by FDA in 2007, PTNS showed considerable promise in the management of OAB. There were several non-comparative and comparative studies comparing PTNS with sham procedure, anti-muscarinic therapy, and multimodal therapy consisting of PTNS and anti-muscarinic for the treatment of OAB.

In non-comparative studies, Burton et al19 found in their meta-analysis that the PTNS pooled objective success rate was 60.6% (95% CI=49.2–74.7) and subjective success rate was 61.4% (95% CI=57.5–71.8).

Efficacy of PTNS in OAB has also been proven in sham-controlled trials. When compared to sham procedure, the number or percentage of responders in PTNS group were statistically higher (p<0.01), not only in objective responses32-34 but also in global response assessment (GRA) improvement or cure.35 The significant reduction of symptoms in PTNS group compared to sham group were found in frequency, urinary incontinence, and nocturia episodes.32,33,35 In the matter of voided volume, two studies comparing PTNS and sham procedure by Agro et al33 and Agro et al35 showed significant increase of voided volume in PTNS group (p≤0.001) but not

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significant in sham group. The efficacy of PTNS in OAB is supported in meta-analysis by Burton et al19 which concluded that patients who received PTNS treatment have seven times possibility to get improvement compared to sham treatment (RR=7.02 [95% CI=1.69–29.17]). The same result was shown in another meta-analysis by Wibisono36 with overall risk ratio of 7.32 (95% CI=1.69–32.16) favouring PTNS over sham procedure and with number needed to treat 4.28 (95% CI=3.19–6.49).

In studies comparing PTNS and anti-muscarinic, there were variable results in changes of voiding diaries symptoms. Most of them showed significant reduction of symptoms in both groups with no significant difference.37–39 Peters et al38, with study subjects of 100 OAB patients, compared PTNS with tolterodine 4 mg/day with similar results in voiding diaries symptom reduction. However, symptoms improvement was reported by more subjects treated with PTNS (79.5%) than tolterodine (54.8%) (p=0.010).

In comparison of PTNS and multimodal therapy, there was significant difference of symptoms episodes favouring multimodal therapy in a study by Sancaktar et al40 and significantly higher response rate of multimodal therapy group compared to PTNS reported by Karademir et al.31

In the treatment of neurogenic OAB specifically, PTNS showed similar results. Kabay et al41 evaluated 32 patients with Parkinson’s disease accompanied with neurogenic detrusor DO. They found significant improvement of urodynamic parameters in patients treated with PTNS including mean first involuntary detrusor (first IDCV) contraction and mean maximum cystometric capacity (MCC) after PTNS.41 It was supported by the study of PTNS in multiple sclerosis patients with OAB symptoms where there were significant reduction of daytime frequency, nocturia, and mean post-micturition residual, as well as increase of mean voided volume. Approximately, 89% patients had a treatment satisfaction of 70%.42

In children, PTNS is also proved feasible, safe and minimally painful. De Gennaro et al43 reported that 80% of children with OAB had improved their symptoms which were 44% totally dry. Children had significant improvement in bladder capacity and urinary retention symptoms as much as

62.5% and 71%, respectively. The faces rating scale results introduce no severe pain face with concurrent visual analog scale results showing a low score with a further decrease during the first, sixth, and twelfth sessions (p<0.05).43 In another study, Hoebeke et al44 prospectively demonstrated similar success with 32 therapy-resistant children, non-neurogenic bladder sphincter dysfunction. Out of the 28 children with pretreatment urgency, seven children experienced disappearance of their urgency and 10 children had improvement. After stimulation, four out of 23 children with daytime incontinence became dry, 12 of them had their incontinence decreased. A daily normal one of four to six voids achieved in 16 out of 19 children with abnormal voiding frequency.10,44

Long-term efficacyFrom the information above, the short-term therapy of PTNS should be beneficial in the treatment of OAB. However, symptoms deteriorate within 6–12 weeks without ongoing treatment. Thus, it will be necessary to explore more information on long-term therapy.19 Macdiarmid et al45 evaluated the durability in long-term of PTNS in OAB by the continuation of the second phase of the overactive bladder innovative therapy trial (OrBIT) where 33 responders of the PTNS group received an additional nine months of PTNS treatment. There was significant (statistically) improvement of OAB symptoms with 12 weekly PTNS that demonstrated good durability of 12 months through. This conclusion was obtained from 12 months mean improvements from the baseline in frequency, urge incontinence, nocturia, and voided.45 In addition, there was sustained therapeutic effects of PTNS (STEP) study which was the continuation from Study of Urgent PC versus Sham Effectiveness in Treatment of Overactive Bladder Symptoms (SuMIT) evaluating long-term efficacy of PTNS. After successful 12 weekly treatments, patients continued with 14-week tapering protocol and personalized treatment plan. Frequency, urge incontinence, nocturia, and urgency episodes improvements were statistically significant compared to baseline at six, 12, 18, and, 24 months.46 At three years, they found 77% (95% CI=64%–90%) of subjects with maintained or marked OAB improvements.47 All in all, PTNS is durable and can be a long-term treatment option for OAB.



Adverse eventsThe side effects of PTNS are not frequent and considered minimal. They include minor irritation (redness or inflammation), ankle bruising, discomfort or pain near the stimulation site or around ankle, tingling in leg, generalized swelling, toe numbness, stomachache, headache, hematuria, bleeding where needle has been inserted, and inability to tolerate stimulation.19,29,35,38 They were found in a few number of patients and considered rare. In long-term PTNS therapy (STEP study), the events reported were slow stream, UTI, bladder pressure, pulling feeling on feet, and pinched nerve, with no direct relationship to PTNS.46 In patients receiving anti-muscarinic, the common adverse events were constipation, infection, dizziness, visual disturbance, and fatigue.38

In a study comparing PTNS and anti-muscarinic, a well toleration with negligible serious adverse events reported in both treatments. It was stated that after 12 weeks of therapy, several symptoms were reported significantly less in the PTNS group compared to the anti-muscarinic group, including dry mouth and constipation.38 A study of Peter et al35 showed no serious adverse events reported in sham group and PTNS group.

In conclusion, many of millions people suffering from OAB do not respond well to the medical and behavioral therapy. PTNS offers these refractory patients a safe, effective and minimally invasive treatment. AcknowledgmentsNo benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.


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39. Vecchioli-Scaldazza C, Morosetti C, Berouz A, Giannubilo W, Ferrara V. Solifenacin succinate versus percutaneous tibial nerve stimulation in women with overactive bladder syndrome: results of a randomized controlled crossover study. Gynecologic and obstetric investigation. 2012;75(4):230–4.

40. Sancaktar M, Ceyhan ST, Akyol I, Muhcu M, Alanbay I, Mutlu Ercan C, et al. The outcome of adding peripheral neuromodulation (stoller afferent neuro-stimulation) to anti-muscarinic therapy in women with severe overactive bladder. Gynecological Endocrinology. 2010;26(10):729–32.

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Lestari and Rizki.Epigenetic as an update approach of infertility

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Epigenetic: A new approach to etiology of infertility

Keywords: epigenetic alteration, epigenetic modification, female infertility, male infertility

pISSN: 0853-1773 • eISSN: 2252-8083 • http://dx.doi.org/10.13181/mji.v25i4.1504 • Med J Indones. 2016;25:255–62• Received 20 Jul 2016 • Accepted 19 Dec 2016

Corresponding author: Silvia W. Lestari, [email protected]


Silvia W. Lestari,1 Meidika D. Rizki21 Department of Medical Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia2 Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Review Article


ABSTRAK

Infertilitas merupakan penyakit yang kompleks dan dapat disebabkan oleh faktor laki-laki dan perempuan. Etiologi dari kedua faktor infertilitas tersebut perlu ditelusuri lebih lanjut. Terdapat beberapa pendekatan untuk memahami infertilitas, salah satunya adalah epigenetik. Modifikasi epigenetik terdiri dari metilasi DNA, modifikasi histon, dan pembentukan kromatin. Sel germinal laki-laki dan perempuan mengalami modifikasi epigenetik secara dinamis dan membentuk sel sperma dan oosit yang matang. Pada laki-laki, perubahan metilasi DNA pada spermatogenesis dapat menyebabkan kelainan berupa oligo/astenozoospermia. Selain itu, metilasi dan asetilasi histon serta modifikasi histon lainnya dapat menyebabkan sperma tidak mampu untuk membuahi oosit. Serupa dengan laki-laki, pada perempuan pun dapat terjadi perubahan metilasi DNA dan modifikasi histon yang dapat mempengaruhi oogenesis, menciptakan anueploidi pada oosit yang terbuahi, dan mengakibatkan kematian janin di uterus. Perubahan pada pola modifikasi epigenetik dapat mengakibatkan infertilitas, baik pada laki-laki maupun perempuan.

ABSTRACT

Infertility is a complex disease which could be caused by male and female factors. The etiology from both factors needs further study. There are some approaches to understanding the etiology of infertility, one of them is epigenetic. Epigenetic modifications consist of DNA methylation, histone modifications, and chromatin remodelling. Male and female germinal cells undergo epigenetic modifications dynamically during differentiation into matured sperm and oocyte cells. In a male, the alteration of DNA methylation in spermatogenesis will cause oligo/asthenozoospermia. In addition, the histone methylation, acetylation, or other histone modification may lead sperm lose its ability to fertilize oocyte. Similarly, in a female, the alteration of DNA methylation and histone modification affects oogenesis, created aneuploidy in fertilized oocytes and resulted in embryonic death in the uterus. Alteration of these epigenetic modification patterns will cause infertility, both in male and female.



Infertility is defined as the inability to conceive after one year of regular unprotected sexual intercourse by a couple.1 Identifying factors involved in the etiology of infertility is a necessary. There are several approaches to understanding the etiology of infertility, one of which is epigenetics.

Epigenetics is a modified form of chromatin which may alter the structure of chromatin constituent. Changes in chromatin structure will affect the activation or repression of gene expression. Epigenetic changes affect gene expression or phenotype that can be inherited to the next generation without any modification of its deoxyribonucleic acid (DNA) sequences.

Epigenetics regulates a variety of important processes in the body. One of them is fertility which is associated with spermatogenesis or oogenesis. Spermatogenesis is the process of formation, development, and maturation of male germinal cells that occurs in the testis. Oogenesis is the process of formation, development, and oocyte maturation that occurs in the ovary. The newly formed sperms and oocytes undergo epigenetic modifications during the process of differentiation into matured sperms and oocytes. Epigenetics on spermatogenesis and oogenesis is a new issue and there are still many which have not been revealed in the research.

Alteration of these epigenetic patterns can cause infertility, both in male and female. This paper will discuss some of the epigenetic modifications that occur during the process of spermatogenesis and oogenesis as well as some alteration patterns of epigenetic modifications.

Overview of epigenetic mechanismEpigenetics is defined as the changes that affect gene expression or phenotype and can be inherited to the next generation through meiosis or mitosis without modification in its DNA sequence.2–4 Epigenetics occurs at the level of chromatin, the constituent subunits of nucleosome. The placement of DNA strands that wrapped around histone proteins in chromatin determines whether a gene is transcribed or not. Chromatin is functional in two forms, namely 1) the DNA strand which is compacted tightly during mitosis and meiosis and called heterochromatin. This section is not actively transcribed; and 2)

DNA strands that loosely bind to histones and called euchromatin. This section is actively transcribed.5,6

Related to this, epigenetic alteration plays an important role in determining which genes will be expressed in a specific cell. The mechanism of epigenetic modifications in determining the activation and inactivation of genes, occurs through DNA methylation, histone modifications, and chromatin remodeling as described in Figure 1.

DNA methylationDNA methylation occurs through the addition of a methyl group of S-adenosylmethionine (SAM) donor to fifth carbon position (C5) major groove of cytosine residues that are in the 5’-G-phosphate-C-3’ (CpG) dinucleotide. CpG dinucleotide consists of cytosine that binds to guanine through phosphodiester bonds and can be found as a group called the CpG island (CGI). CGI associated with gene promoter and regulatory regions in the transcription initiation.7,8

The mechanism of DNA methylation, conducted by the addition of a methyl group to DNA, was catalyzed by the DNA methyltransferase (DNMT) enzymes, whereas DNA demethylation was catalyzed by the DNA demethylation enzymes. In mammals, there are three kinds of DNMT, namely i) DNMT1 which plays role in maintaining methylation, ii) DNMT3a and iii) DNMT3b which is de novo methyltranferases that methylates the genomic DNA during early embryonic development.9 The DNA methylation regulates gene expression by inhibiting the interaction of

Figure 1. Epigenetic mechanism namely 1) DNA methyla-tion; 2) histone modification; 3) chromatin remodelling

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transcription factors to DNA by compressing the structure of chromatin so transcription factors do not have access to DNA.5,6,10

Histone modificationHistone modification involves nucleosomal histone that has a function in DNA packaging. The visible strands of DNA are wrapped around octamer on two subunits per each core histone (H2A, H2B, H3 and H4) and are connected by connecting histones (H1) to form nucleosomes which become the basic subunit of chromatin. The N-terminal tail of histone consists of amino acid residues which can be affected by methylation, acetylation, phosphorylation, ubiquitylation, and sumoylation.11

Histone methylation is the addition of methyl groups on lysine (K) and arginine (R) residues which is regulated by histone methyltransferase (HMTase). In general, histone methylation is associated with gene silencing, depends on the position of the methylated residue. For example, the lysine 9 on H3 (H3K9) which is methylated, will suppress the gene expression.5,7 On the other hand, the methylation of lysine 4 residue in H3 (H3K4) leads to gene expression.12

Histone acetylation is the transfer of an acetyl group from acetyl-CoA to the cluster ε-NH2 to lysine N-terminal histone residues, catalyzed by the histone acetyltransferase (HAT) enzyme. It activates gene expression and histone deacetylase (HDAC) enzyme that play a role in the deacetylation of histones which will inhibit the gene expression.5,6,11,13,14

Histone phosphorylation is the addition of a phosphate group to the histone proteins. Phosphorylation occurs on serine residues and generally leads to gene activation.15 Histone ubiquitylation the addition of ubiquitin group on the histone proteins. Ubiquitin is a polypeptide consisting of 76 amino acids which can be attached enzymatically to all kinds of proteins. Sumoylation is one of histone modifications with the attachment of small ubiquitin-related modifier protein (SUMO).11 The histone modification by sumoylation may cause gene expression and inhibits other types of histone modification.16

Chromatin remodellingThe third epigenetic mechanism is chromatin remodelling that controls the position of

nucleosomes. Chromatin remodelling is regulated by ATP-dependent chromatin remodeling complex, which mediates the access of transcription factors to DNA chromosome as its main function, so it can control gene expression, DNA replication, and DNA repair.5,17

ATP-dependent chromatin remodelling complex requires the energy from ATP hydrolysis to change the position and the structure of the nucleosome, causing the windings of DNA on histones of octamer loosened. As a result, the gene becomes accessible to transcription factors that lead to gene expression or silencing.17

Chromatin remodelling is catalyzed by an ATPase enzyme which is Sucrose Non-Fermentable 2 (SNF2) family. Some members of the SNF2 family like Sth1 subunit of Remodel the Structure of Chromatin (RSC) complex in yeast, Brahma in Drosophila, and Brahma-related gene (BRG)-1 and hBRMin humans also have bromodomain, which can be affected by histone acetylation to encourage chromatin remodeling.17–19

Epigenetic in male infertility It is estimated that the male factors that cause infertility is about 30%.1 The male factors in infertility could be the abnormality of sperm count (oligozoospermia), the sperm motility (asthenozoospermia), and the sperm morphology (teratozoospermia) or combination, as a result of disruption in spermatogenesis. Epigenetics becomes one of the important roles in the regulation of male fertility, starts from spermatogenesis until embryo development.

DNA methylation and male infertilityDe novo DNA methylation occurs in the early stages of spermatogenesis. As explained above, DNA methyltransferase (DNMTs) enzymes regulate de novo DNA methylation, including during spermatogenesis stages. A study showed the highest level of DNMT3a and DNMT3b expression in spermatogonia A.20 The expression of DNMT1 exhibited a similar pattern to DNMT3a and DNMT3b expression.

The alteration of the DNA methylation level in the sperm affects male infertility. As in the study Schütte et al21 in men with oligozoospermia, there were several genes that changed in DNA methylation level leading to hypermethylation



or hypomethylation which might affect spermatogenesis.

In addition, the research of Montjean et al22 showed that global DNA methylation in sperm was associated with sperm concentration. Oligozoospermic men had global DNA methylation level which were significantly lower compared to normozoospermic men. Furthermore, the global DNA methylation level in sperm was also associated with sperm motility. Men with severe asthenozoospermia had a global DNA methylation level which was significantly lower compared to normal sperm motility.

Histone modification and male infertilityHistone modification that occurs in spermatogenesis could modify the accessibility of DNA to transcription factors. The histone methyltransferase (HMT) and histone demethylase (HDM) enzymes regulate the methylation patterns in histones. Methylation at H3K4 is generally associated with gene expression. In a study conducted by Godmann et al.23 showed the level of H3K4 methylation that had a peak in spermatogonia and diminished in spermatocytes stage. This modification may be required in the protein forming which needed to begin the development phase to become spermatocytes.

H3K9 methylation occurs during meiosis,24 and the process is continued to H3K9 demethylation at the end of meiosis. In his study, Okada et al25 did knockout on Jumonji C (JmjC) domain–containing histone demethylase 2A (jhdm2a) and found that jhdm2a was expressed at round spermatid stage. Furthermore, it was found that jhdm2a deficiency would cause spermatid failure to elongate because of the disruption of chromatin condensation, smaller size of testis, and infertility. Thus, it can be concluded that H3K9 demethylation has an essential role during spermiogenesis process.

During spermatogenesis, H4 hyperacetylation lysine residue that is regulated by the Histone Acetyltransferase (HAT) and Histone Deacetylase (HDAC) enzymes plays an important role in the transition of histone to protamine and allows nucleosomes to experience the demolition at spermatid elongation. The decreased level of H4 hyperacetylation was found in infertile men with impaired spermatogenesis.26 Thus, the ubiquitylation of histones H2A through RNF8-

dependent is also an important factor to regulate the demolition of nucleosomes in the post-meiotic stages and in the incorporation of transition protein into chromatin. The ubiquitylation of histone H2A with RNF8 will open conformation of chromatin to become less tight, so as to provide access for the exchange of histone proteins with a protein transition.27 Meanwhile, a study by Lu et al,28 indicated an association between the ubiquitylation of histones H2A and H2B by RNF8 in induces of histone acetylation of H4K16 that facilitated the transition of histone-protamine on spermatid elongation.

Deficiency of RNF8 can cause sperm to become immotile, decrease the number of sperm in the epididymis, make the head of the sperm less condensed, and cause the residual body unseparated completely from the sperm. Furthermore, the results of in vitro fertilization test conducted by Lu et al28 showed that abnormal sperm had no capability to fertilize the normal oocyte.

Sumoylation and male infertilityAnother form of epigenetic modifications that occurs during spermatogenesis is sumoylation. Some important proteins in spermatogenesis are known to be SUMO interaction target. Sumoylation has a role as a major factor in the regulation of transcription, response to stress, regulation of enzymatic main pathway, nuclear - cytoplasmic transport, cell cycle control, and other functions in spermatogenesis. Proteins involved in ubiquitylation, DNA repair, and chromatin remodeling experience sumoylation high levels in spermatocytes. In contrast, the cytoskeleton proteins is modified by SUMO in spermatid.29 One of the proteins involved in epigenetic modifications in spermatogenesis is SUMO-1. SUMO-1 expression in testis showed another specific function, namely to help the inactivation of sex chromosomes during meiosis. SUMO-1 is in the chromatin during the zigoten phase when paired homologous chromosomes occurs and during the initiation of sex chromosome condensation.30

The list of epigenetic alterations in male infertility is summarized in Table 1.

Epigenetic in female infertility Similar to the male factors, it is estimated that the female factors can cause infertility by about 25%.

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Study by Alteration and male infertility Reference

Schütte Hyper and hypomethylation affecting spermatogenesis

21

Montjean DNA methylation with oligo/asthenozoospermia

22

Sonnack V H4 hyperacetilation with impaired spermatogenesis

26

Okada jhm2da deficiency in spermatid fail to elongate

25

Table 1. List of epigenetic alteration in male infertility Table 2. List of epigenetic alteration in female infertility

DNA= deoxyribonucleic acidDNA= deoxyribonucleic acid; LHCGR= luteinizing hor-mone/choriogonadotropin receptor; PCOS=polycystic ovary syndrome

Study by Alteration and female infertility Reference

Yue M DNA methylation is lower in 35- to 40-week old oocytes

39

Hamatani Histone deacetylase is downregulated at aged oocytes

38

Naqvi Abnormal methylation pattern act as proinflammatory factors which induce endometriosis

45

Wang Altered DNA methylation of LHCGR gene in PCOS

50

Besides playing a role in male fertility, epigenetics also influence female fertility, i.e. oogenesis. The female infertility factors that are going to be discussed in this paper are ageing oocyte, endometriosis, and polycystic ovarian syndrome (PCOS).31,32 Alterations of epigenetic modification play an essential role in the regulation of gene expression changes in those female factors. Several studies have shown a change in the pattern of DNA methylation and histone modifications in respective genes.

Epigenetic in oogenesisThe oogenesis process in the ovarium is regulated by many factors, i.e. epigenetic mechanism as explain in the Figure 2.

The profile of DNA methylation remains constant at the primordial stage to the stage of primary follicles, but it increases progressively until antrum formation. Studies conducted by Kageyama et al34 in the female mice showed the increase of DNA methylation began on day 10

until day 15. Hiura et al35 added that the profile of DNA methylation depended on the size of oocyte, that DNA methylation occured mostly in oocytes with a diameter around 55-60 μm.

In the development of oocytes, besides DNA methylation, there is also histone acetylation. As illustrated, during the early stages of development, histone acetylation has a low level and begins to rise abruptly along with the follicle development.33 Ling Gu et al36 reported the dynamic changes in the acetylation of histone H4 lysine residues at H4K5, H4K8, H4K12, H4K16 positions and histone H3 at H3K9 and H3K14 positions. At the beginning, the level of acetylation in all six residues decreased at the end of the GV stage, indicating that histone

deacetylase (HDAC) is required. Furthermore, all of H4K5 and H4K16 have deacetylation on stage MI (metaphase I), followed by reacetylation at the AI stage (anaphase I), then they undergo deacetylation at the MII (metaphase II) of oocyte.

In the oocyte development, when the follicle reached antrum formation, histone H3 dimethylated at K4me2 and K9me2 position or trimethylated at K4me3 position, which is regulated by MLL2 methyltransferase.33 Kageyama et al34 showed that methylation in the H3K4 and H3K9 position was


Figure 2. Epigenetic mechanism in the follicle development


also increased.34 Moreover, H3K4me2, H3K4me3, and H3K9me2 experienced a slight increase in the oocyte development on day 10, and then increased significantly on day 15. The increasing of H3K9 di- and tri-methylation may play a role in the inactivation of the whole genome transcription during oocyte development. In contrast, the H3K4 methylation may be involved in the changes of chromatin configuration, which is not related to transcription.37

Epigenetic and ageing oocytesIt has been known that the epigenetic modification of oocytes may be affected by advanced maternal age. One of the reasons is associated with the altered expression of DNMTs and histone acetyltransferases. Hamatani et al38 showed the difference of gene expression profile between the 5- to 6-week-old mouse oocytes, related to DNMT1, 3b and 3I activity which maintains the DNA methylation. In addition, the pregnancy rate of older Kunming mice is lower than that of younger ones, which may be associated with abnormal DNA methylation in oocytes.39 Besides the DNA methylation, histone modifications also affect the ageing oocyte. During meiosis, histone is deacetylated globally at the Metaphase I (MI) and Metaphase II (MII) stages by histone deacetylase activity in mammalian oocytes. Histone deacetylase is downregulated at the transcript level in ageing mouse oocytes. In addition, Akiyama et al40 reported that if meiotic histone deacetylation was inhibited, aneuploidy occurred in fertilized mouse oocytes and resulted in embryonic death in the uterus.

Epigenetic and endometriosisEndometriosis is defined as a disorder in which endometrial tissue grows outside the uterus, which can induce chronic inflammatory reactions.41,42 Epigenetics plays a role in regulating the expression of endometrial genes during the menstrual cycle. The alteration in the epigenetic modification is a mechanism that may occur in endometriosis-induced changes in gene expression in endometriosis, including genes that play a role in the regulation of transcription, proliferation, inflammation, apoptosis, and cell cycle associated with implantation failure.43 In women with eutopic endometriosis, an increased of DNMT3A expression was found, which might play a role in hypermethylation in some conditions of endometriosis.44

Naqvi et al45 found the alteration of DNA methylation in some genes associated with endometriosis. The abnormalities of the methylation pattern of genes that act as proinflammatory factors will cause a change in the signal of the immune response, which induced chronic inflammation in endometriosis. Moreover, the expression of DNA methyltransferase enzyme that plays a role in repairing DNA methylation patterns, has been suppressed due to hypermethylation of its encoding gene, namely MGMT. It will create the possibility of DNA methylation irregularities of candidate genes in endometriosis. Meanwhile, other irregularities of gene expression resulting from changes in DNA methylation become hypermethylated which occured in the progesterone-B, E-cadherin and HOXA10 gene receptor. In addition, estrogen-β receptor, steroidogenic-1factor and aromatase also underwent hypomethylation.46

Inhibition of the E-cadherin expression due to hypermethylation causes disruption of cell adhesion, thus inducing invasion and metastasis of endometrial cells. HOXA10 genes in humans is expressed in the endometrium that is regulated by estrogen and progesterone. HOXA10 plays role in the development of the endometrium to prepare for implantation. Hypermethylation on HOXA10 causes a defect in the endometrium function, hence implantation failure in women with endometriosis. Meanwhile, aromatase is an enzyme that catalyzes the conversion of androgens to estrogens. Hypomethylation on aromatase gene leads to the increase of aromatase expression and of estrogen production that induces proliferation of endometrium cell.46,47

Meanwhile, deviations of other epigenetic modification, such as acetylation of histone modifications are also found in endometriosis. Histone acetylation is generally associated with the activation of a gene expression. The analysis of the chromatin immunoprecipitation (ChIP) conducted by Monteiro et al48 showed the hypoacetylation on the H3/H4 at the promoter regions of candidate genes was down regulated in endometriosis, namely HOXA10, ESR1, CDH1 and p21.48 On the other hand, the steroidogenic factor 1 promoter gene underwent increased acetylation of H3 and H4 which correlated with the high expression of the gene.

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Epigenetic and polycystic ovarian syndrome (PCOS)Polycystic ovarian syndrome (PCOS) is a set of symptoms resulting from the hormonal imbalance in women. It is characterized by the following three conditions: 1) the absence of ovulation, which leads to irregular menstrual periods or amenorrhea, 2) high androgen, and 3) the presence of cysts on one or both ovaries. PCOS is a major cause of infertility as a result of the absence of ovulation.49

The other epigenetic alteration found in PCOS, is a deviation of DNA methylation pattern in multiple genes. Wang et al.50 found an altered level of DNA methylation luteinizing hormone (LH)/choriogonadotropin receptor (LHCGR) gene in patients with PCOS had hypomethylation on its multiple CpG sites. LHCGR is identified as a susceptibility gene for PCOS. LHCGR interaction with its ligands, namely LH, has an important role in the process of ovulation in mammals. Meanwhile, LH induces theca cells in ovarian to secrete androgen precursor.51

Therefore, LHCGR can be considered as a candidate gene of PCOS. Hypomethylation on LHCGR genes causes the expression of LH receptors to increase, which leads to increase of sensitivity to LH.

The list of epigenetic alteration in female infertility is summarized in Table 2.

In conclusion, epigenetic modifications play an important role as regulators in the activation and inactivation of gene expression. Epigenetic modifications during spermatogenesis occur with various dynamic intensities. This review shows that epigenetic modifications play specific roles at certain stages in the spermatogenesis process, from DNA methylation and histone modification to sumoylation. Epigenetic modifications during oogenesis also affect the oocyte quality as well as in the other etiology of female infertility, i.e. endometriosis and PCOS. Alteration in the epigenetic modification patterns is associated with disturbances in spermatogenesis and oogenesis which might lead to infertility.

Acknowledgment Authors would like to express gratitude to Indonesian Reproductive Medicine Research and Training Center (Ina-Repromed) for supporting this review.

Conflict of interest The authors affirm no conflict of interest in this study.

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Syam.Helicobacter pylori infection in Indonesia

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Current situation of Helicobacter pylori infection in Indonesia

Keywords: current situation, Helicobacter pylori infection, prevalence, risk factor

pISSN: 0853-1773 • eISSN: 2252-8083 • http://dx.doi.org/10.13181/mji.v25i4.1408 • Med J Indones. 2016;25:263–6• Received 20 Mar 2016 • Accepted 01 Nov 2016

Corresponding author: Ari F. Syam, [email protected]


Ari F. SyamDepartment of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia

Brief Communication


ABSTRAK

Data epidemiologi infeksi Helicobacter pylori (H. pylori) terus berubah dalam beberapa dekade terakhir. Indonesia dilaporkan memiliki prevalensi infeksi H. pylori yang rendah dibandingkan dengan negara lain di Asia. Beberapa penelitian di Indonesia melaporkan bahwa sanitasi yang buruk, usia, agama, etnis merupakan faktor risiko untuk infeksi H. pylori. Dibandingkan dengan tes diagnostik lainnya, tes urine merupakan tes yang dapat diandalkan untuk mendeteksi H. pylori di Indonesia karena tes tersebut bersifat non-invasif dengan harga yang cukup terjangkau dan memiliki akurasi yang tinggi. Meskipun banyak penelitian telah dilakukan mengenai prevalensi infeksi H. pylori pada beberapa etnis di Indonesia, peneliti masih memiliki beberapa pertanyaan yang belum terjawab mengenai infeksi H. pylori di Indonesia. Oleh karena itu, diperlukan untuk membangun pusat penelitian H. pylori yang menyediakan fasilitas untuk kultur, evaluasi resistensi antibiotik, dan memperoleh informasi genotipe yang dapat menjelaskan perbedaan dalam infeksi H. pylori di antara berbagai etnis di Indonesia.

ABSTRACT

The epidemiology of Helicobacter pylori (H. pylori) has been changing over the past decades. Indonesia was reported have a low prevalence of H. pylori infection compared to other countries in Asia. Some studies in Indonesia have evaluated that poor sanitation, age, religion, ethnicity are the risk factors for H. pylori infection. Compared to other diagnostic tests, the urine test will be reliable for the detection of H. pylori in Indonesia because it is non-invasive and low cost with high accuracy. Although we have already performed studies on the prevalence of H. pylori infection in several ethnics, we still have some questions that remain unclear regarding H. pylori infection in Indonesia. Therefore, we have a need to build a H. pylori center that provide facilities for culturing, evaluating antibiotic resistance, and obtaining the genotype information that may explain the differences in H. pylori infection among ethnic groups in Indonesia.



As a developing country in Southeast Asia, Indonesia is an archipelago with a multi-ethnic society (more than 1,000 ethnic and sub-ethnic groups). The age-standardized incidence of gastric cancer in Indonesia has been reported to be 2.8/100,000, which is low compared with the one recorded among other Asian countries.1 Indonesia, with its different ethnicities, cultures, life-styles and religions, presents an appropriate model to examine the effects of migration and co-evolution on the bacteria-host interactions involved in the Helicobacter pylori (H. Pylori) infection

Until March 2013, only 313 hospitals provided gastrointestinal endoscopy services in Indonesia. Although these hospitals were distributed in 33 provinces in the country, 72% (98/136) of them were located on Java island.2 This uneven distribution makes it difficult for our physicians to establish a diagnosis of H. pylori.

The epidemiology of H. pylori has been changing over the past decades, with a decrease in the prevalence of infection in most countries. The changing epidemiology of the bacterium has been associated with a decline in peptic ulcer disease and gastric cancer. A Malaysian study reported by Leow et al3 found that in the period of 1989–1990, the overall prevalence of H. pylori was 51.7%. This prevalence decreased to 30.3% in the second period, 1999–2000, and to 11.1% in the third period, 2009–2010 (p<0.001).3

Syam et al reported a prevalence of H. pylori infection of 22.1% (59/267).4 In contrast to other countries in Asia, we have a low prevalence of H. pylori infection. We attempted to identify the risk factors for H. pylori infection, but found inconsistencies in terms of locations. Hence, questions remain about H. pylori infection in Indonesia are, first, what are the risk factors for the H. pylori infection in Indonesia? And second, why does the incidence of the H. pylori infection remain low in Indonesia?

In addition to these problems, we aim to identify the best test for H. pylori screening in Indonesia.

What are the risk factors for the H. pylori infection in Indonesia?Some studies have evaluated the risk factors for H. pylori in Indonesia. The latest study reported

by Darnindro et al5 found that the incidence of H. pylori infection in patients with poor sanitation was higher compared with patients who had a good sanitation status. The lower the sanitation status, the higher the risk of H. pylori infection (OR=2.5; 95% CI=1.01–6.19; p=0.044). Furthermore, the use of public toilets can increase the spread of infection because of individual’s lack of hygienic behaviour. Also, the use of wells, especially shallow wells, as a water source increases the risk of transmission of H. pylori.5 Darnindro et al5 reported that the incidence of H. pylori infection in areas with a low clean water index was higher than in areas with a high clean water index. The lower the index, the greater the risk of H. pylori infection (OR=1.524; 95% CI=0.57–4.04; p=0.396). Darnindro et al5 also showed that 13.5% of their sample had a high crowding index, although the association between H. pylori infection and crowding did not achieve statistical significance (OR=1.2; 95% CI=0.37–4.49). 5

Our study, conducted from January 2014 to February 2015, involved a total of 267 patients with dyspeptic symptoms on Java, Papua, Sulawesi, Borneo, and Sumatra islands. The patients aged 50–59 had a significantly higher H. pylori infection rate than did the 30–39-year-old group (OR=2.98; p=0.05). The Protestants had a significantly higher H. pylori infection rate than did the Catholics (OR=4.42; p=0.008). The infection rate was also significantly lower among people who used tap water as their source of drinking water compared with those who drank from wells or rivers (OR=9.67; p=0.03).4

The major ethnicities in the study of Darnindro et al5 were Javanese (45.9%), Bugis (18%), and Sundanese (15.3%). The incidence of H. pylori infection among Javanese in their study was 17.9%, whereas it was 30% among Bugis, and 29.4% among Sundanese.5 Our epidemiology study in five major islands in Indonesia yielded a similar trend.4 There were significant differences in the prevalence of H. pylori infection in terms of ethnic groups (p<0.001). The highest prevalence of H. pylori infection was found among Papuan patients: 9/21 (42.9%) tested positive for H. pylori. This compared with 28/70 (40.0%) of Batak patients, 11/30 (36.7%) of Buginese patients and just 7/54 (13.0%) of Chinese patients. There was

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no significant difference in the H. pylori infection rate between Surabayan Chinese (15.4%, 4/26) and Pontianak Chinese (12.5%, 3/24; P=0.93). Three Dayak patients (7.5%) were positive for H. pylori infection. The comparison of these two studies allowed us to conclude that ethnicity is one of the risk factors for H. pylori infection.4

Why does the incidence of H. pylori infection remain low in Indonesia?The causes of the low H. Pylori infection rate in Indonesia remain unknown. We would expect H. pylori infection to be as high in this country as in other developing countries, because the sanitation infrastructure remains to be constructed. Most of Indonesians still do not have direct access to clean water. Table 1 presents a summary of previous studies that examined the prevalence of H. pylori infection and gastric cancer in Indonesia. Although a number of researchers have investigated this, the results are controversial and contradictory (0–68%).6,7 This could be attributable to the different study populations and different tests used for H. pylori diagnosis.8 Moreover, most of these studies investigated only the largest ethnic group, the Javanese.6,7,9,10 In a previous study, Miftahussurur et al confirmed that the prevalence of H. pylori infection in Surabaya (Java island) was low, only 11.5%, diagnosed using five different methods.8

It is still to be expected that our genetic-polymorphism evaluation of the possibility that some ethnic groups, such as the Javanese and most of the ethnic groups in Sumatra (Acehnese, Minang, and Palembang), might have immunity against H. pylori infection. It is also possible that some diets may be useful in preventing H. pylori infection. In addition to the remaining question of why the prevalence of H. pylori is low in Indonesia, another enigma emerges: why do some ethnic groups, such as the Papuans, Bugis (South Celebes) and Batak (North Sumatra), have

Author Study period Area n Gastric cancer H. pylori positive rateSyam AF et al10 2003–2004 5 cities 550 1/550 (0.2%) 10.2%Syam AF et al11 2001 Jakarta 63 1/63 (1.6%) 9.5%Syam AF et al4 2014–2015 6 cities 267 1/267 (0.4%) 22.1%Saragih et al9 1998–2005 Jakarta 2903 47/2903 (1.61%) 9.0%Siregar G et al12 2003 Medan 50 3/50 (6%) 60%

Table 1. Summary of the prevalence of gastric cancer and H. pylori infection reported by several studies

a high prevalence of H. pylori infection compared with other ethnicities?

What is the best tool for establishing the diagnosis of H. pylori infection in Indonesia?We performed several studies to identify the best H. pylori diagnostic test for the Indonesian population, such as Pronto Dry, H. pylori stool antigen (HpSA), or a urine test.

In 2003–2004, we evaluated the rapid urease test (Pronto Dry) in patients with dyspepsia in several endoscopic centers in Indnesia. The study was conducted in patients with dyspepsia who underwent endoscopic examination in the endoscopic centers of several cities in Indonesia, as follows: Cipto Mangunkusumo Hospital Jakarta, Hasan Sadikin Hospital Bandung, Sardjito Hospital Yogyakarta, Soetomo Hospital Surabaya, Adam Malik Hospital Medan and Sanglah Hospital Denpasar. The study took place from January 2003 to April 2004. Out of 525 cases, 56 cases were considered H. pylori positive based on the criteria used, 39 tested positive using Pronto Dry, and 17 tested negative using Pronto Dry. The overall sensitivity and specificity of Pronto Dry were 69.7% and 95.7%, respectively. Its positive predictive value was 66.1%, its negative predictive value was 96.4% and its overall accuracy rate was 92.9%. When we calculated the sensitivity and specificity at each endoscopic center, the results varied.11

Our study evaluating the HpSA test found that the area under the receiver operating characteristic (ROC) curve was 0.722 (95% CI=0.518–0.927). Using a cut-off value of 0.274 instead of 0.16 (as recommended by the manufacturer), the sensitivity and specificity were 66.7% and 78.9%, respectively.9

To confirm the accuracy of the urine test (RAPIRUN), we compared the results



of the test with histology confirmed by immunohistochenmistry (IHC) and culture. From this study, we could confirm the high accuracy of RAPIRUN, which is a rapid immunochromatographic method for the determination of anti-H. pylori IgG in urine. We found identical results between histology confirmed by IHC and culture (12/88, 13.6%). Only two samples tested positive using the urine test, but were found to be negative using the two gold-standard tests. Conversely, four samples tested negative using the urine test but were found to be positive using histology confirmed by IHC and culture. The overall sensitivity and specificity of RAPIRUN were 83.3% and 94.7%, respectively. Its positive predictive value was 71.4%, its negative predictive value was 97.3% and its overall accuracy rate was 93.2%. This urine test will be reliable for the detection of H. pylori in Indonesia.12 This result was also in agreement with a previous report from North Sulawesi which found that the results of urine tests using a similar kit were identical to those obtained using the anti-H. pylori antibody serum test.13 As a non-invasive test, the urine test is user friendly and combines low cost with high accuracy; therefore, it is the best option for measuring H. pylori status in remote areas that lack of an endoscopy system, such as much of Indonesia. This urine test can also be very useful for mass screening.

What should be done?We have the means to establish a center dedicated to the study of H. pylori infection in Indonesia. We expect to collect samples from the five largest islands in Indonesia. We have a system to manage the samples, including their storage at -20 or -80°C. We have experience in culturing other bacteria and using them in molecular biology studies. We have already performed studies and obtained updates on the prevalence of H. pylori infection in several ethnic groups; we now need to build a H. pylori center to address the remaining unanswered questions, based on the following steps (1) providing a facility for culturing H. pylori and evaluating antibiotic resistance to H. pylori and (2) providing a facility to perform molecular biology studies and to obtain genotypic information that may partly explain the differences in H. pylori infection rates among ethnic groups in Indonesia.

REFERENCES

1. Indonesia: Estimated age-standardized incidence and mortality rates: both sexes. [Internet] 2012 [cited 2016 Jan 20]. Available from: www. http://globocan.iarc.fr/Pages/fact_sheets_population.aspx

2. Makmun D. Present status of endoscopy, therapeutic endoscopy and the endoscopy training system in Indonesia. Dig Endosc. 2014;26(Suppl2):2–9.

3. Leow AH, Lim YY, Liew WC, Goh KL. Time trends in upper gastrointestinal diseases and Helicobacter pylori infection in a multiracial Asian population--a 20-year experience over three time periods. Aliment Pharmacol Ther. 2016;43(7):831–7.

4. Syam AF, Miftahussurur M, Makmun D, Nusi IA, Zain LH, Zulkhairi, et al. Risk factors and prevalence of Helicobcater pylori in five largest islands of Indonesia: a preliminary study. PLoS ONE;10(11):e0140186.

5. Darnindro N, Syam AF, Fauzi A, Rumende CM. Seroprevalence and socio-demographic factors of Helicobcater pylori infection in patients with dyspepsia in Kalibaru primary health care north Jakarta. Acta Med Indones. 2015;47(4):297–303.

6. Tokudome S, Samsuria Soeripto WD, Triningsih FX, Suzuki S, Hosono A, Triono T, et al. Helicobacter pylori infection appears essential for stomach carcinogenesis: observations in Semarang, Indonesia. Cancer Sci. 2005;96(12):873–5.

7. Abdullah M, Ohtsuka H, Rani AA, Sato T, Syam AF, Fujino MA. Helicobacter pylori infection and gastropathy: a comparison between Indonesian and Japanese patients. World J Gastroenterol. 2009;15(39):4928–31.

8. Miftahussurur M, Shiota S, Suzuki R, Matsuda M, Uchida T, Kido Y, et al. Identification of Helicobacter pylori infection in symptomatic patients in Surabaya, Indonesia, using five diagnostic tests. Epidemiol Infect. 2015;143(5):986–96.

9. Syam AF, Rani AA, Abdullah M, Manan C, Makmun D, Simadibrata D, et al. Accuracy of Helicobacter pylori stool antigen for the detection of Helicobacter pylori infection in dyspeptic patients. World J Gastroenterol. 2005;11(3):386–8.

10. Saragih JB, Akbar N, Syam AF, Sirait S, Himawan S, Soetjahyo E. Incidence of Helicobacter pylori infection and gastric cancer: an 8-year hospital based study. Acta Med Indones. 2007;39(2):79–81.

11. Syam AF, Abdullah M, Rani AA, Nurdjanah S, Adi P, Djumhana A, et al. Evaluation of the use of rapid urease test: Pronto Dry to detect H. pylori in patients with dyspepsia in several cities in Indonesia. World J Gastroenterol. 2006;12(38):6216–8.

12. Syam AF, Miftahussurur M, Uwan WB, Simanjuntak D, Uchida T, Yamaoka Y. Validation of urine test for detection of Helicobacter pylori infection in Indonesian population. BioMed Res Int. 2015;2015(152823):1–6.

13. Miſtahussurur M, Tuda J, Suzuki R, Kido Y, Kawamoto F, Matsuda M, et al. Extremely low Helicobacter pylori prevalence in North Sulawesi, Indonesia, and identification of a Maori-tribe-type strain: a cross-sectional study. Gut Pathog. 2014;6(42):1–8.

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