Disertasi

Studi eksplorasi mekanisme epigenetik pada Steroidogenesis subjek Sindrom Ovarium Polikistik. Fokus pada peran asam folat terhadap gen CYP19A1, HSD17B1 dan FSHR. = Exploration study epigenetic mechanism Steroidogenesis Polycystic Ovary Syndrome subject. Focus in folic acid in CYP19A1, HSD17B1 and FSHR gene.

Latar belakang: Sindrom Ovarium Polikistik (SOPK) merupakan salah satu kelainan endokrin paling umum pada 7-15% wanita usia reproduksi yang menyebabkan infertilitas anovulatori. SOPK sering ditemukan pada wanita gemuk, sekitar 50-75% tetapi sindrom ini juga dapat ditemukan pada wanita kurus sebesar 5,5%. Penyebab dan patogenesis SOPK multifaktorial, asam folat dianggap sebagai salah satu penyebab SOPK, penelitian epigentik dipandang perlu dilakukan pada SOPK. Gen-gen dan hormon yang berperan dalam steroidogenesis menjadi fokus pada penelitian ini, karena bila salah satu atau sebagian gen tersebut tidak bekerja optimal maka produksi estradiol tidak adekuat sehingga akan memengaruhi kerja dari hormon steroidogenesis yang akan berujung pada anovulasi/ infertilitas. Tujuan: Untuk mengetahui status metilasi DNA, ekspresi mRNA dan korelasi metilasi DNA dan ekspresi mRNA pada gen CYP19A1, HSD17B1dan FSHR, serta kadar asam folat dan korelasi asam folat dan metilasi DNA. Selain itu diperiksa juga kadar estradiol, AMH serta korelasi AMH dengan FSHR dan korelasi AMH dan ekspresi mRNA FSHR. Metode: Penelitian deskriptif analitik dengan rancangan potong lintang. Sampel berupa sel granulosa dari folikel ovarium wanita dan serum darah di klinik Yasmin RSCM dan IRSI Bunda Jakarta, April 2018 sampai Februari 2019, kemudian dilakukan isolasi DNA dan RNA. Pada isolat DNA dilakukan konversi bisulfit, MSP, elektroforesis dan analisis ketebalan pita dengan perangkat lunak ImageJ untuk mendapatkan data metilasi DNA. Pada isolat RNA dilakukan qPCR untuk mendapatkan ekspresi relatif mRNA gen CYP19A1, HSD17B1dan FSHR. Pemeriksaan kadar asam folat menggunkan LC-MS/MS. Data karakteristik dikumpulkan saat informed consent data hormon didapatkan dari rekam medik. Hasil: Analisis data dari 30 subjek SOPK dan 30 subjek nir-SOPK konsentrasi asam folat tidak berbeda bermakna antara kedua kelompok (p=0,341), terjadi peningkatan metilasi DNA pada gen CYP19A (p=0,000) dan FSHR (p=0,000), terjadi penurunan metilasi pada gen HSD17B1 (p=0,160). Terjadi penurunan ekspresi mRNA pada gen CYP19A1 (p=0,040), HSD17B1 (0,003) dan FSHR (p=0,015). Peningkatan metilasi DNA FSHR berhubungan dengan penurunan ekspresi mRNA (p=0,001). Peningkatan metilasi DNA tidak berhubungan dengan kadar asam folat pada wanita SOPK gen CYP19A1 (p=0,209), HSD17B1 (0,567) dan FSHR (p=0,835). Konsentrasi estradiol wanita SOPK (25.78 pg/ml) lebih rendah dibandingkan dengan wanita nir-SOPK (36.74pg/ml). Konsentrasi AMH wanita SOPK (6,49 ng/ml) lebih tinggi dibandingkan dengan wanita nir-SOPK (3,98 ng/ml). Semakin tinggi konsentrasi AMH wanita SOPK, semakin rendah konsentrasi FSH dan mRNA FSHR wanita SOPK. Kesimpulan: Asam folat pada pasien SOPK bukan merupakan penyebab terjadinya metilasi. Peningkatan metilasi pada gen CYP19A1 dan FSHR menyebabkan penurunan ekspresi mRNA CYP19A1, HSD17B1 dan FSHR pada berakibat pada terganggunya kerja dari steroidogenesis sehingga produksi estradiol yang tidak adekuat yang pada akhirnya menyebabkan anovulasi pada SOPK.
Kata kunci: Steroidogenesis, SOPK, asam folat, metilasi DNA, ekspresi mRNA, gen CYP19A1, HSD17B1 dan FSHR.


Background: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in (7- 15%) of gestational women which causes anovulatory infertility. PCOS is oftenly found in obese women (50-75%) but this syndrome can also be found in lean women at (5.5%). PCOS is a multifactorial disease in which folic acid has been suspected as a causal factor. Genes and hormones that play a role in steroidogenesis are the focus of this study, because if one or more of these genes do not work optimally, it will produce inadequate estradiol which will affect the work of steroidogenesis hormones and lead to anovulation / infertility. Objective: To determine the concentration of folic acid in female serum PCOS, to find out DNA methylation and mRNA expression and correlation methylation and expression on CYP19A1, HSD17B1 and FSHR genes, to determine the related methylation of DNA and folic acid. To investigate estradiol and AMH levels, to find out correlation between AMH and FSH and correlation between AMH and mRNA FSHR subject PCOS. Method:This is a descriptive analytical study with a cross-sectional design. The sample consisted of granulosa cells from ovarian follicles and blood serum were obtained from women at the Yasmin RSCM Clinic and IRSI Bunda Jakarta during April 2018 until February 2019. DNA isolation was carried out by bisulfite conversion, MSP, electrophoresis and band intensity analysis with ImageJ software. qPCR was performed on RNA isolates to obtain relative expression of mRNA genes CYP19A1, HSD17B1 and FSHR. Serum folic acid were analyzed by LC-MS / MS. Characteristic data was collected during informed consent session and hormonal data was obtained from medical records. Results: Analysis data from 30 subjects with PCOS and 30 subjects without PCOS folic acid levels did not differ between the two groups (p = 0.341), there was incerased methylation of DNA in the CYP19A gene (p = 0,000) and FSHR (p = 0,000), but decreased methylation HSD17B1 gene (p = 0.160). There was a decrease in mRNA levels in the CYP19A1 gene (p = 0.040), HSD17B1 (0.003) and FSHR (p = 0.015). Increased methylation of DNA FSHR was associated with a decreased mRNA (p = 0.001). Increased methylation of DNA is not related to folic acid serum in women PCOS CYP19A1 genes (p = 0.209), HSD17B1 (0.567) and FSHR (p = 0.835). The female estradiol level of PCOS (25.78 pg / ml) was lower than that of non-PCOS women (36.74pg / ml). AMH levels of PCOS women (6.49 ng / ml) were higher than those of non-PCOS women (3.98 ng / ml). The higher the AMH level in women with PCOS, the lower the FSH and mRNA levels of female FSHR PCOS. Conclusion: Folic acid levels in PCOS patients are not a cause of methylation. Incerased methylation of CYP19A1 and FSHR genes and decreased CYP19A1, HSD17B1 and FSHR mRNA expression result in impaired work of steroidogenesis which lead to inadequate estradiol, thereby contributes to the etiology of PCOS.
Keywords: Steroidogenesis, PCOS, folic acid, DNA methylation, mRNA expression, CYP19A1, HSD17B1 and FSHR genes.