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"Introduction: The prevalence of HIV in Indonesia is estimated to be up to 640,000 [550,000 – 750,000] in the year 2018 and is still a global problem until today. With improvement in management of this condition throughout the years, life expectancies of HIV-1 patients have been improving. Therefore more and more patients have been interested in growing families and having offsprings, however it is known that HIV infection and it’s management of ARV may lead to infertility. Sperm Analysis is a method to evaluate the sperm abnormality, namely oligozoospermia, asthenozoospermia and teratozoospermia. This study compares the frequency of the aforementioned abnormalities of HIV-1 infected males in Indonesia to control individuals. Methods: The research is an experimental study using categorical data of HIV positive and control healthy men, comparing their fertility status using a numeric data output for semen parameters including sperm concentration, sperm motility and sperm morphology. Our 30 samples will be processed using Computer-Assisted Sperm Analyzer (CASA) for sperm concentration and motility along with Giemsa staining for sperm morphology, and then a statistical analysis will be carried out by the Shapiro-Wilk test, Unpaired T-Test and Mann-Whitney by using the SPSS programme. Results: This study found that sperm concentration is significantly lower in HIV positive samples, along with sperm progressive motility, and normal sperm morphology (p < 0.05). Frequency of normal semen concentration, motility, and morphology have been found to be higher in control samples as compared to HIV-positive samples. Conclusion: The study shows that sperm quality abnormalities such as oligozoospermia, asthenozoospermia and teratozoospermia happens more frequently in HIV-1 infected Indonesian men compared to controls.

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Latar Belakang: Prevalensi HIV di Indonesia diperkirakan mencapai 640.000 [550.000 - 750.000] pada tahun 2018 dan masih menjadi masalah global hingga saat ini. Dengan perbaikan dalam pengelolaan HIV selama bertahun-tahun, life expectancy pasien HIV-1 telah meningkat. Oleh karena itu, semakin banyak pasien yang ingin berkeluarga dan memiliki keturunan, namun diketahui bahwa infeksi HIV serta pengobatannya dengan ARV dapat menyebabkan infertilitas. Analisis Sperma adalah metode untuk mengevaluasi abnormalitas sperma, berupa oligozoospermia, asthenozoospermia dan teratozoospermia. Pada studi ini, dilakukan perbandingan abnormalitas sperma tersebut dari laki-laki yang terinfeksi HIV-1 di Indonesia dengan laki-laki kontrol.

Metode: Penelitian ini merupakan penelitian eksperimental dengan menggunakan sampel sperma dari pasien HIV-positif dan kontrol, dengan keluaran data numerik untuk parameter semen termasuk konsentrasi sperma, motilitas sperma dan morfologi sperma. 30 sampel kami akan diolah menggunakan Computer-Assisted Sperm Analyzer untuk konsentrasi dan motilitas sperma serta dengan pewarnaan Giemsa untuk morfologi sperma. Kemudian dilakukan analisis statistik melalui uji Shapiro-Wilk, Unpaired T-Test dan Mann-Whitney dengan menggunakan program SPSS.

Hasil: Dalam penelitian ini didapatkan konsentrasi, motilitas, dan morfologi sperma (p < 0,05) secara signifikan lebih rendah pada sampel HIV-positif dibandingkan dengan kontrol. Frekuensi konsentrasi, morfologi dan motilitas sperma normal ditemukan lebih tinggi pada sampel kontrol dibandingkan dengan sampel HIV-positif.

Kesimpulan: Data penelitian menunjukkan bahwa abnormalitas kualitas sperma seperti Oligozoospermia, Asthenozoospermia dan Teratozoospermia lebih tinggi pada laki-laki yang terinfeksi HIV-1 dibandingkan kontrol. "

"ABSTRAKLatar Belakang: Untuk meningkatkan kemungkinan konsepsi pada pasangan yang menjalani inseminasi intrauterin (IIU), dilakukan preparasi spematozoa dengan metode pencucian swim-up (SU) yang dapat meningkatkan kualitas spermatozoa. Aktivitas dari dinein ATPase dapat terlibat dalam proses preparasi spermatozoa, namun nilai yang pasti dari aktivitas dinein ATPase pada spermatozoa kelompok astenozoosperma yang menjalani pencucian SU belum diketahui. Tujuan: Studi ini dilakukan untuk melakukan evaluasi terhadap efisiensi dari metode preparasi spermatozoa dengan pencucian SU pada sampel astenozoospermia pada laki-laki infertil. Metode: Sampel semen didapatkan dari 6 laki-laki pasangan infertil (astenozoospermia) yang akan menjalani terapi inseminasi intrauterin. Analisis semen dilakukan sebelum dan sesudah dilakukannya preparasi spermatozoa. Preparasi spermatozoa dilakukan dengan metode swim-up (SU). Kemudian, aktivitas dinein ATPase diuji dengan metode Vivenes setelah fraksi aksonem sperma dikumpulkan dengan metode Olson. Dilakukan uji statistik paired t-test atau uji Wilcoxon Signed Rank untuk melihat derajat kemaknaan, dengan nilai bermakna jika p<0,05. Hasil: Berdasarkan analisis semen, ditemukan peningkatan signifikan terhadap motilitas dan morfologi progresif spermatozoa kelompok astenozoospermia setelah dilakukannya preparasi sperma dengan metode swim-up (p<0,05). Didapatkan pula peningkatan pada aktivitas spesifik dinein ATPase pasca-pencucian (p>0,05). Walaupun begitu, terdapat penurunan pada nilai konsentrasi sperma (p>0,05). Kesimpulan: Terdapat peningkatan kualitas spermatozoa kelompok astenozoospermia yang signifikan disertai peningkatan aktivitas spesifik dinein ATPase setelah pencucian dengan metode swim-up.

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ABSTRACT

Background: To increase the likelihood of conception in couples undergoing intrauterine insemination (IIU), spematozoa preparation was carried out with a swim-up (SU) washing method that could improve the quality of spermatozoa. The activity of dinein ATPase can be involved in the preparation process of spermatozoa, but the exact value of dinein ATPase activity in the spermatozoa of the astenozoosperm group undergoing SU washing is unknown. Objective: This study was conducted to evaluate the efficiency of the spermatozoa preparation method by swim-up washing in the asthenozoospermia sample in infertile men. Methods: Semen samples were obtained from 6 men from infertile couples (asthenozoospermia) who would undergo intrauterine insemination therapy. Cement analysis was carried out before and after the preparation of spermatozoa. Preparation of spermatozoa is carried out by the Swim-up (SU) method. Then, the dinein ATPase activity was tested by the Vivenes method after the axoneme fraction of the sperm was collected by the Olson method. Paired t-test statistics or the Wilcoxon were conducted to see the degree of significance, with a significant value if p <0.05. Results: Based on semen analysis, it was found a significant increase in the progressive motility and morphology of the asthenozoospermia spermatozoa after swim-up method of sperm preparation (p <0.05). There was also an increase in post-washing dinein ATPase specific activity (p> 0.05). However, there was a decrease in the value of sperm concentration (p> 0.05). Discussion: There was an increase in the quality of the asthenozoospermia spermatozoa and significant specific dinein ATPase activity after spermatozoa preparation with swim-up method."

"LATAR BELAKANG : Salah satu hambatan dalam program reproduksi berbantu adalah rendahnya viabilitas dan motilitas sel spermatozoa. Analisis proteomik menunjukkan adanya banyak protein yang diduga berperan dalam regulasi motilitas dan viabilitas spermatozoa antara lain progesteron. Penelitian ini bertujuan untuk menganalisis efek prosurvival progesteron terhadap spermatozoa melalui penekanan apoptosis.BAHAN DAN CARA KERJA : Sampel spermatozoa dicuci dengan sentrifugasi gradient. Sampel spermatozoa di tambahkan progesteron dengan konsentrasi 0 kontrol , 250, 500, 750 dan 1000 ng/mL. Setelah perlakuan, sampel dilakukan pemeriksaan integritas membran dengan metode HOS dan pemeriksaan motilitas dengan Computer Assisted Sperm Analyzer CASA . Deteksi protein fosforilasi tirosin dan Akt serta aktivitas kaspase dilakukan dengan metode western blot.HASIL : Efek penambahan progesteron meningkatkan rerata motilitas spermatozoa namun berbeda tidak bermakna p>0.05 . Integritas membran spermatozoa tidak berpengaruh pada pemberian progesteron. Analisis western blot menunjukkan peningkatan fosforilasi protein tirosin antara kelompok kontrol dan setelah diberikan progesteron p>0.05 . Demikian halnya dengan hasil fosforilasi protein Akt juga mengalami peningkatan pada kelompok kontrol dan setelah diberikan progesteron berbagai dosis. Aktivitas kaspase-3 mengalami penurunan bila dibandingkan antara kelompok kontrol dan setelah diberikan progesteron p

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BACKGROUND One of the obstacles in assisted reproduction programs is the low viability and motility of spermatozoa cells. Proteomic analysis indicates that many proteins are thought to play a role in the regulation of motility and viability of spermatozoa, among others, progesterone. This study aims to analyze the prosurvival effect of progesterone against spermatozoa through apoptosis suppression.METHODS Spermatozoa was washed with gradient centrifugation. Progesterone is added to each sample with a final concentration 0 control , 250, 500, 750 and 1000 ng mL. After the sample treatment was done, membrane integrity checking with hypoosmotic swelling test and motility examination with Computer Assisted Sperm Analyzer CASA . Detection of protein in the western blot will be done that recognizes the phosphorylation of tyrosine residues and Akt and caspase activity.RESULT The effect of addition of progesterone increases sperm motility but not signicantly different p 0.05 . The integrity of the spermatozoa membrane is no effect in progesterone. Western blot analysis revealed an increase of tyrosine phosphorylation protein levels between control and after progesterone group p 0.05 . Similarly, the results of Akt protein phosphorylation also increased in control and after progesterone group. Caspase 3 activity decreased when compared between control and after progesterone group p "

"Infeksi HIV-1 merupakan salah satu permasalahan kesehatan di Indonesia yang perlu diteliti untuk mendapatkan strategi intervensi yang sesuai dengan galur virus yang bersirkulasi di Indonesia. Untuk pengembangan kultur galur HIV Indonesia, diperlukan antibodi spesifik terhadap protein awal HIV-1 yaitu Tat dan Rev sebagai marka infeksi HIV pada sel yang terinfeksi HIV-1. Penelitian ini bertujuan untuk mendapatkan plasmid pengekspresi bagian immunodominant protein fusi Tat dan Rev pada sistem ekspresi mamalia agar dapat dimanfaatkan untuk menginduksi pembentukan antibodi spesifik pada hewan coba mamalia. Susunan DNA penyandi protein fusi Tat dan Rev didapat dengan melakukan analisis optimasi kodon penyandi susunan asam amino protein fusi menggunakan piranti lunak GenScript, untuk mendapatkan nilai Codon Adaptation Index CAI > 0.80 pada sistem ekspresi mamalia. Susunan nukleotida yang diperoleh kemudian diberikan tambahan susunan nukleotida situs restriksi pada bagian 5 rsquo; dan 3 rsquo; untuk memudahkan pengklonaan ke dalam vektor ekspresi dan dipesan ke penyedia jasa sintesis asam nukleat. Potongan DNA sisipan TatRev, penyandi protein fusi TatdanRev diperoleh dari penyedia jasa sintesis dalam bentuk terklona dalam plasmid pUC19. Agar dapat terekspresi dalam sistem ekspresi mamalia, DNA sisipan dipotong dengan enzim restriksi KpnI dan HindIII dari plasmid pUC19 dan disubklona ke dalam situs restriksi KpnI dan HindIII pada plasmid pTriEx-4. Analisis restriksi enzim Kpn I dan Hind III plasmid rekombinan yang diperoleh dengan elektroforesis jel agarosa menunjukkan adanya dua pita yang bermigrasi sesuai ukuran plasmid pTriEx-4 dan DNA sisipan TatRev, yaitu sebesar 5000 pb dan 600 pb.

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HIV 1 infection is one of the health problems in Indonesia that is necessary to be studied in order to obtain intervension strategies that is in accordance with the circulating viral strains in Indonesia. In order to develop culture of Indonesian HIV strainss, specific antibodies towards early proteins of HIV, namely Tat and Rev, that are markers of HIV infection in HIV 1 infected cells. This study is aimed at obtaining a plasmid for expression of immunodominant region of Tat and Rev fusion protein in mammalian expression system to be used for stimulation of specific antibody formation in mammals as experimental animal. Nucleotide sequence of DNA encoding the Tat and Rev fusion protein was obtained by codon optimization analysis of the amino acid sequence of the fusion protein using the GenScript software, to obtain a Codon Adaptation Index CAI 0.80 for mammalian expression system. The nucleotide sequence that is obtained was then added with recognition sequences for enzymatic restriction at the 5 rsquo and 3 rsquo end to facilitate cloning into expression vector, and requested to a service provider for nucleic acid synthesis. The TatRev insert DNA, encoding the fusion protein of Tat and Rev was obtained from the nucleic acid synthesis service provider in the formed of cloned DNA in the plasmid pUC19. For expression in mammalian expression system, the insert DNA was restricted using restriction enzymes KpnI and HindIII from the pUC19 plasmid and subcloned into the KpnI and HindIII restriction sites in plasmid pTriEx 4. Agarose gel electrophoresis analysis of KpnI and HindIII enzymatic restriction of the resulting recombinant plasmid showed the presence of two bands that migrated according to the sizes of the plasmid pTriEx 4 and the TatRev insert DNA, namely 5000 bp and 600 bp."

"Latar Belakang: P24 protein adalah salah satu protein milik HIV-1 yang membentuk kapsid bagian dalam dan dirilis ketike Gag protein di belah oleh protease virus. Kadar antien p24 umumnya akan meningkat di darah pada fase akut infeksi. Oleh sebab ini, produksi protein rekombinan p24 yang efektif dan memiliki imunogenisitas serta imunoreaktifitas seperti protein p24 natural merupakan hal yang penting, untuk deteksi antibodi p24 dan membuat antibodi monoklonal p24. Dalam penelitian ini, dilakukan optimasi ekspresi protein p24 subtype HIV-1 CRF01_AE. Metode : Penelitian ini menguji beberapa factor untuk ekspresi optimal dari protein rekombinan p24 pada plasmid pQE-80L milik Eschericia coli, termasuk konsentrasi inductor, durasi induksi, dan kultur media yang digunakan. Deteksi, quantifikasi, dan analisis protein dilakukan dengan SDS Page dan di analisis menggunakan Imagelab. Hasil : p24 recombinant protein from HIV-1 Subtype CFR01_AE terekspresikan secara optimal menggunakan media Terrific Broth dengan di induksi 1mM Isopropyl-beta-D-thiogalactopyranoside (IPTG) selama 6 hours pada suhu 37°lC. Kesimpulan : Strategi dan Metode yang digunakan untuk mengekspresikan protein rekombinan dapat berbeda antar sistem dan antar jenis protein yang diekspresikan. Penelitian ini menunjukkan bahwa plasmid pQE80L-p24 yang sudah dikembangkan di PRVKP FKUI memiliki kemampuan untuk mengekspresikan protein rekombinan p24, dan teroptimasi pada media ekspresi Terrific Broth, konsentrasi Isopropyl-beta-D- thiogalactopyranoside (IPTG) 1mM, selama 6 jam pada suhu 37°C.

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Background: P24 protein is one of HIV-1 protein that forms inner capsid and is released during cleave of Gag protein by viral protease. P24 antigen level will increase in the blood during the acute stage of infection. Therefore, an effective production of p24 recombinant protein that have the same immunogenicity and immunoreactivity of natural p24 protein are very important for detecting of p24 antibody and producing monoclonal p24 antibody. In this research, expression of p24 obtained from HIV-1 Subtype (HIV-1 CRF01_AE) was optimized to find the best expression system for the above protein.

Methods: In this research, several factors will be tested for optimal expression of recombinant p24 protein in plasmid pQE-80L of Eschericia coli, including the inducer concentration, duration of induction, and culture medium. Detection, quantitation, and analysis of the protein will be done using SDS Page and documented using Gel Documentation System to compare the effect of these factors.

Result: p24 recombinant protein from HIV-1 Subtype CFR01_AE is optimally expressed in Terrific Broth Medium in 1mM Isopropyl-beta-D-thiogalactopyranoside (IPTG), for 6 hours.

Conclusion: The strategies and method for protein expression may vary between a system, and a protein, to another. From this research it could be concluded that pQE80L-p24 plasmid that has been developed in PRVKP FKUI is able to express p24 recombinant protein. Protein expression is optimized in Terrific Broth Medium in 0.5mM Isopropyl-beta-D-thiogalactopyranoside (IPTG), for 4 hours in 37°C."

"Protein pol termasuk dalam tiga gen signifikan yang mengkode protein struktural HIV-1. Namun, sebagian besar tes diagnostik HIV hanya melibatkan dua produk HIV yang signifikan yaitu, protein Env atau Gag, yang berarti bahwa sebagian besar tes diagnosa HIV dilakukan atau dikembangkan menggunakan antigen yang sama. Hal ini pada akhirnya dapat menghasilkan hasil positif palsu yang berulang jika ada antibodi yang bereaksi silang karena sebagian besar tes disiapkan hanya dengan antigen umum yang sama. Solusi untuk masalah ini adalah mengembangkan uji diagnostik alternatif yang berbeda menggunakan antigen utama lain, seperti protein Pol. Salah satu produk protein Pol, enzim Integrase, termasuk dalam salah satu protein imunogenik HIV, yang berarti dapat digunakan untuk meningkatkan spesifisitas tes diagnostik HIV. Menggunakan protein Pol Immunodominant 2 (Pol ID2), sebuah protein yang terdiri dari Integrase dan RNAse H, yang sudah dikembangkan sebelumnya menggunakan subtipe HIV-1 yang paling menonjol di Indonesia (HIV-1 CRF01_AR), penelitian ini bertujuan untuk mendapatkan pengetahuan tentang kondisi optimal untuk mengekspresikan protein rekombinan Pol ID2 dalam plasmid pQE-80L Escherichia coli (E. coli) dengan harapan dapat berkontribusi terhadap pengembangan dan peningkatan kemandirian tes diagnostik HIV-1 di Indonesia. Metode: Penelitian ini dilakukan menggunakan metode desain studi eksperimental analitik. Dalam penelitian ini, protein rekombinan Pol Immunodominant 2 (ID2) dalam plasmid pQE-80L E. coli diekspresikan pada beberapa variabel media kultur, konsentrasi penginduksi, dan waktu induksi. Kultur ekspresi divisualisasikan menggunakan elektroforesis SDS PAGE dan didokumentasikan menggunakan mesin ImageQuant Las 4000. Meskipun tidak ada analisis statistik yang dilakukan, software analisis gel Image Lab 6.1 digunakan untuk menganalisis, mengukur, dan mendapatkan rasio kuantitas absolut dari konsentrasi protein pada setiap variabel. Hasil: Protein rekombinan Pol ID2 yang diperoleh dari HIV-1 subtipe CFR01_AE dapat diekspresikan secara optimal menggunakan media Terrific Broth dengan menggunakan 1mM Isopropil- beta-D-thiogalactopyranoside (IPTG) selama 3 jam induksi. Kesimpulan: Dapat disimpulkan bahwa plasmid pQE80L-Pol ID2 yang sebelumnya sudah dikembangkan di laboratorium PRVKP FKUI-RSCM dapat mengekspresikan protein rekombinan Pol ID2 dari HIV-1 subtipe CFR01_AR yang dikembangkan di bawah laboratorium yang sama. Protein dapat diekspresikan secara optimal dalam media kultur Terrific Broth menggunakan IPTG 1mM selama 3 jam induksi pada suhu 37oC.

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Background: Pol protein is included in the three significant genes that encode a structural protein of HIV-1. However, most HIV diagnostic tests involve only the two significant HIV products, Env or Gag protein, which means that most manufacturers use the same antigen sequence to develop these tests. This may eventually result in repetitive false-positive results if there is any cross-reacting antibody since the test is prepared only with the same common antigens. A solution to this problem is developing a different alternative diagnostic assay using a different major antigen such as Pol genes. One of the Pol gene products, viral enzyme integrase, is included in one of the immunogenic proteins of HIV, meaning that it may be used to improve the specificity of HIV diagnostic assays. Using a previously generated Pol Immunodominant 2 (Pol ID2) protein, comprised of Integrase and RNAseH, obtained from the most prominent HIV-1 subtype in Indonesia (HIV-1 CRF01_AR), this research aims to gain knowledge regarding the optimal conditions to express Pol ID2 recombinant protein in pQE-80L plasmid of Escherichia coli (E. coli) in hopes to contribute towards the development and self-reliance of HIV-1 diagnostic tests in Indonesia. Experimental, analytical study design was used for this research. The Recombinant Pol Immunodominant 2 (ID2) protein in the pQE-80L plasmid of E. coli was expressed under several variables of culture media, inducer concentration, and induction time. The expression culture was visualized using SDS PAGE and documented using ImageQuant Las 4000 machine. Although no statistical analysis was done, Image Lab 6.1 gel analysis software was used to analyze, quantify, and obtain the absolute quantity ratio of protein concentration of each variable.

Result: Pol ID2 recombinant protein obtained from HIV-1 subtype CFR01_AE are optimally expressed using Terrific Broth media using 1mM Isopropyl-beta-D-hiogalactopyranoside (IPTG) for 3 hours of induction.

Conclusion: It could be concluded that pQE80L-Pol ID2 plasmid previously developed in IHVCB FMUI-RSCM Laboratory can express Pol ID2 recombinant protein from HIV-1 subtype CFR01_AR that is constructed under the same laboratory. The protein expression is optimized in Terrific Broth culture media using 1mM IPTG inducer for 3 hours of induction in 37oC."

"Men who have sex with men living with HIV (MSM-LWH) experience psychological and social issues, including depression, anxiety, fear of infecting others, frustration, and social isolation. They may also experience problems in their relationships due to a fear of social stigma, such as marital issues, family conflicts, a lack of family support, economic difficulties, and social rejection by the family. This research aimed to assess the relationship between HIV status disclosure and stress in MSM-LWH in Medan, Indonesia. Here, a cross-sectional design and the convenience sampling technique were used. A total of 176 respondents who were MSM, HIV positive, and residents of Medan City were included in this work. Data were collected by means of HIV Status Disclosure questionnaires and a Perceived Stress Scale (PSS). Overall, 70.9% respondents reported disclosing their status to others and approximately half revealed experiencing stress. Moreover, HIV status disclosure was significantly associated with stress (p= 0.025). This study reveals that HIV status disclosure may result in negative effects on MSMLWH, represent a barrier to medical treatment, and increase internal stress."