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"ABSTRAKLatar Belakang: Metode seleksi embrio terbaik banyak dikembangkan untuk memperoleh transfer embrio tunggal dalam siklus fertilisasi in vitro (FIV). Blastokista merupakan tahap embrio terbaik yang didapat dari embrio dengan morfologi normal atau 2 pro-nukleus (2PN) maupun morfologi abnormal atau 3 pro-nukleus (3PN). Transfer blastokista masih memberikan tingkat keberhasilan implantasi dan kehamilan yang rendah karena penilaian morfologi yang baik tidak selalu mempunyai status kromosom yang baik. Aneuploidi dan mosaik diketahui masih ditemukan dalam populasi blastokista, sehingga perlu diketahui faktor-faktor yang menyebabkan kelainan tersebut. Tujuan dari penelitian ini adalah mengetahui hubungan antara morfologi dan perkembangan blastokista dengan kejadian aneuploidi dan mosaik, serta faktor yang mempengaruhinya.Metode: Penelitian dilakukan dengan metode potong lintang. Embrio diperoleh dari siklus FIV dan dikultur sampai tahap blastokista, penilaian morfologi blastokista dilakukan menggunakan skoring Gardner dengan 3 parameter yaitu grade ekspansi blastokista, inner cell mass, dan tropektoderm. pre-implantation genetic testing for aneuploidi (PGT-A) dilakukan dengan cara biopsi blastokista pada hari ke 5 atau 6 dan analisis kromosom dilakukan menggunakan metode Next Generation Sequencing (NGS). Hubungan antara morfologi blastokista dan status kromosom serta faktor yang mempengaruhinya kemudian dianalisis.Hasil: Didapatkan 70 embrio dari 25 pasien, dengan pembagian 45 embrio 2PN dan 25 embrio 3PN. Frekuensi aneuploidi pada sampel 2PN dan 3PN berturut turut 42% dan 4%, sedangkan mosaik 2PN 16% dan 3PN 8%, pada embrio 3PN juga didapati kromosom triploid sebesar 52%. Didapatkan hubungan bermakna antara grade tropektoderm embrio 2PN dengan status kromosom (p<0,05), sedangkan embrio 3PN tidak didapati hubungan antara parameter morfologi dengan kromosom. Blastokista dengan grade ³3AA memiliki persentase euploidi sebesar 46%. Usia maternal, usia paternal, jumlah oosit, laju fertilisasi, volume dan motilitas sperma berhubungan dengan status kromosom (p<0,05).Kesimpulan: Grading blastokista berhubungan dengan status kromosom, dan usia maternal merupakan faktor yang paling berhubungan dengan kejadian aneuploidi dan mosaik.

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ABSTRACTBackground: Several method of assessing embryo viability have been employed over these year to obtain single embryo transfer in IVF cycle. Blastocyst, known as the best morphological embryo derived from both normal or 2 pronucleus (2PN) and 3 pronucleus (3PN) still not give the best implantation and pregnancy rate. However, morphology assesment only did not properly evaluate chromosomal status of the embryos. A good morphology blastocyst still can harbour aneuploidy and mosaic, and it is needed to find factor that effect aneuploidy and mosaic. Therefore, the aim of this study was to investigate the correlation between blastocyst morphology with aneuploidy, mosaicism and other related factor.Methods: A cross-sectional study to compare blastocyst morphology with chromosomal status. Embryo collected from IVF-ICSI cyles then cultured until blastocyst stage, Blastocyst scoring was done by Gardner scoring system with 3 parameter including blastocyst expansion, inner cell mass and tropectoderm. Preimplantation genetic testing for aneuploidi (PGT-A) was done by tropectoderm biopsy on day 5 or 6 which were the screened for chromosomal status by Next Generation Sequencing (NGS) method. The relationship between blastocyst morphology and chromosomal status and other related factor were evaluated.Results: A total 70 blastocyst were collected, 45 derived from 2PN zygot and the other 25 from 3PN zygot. The frequency of aneuploid in 2PN and 3PN continuosly 42% and 4%, meanwhile mosaic frequency were 15% and 8%. Triploidy chromosome were found 52% in 3PN embryo. Tropectoderm grading and chromosomal status was found significally different (p<0.05), while in 3PN embryo there in no correlation between blastocyst morphology and chromosomal status. >3AA blasctocyst grading had higher euploidy percentage compare to <3AA grading. Maternal age, oocyte number, fertilization rate, sperm volume and sperm motility have correlation with chromosomal status (p<0.05).Conclusions: Blastocyst grading had correlation with chromosomal status, and maternal age is the key factor that affect aneuploidy and mosaicism."

"ABSTRAKHipospadia merupakan salah satu kelainan genitalia paling umum yang ditemukan pada bayi lelaki baru lahir, yang ditandai adanya meatus uretra di ventral dan bentuk abnormal dari kulup penis. Etiologi hipospadia sebagian besar masih belum diketahui, tetapi dilaporkan dipengaruhi oleh faktor genetik dan lingkungan. Salah satu kelainan genetik adalah defek gen steroid 5 alpha-reductase type 2 (SRD5A2). Belum banyak laporan mengenai faktor genetik pada gen SRD5A2 yang melatarbelakangi hipospadia di Indonesia. Teknik PCR-sekuens dilakukan pada ekson 1-5 gen SRD5A2 pada 40 sampel DNA arsip penyandang hipospadia, dan PCR-RFLP pada ekson 1 dan 4 untuk mendeteksi mutasi p.Val89Leu dan p.Arg227Glu. Hasil penelitian mendapatkan 8 mutasi pada gen SRD5A2, yaitu mutasi p.Gly34Fs, p.Arg50His, p.Val89Leu 3 di ekson 1, p.Tyr128Cys di ekson 2, p.Asn193Ser dan p.Arg227Gln di ekson 4, p.Ile253Val di ekson 5, dan c.281+15T>C di intron 1. Studi in silico untuk memprediksi fungsi dan struktur protein adalah possibly dan/atau probably damaging untuk p.Gly34Fs, p.Arg50His, p.Tyr128Cys, p.Asn193Ser, dan p.Arg227Gln, dan benign untuk p.Val89Leu, Ile253Val, dan c.281+15T>C. Mutasi p.Arg50His dan p.Ile253Val merupakan mutasi baru yang belum pernah dilaporkan di populasi lain. Penelitian ini mendapatkan 8 mutasi pada penyandang hipospadia di Indonesia dan 2 diantaranya merupakan mutasi baru. Selain itu penelitian ini berhasil mengembangkan teknik PCR-RFLP untuk mendeteksi substitusi p.Val89Leu dan p.Arg227Glu. Teknik tersebut dapat diterapkan untuk

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ABSTRACT

Hypospadias is one of the most common external genitalia congenital abnormalities found in newborn baby boys, which is characterized by urethral opening, penile curvature, and abnormal distribution of the penis foreskin. The etiology of hypospadia is mostly unknown, but it is believed that hypospadias are caused by genetic and environmental factors. There have not been many reports on variations of steroid 5 alpha-reductase type 2 (SRD5A2) gene underlying hypospadias in Indonesia. The PCR-sequencing technique on exon 1-5 SRD5A2 gene were performed on 40 archived DNA samples from hypospadias cases of aged 0-29 years, and PCR-RFLP on exon 1 and 4 to detect mutation p.Val89Leu and p.Arg227Glu. The sequencing result showed that there were eight different mutations identified in the SRD5A2 gene, p.Gly34Fs, p.Arg50His, p.Val89Leu 3 in exon 1, p.Tyr128Cys in exon 2, p.Asn193Ser dan p.Arg227Gln in exon 4, p.Val89Leu in exon 5, and c.281+15T>C in intron 1. In silico analysis showed 5 mutations predicted to be possibly and/or probably damaging (p.Gly34Fs, p.Arg50His, p.Tyr128Cys, p.Asn193Ser, and p.Arg227Gln) and 3 benign mutations (p.Val89Leu, Ile253Val, and c.281+15T>C). p.Arg50His and p.Ile253Val are new mutations that have never been reported before. This study found 8 mutations obtained from hypospadias patients and successfully developed the PCR-RFLP technique to detect p.Val89Leu and p.Arg227Gln mutations, which can be applied as a starting point for mutation detection in places where the mutations frequently detected and access to sequencing technique is limited."

"[ABSTRAKLatar Belakang: Kelahiran prematur masih menjadi salah satu penyebabutama kematian pada neonatus. Diseluruh dunia kematian akibat kelahiranprematur menempati posisi kedua pada anak usia dibawah lima tahun. Kelahiranprematur dapat disebabkan oleh komplikasi dari ibu, janin dan plasenta.Insufisiensi plasenta merupakan komplikasi kehamilan dimana plasenta tidakdapat membawa oksigen dan nutrisi yang diperlukan untuk pertumbuhan janindalam uterus, sehingga menyebabkan berkurangnya suplai oksigen yangdiperlukan janin dan terjadi keadaan hipoksia dalam uterus. Cygb yang terdapatdalam plasenta yang berfungsi dalam metabolisme oksigen akan berusahamenkompensasi keadaan ini agar suplai oksigen kembali normal. Hipoksia yangterus menerus ini dapat menyebabkan meningkatnya reactive oxygen species(ROS). Pada neonatus prematur terjadi peningkatan ROS dapat melalui dua jalur,yaitu : pertama, tidak tersedianya antioksidan. Kedua, berkurangnya kemampuanuntuk meningkatkan pembentukan antioksidan sebagai respons dari hiperoksiaatau oksidan lain. ROS yang terbentuk akan ditanggulangi oleh antioksidan yangada sel baik yang enzimatik maupun nonenzimatik.Metode: Plasenta bayi prematur dibagi dalam dua kelompok berdasarkan statusoksigennya menjadi hipoksia dan non hipoksia. Kemudian dilakukan pengukuranekspresi mRNA dan protein Cygb, serta aktivitas antioksidan MnSOD, CAT, danGpx.Hasil: Terjadi peningkatan protein Cygb, akan tetapi terjadi penurunan ekspresimRNA Cygb. Terjadi penurunan aktivitas spesifik MnSOD, sedangkan CAT danGPx tidak berbeda bermakan. Analisis statistik menunjukan hubungan bermaknaantara aktivitas spesifik MnSOD dengan aktivitas spesifik GPx dan terdapathubungan yang bermakana antara mRNA Cygb dengan aktivitas spesifik MnSODpada neonatus prematur hipoksia dan tidak hipoksiaKesimpulan: Terjadi peningkatan protein Cygb dan penurunan mRNA Cygbuntuk mempertahankan homeostasis janin dalam keadaan hipoksia. Antioksidanpada bayi prematur lebih rendah, akan tetapi hal ini akan dibantu oleh Cygb dalammengeliminasi ROS yang ada dalam tubuh, terlihat dari penurunan aktivitasspesifik MnSOD pada plasenta prematur hipoksia, sedangkan aktivitas spesifikkatalase dan GPx relatif sama.

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ABSTRACTBackground: Preterm birth is still one of the main causes of mortality inneonates. Nowadays, preterm birth is the second most common cause of death inchildren younger than five years. Preterm birth can be caused by complications ofthe mother, fetus and placenta. Placenta insufficiency is complication ofpregnancy, where the placenta can not carry oxygen and nutrients for fetus growthin uterus, that lead to decrease oxygen supplies for the fetus and hypoxia inuterus. Cygb in placenta, that have function in oxygen metabolism will try tocompensate this situation, so the oxygen suplies will back to normal. The hypoxiawill increase reactive oxygen species (ROS). In preterm neonates the increase ofROS is cause by: First, there is no antioxidant supplies. Second, the lack ofantioxidant respon to hyperoxsia or other oxidan ROS will be eliminate byantioxidant system with in the cell.Methods: Placenta from preterm neonates divided in teo groups, hypoxia and nonhypoxia. And the sample is measure for mRNA Cygb expression, Cygb proteins,and antioxidant activity for MnSOD, CAT and GPx.Results: The Cygb protein increase in placenta neonates hypoxia, but theexpression of mRNA Cygb decrease in placenta neonates hypoxia. There isdecrease of MnSOD specific activity in placental neonates hypoxia, but not inCAT and GPx. Statistical analysis show correlation between MnSOD specificactivity with GPx specific activity, and correlation between mRNA Cygb withMnSOD specific activity.Conclusion: There is an increase of Cygb protein and decrease of Cygb mRNA inplacental neonates hypoxia, to maintain the neonates homeostasis in hypoxiaenvironment. Antioxidant is lower in preterm, Cygb with the capability toeliminate free radical will help antioxidant to reduce the ROS. It was seen at thedecrease of MnSOD specific activity, and the katalase and GPx specific activity isrelatively the same in plasenta hipoksia and non hipoksia, Background: Preterm birth is still one of the main causes of mortality inneonates. Nowadays, preterm birth is the second most common cause of death inchildren younger than five years. Preterm birth can be caused by complications ofthe mother, fetus and placenta. Placenta insufficiency is complication ofpregnancy, where the placenta can not carry oxygen and nutrients for fetus growthin uterus, that lead to decrease oxygen supplies for the fetus and hypoxia inuterus. Cygb in placenta, that have function in oxygen metabolism will try tocompensate this situation, so the oxygen suplies will back to normal. The hypoxiawill increase reactive oxygen species (ROS). In preterm neonates the increase ofROS is cause by: First, there is no antioxidant supplies. Second, the lack ofantioxidant respon to hyperoxsia or other oxidan ROS will be eliminate byantioxidant system with in the cell.Methods: Placenta from preterm neonates divided in teo groups, hypoxia and nonhypoxia. And the sample is measure for mRNA Cygb expression, Cygb proteins,and antioxidant activity for MnSOD, CAT and GPx.Results: The Cygb protein increase in placenta neonates hypoxia, but theexpression of mRNA Cygb decrease in placenta neonates hypoxia. There isdecrease of MnSOD specific activity in placental neonates hypoxia, but not inCAT and GPx. Statistical analysis show correlation between MnSOD specificactivity with GPx specific activity, and correlation between mRNA Cygb withMnSOD specific activity.Conclusion: There is an increase of Cygb protein and decrease of Cygb mRNA inplacental neonates hypoxia, to maintain the neonates homeostasis in hypoxiaenvironment. Antioxidant is lower in preterm, Cygb with the capability toeliminate free radical will help antioxidant to reduce the ROS. It was seen at thedecrease of MnSOD specific activity, and the katalase and GPx specific activity isrelatively the same in plasenta hipoksia and non hipoksia]"

"Penilaian morfokinetik embrio dipakai untuk seleksi embrio. Penelitian cohort ini bertujuan untuk evaluasi hubungan antara jumlah salinan mtDNA di cumulus granulosa cells (CGCs) dengan parameter morfokinetik embrio dan status kromosom. Perhitungan jumlah salinan mtDNA menggunakan real-time PCR pada 129 sample CGCs dari 30 pasien yang mengikuti program IVF-IMSI di Morula IVF Jakarta antara Juli-Oktober 2020. Hubungan antara jumlah salinan mtDNA di CGCs dengan semua parameter menggunakan analisa bivariate dan multiple. Terdapat hubungan signifikan antara jumlah salinan mtDNA di CGCs dengan pencapaian blastokista setelah dikontrol variabel usia maternal dan morfologi sperma (coefficient 0.832, p-value = 0.032, RR value 2.299). Hubungan signifikan pada jumlah salinan mtDNA di CGCs dengan fase awal perkembangan embrio M1 (t2-t8), dengan persamaan M1 adalah 5.702-0.271 jumlah salinan mtDNA di CGCs + 0.017 usia maternal + 0.013 motilitas sperma – 0.115 morfologi sperma (p-value = 0.032). Ditemukan hubungan tidak signifikan antara jumlah salinan mtDNA di CGCs dengan parameter morfokinetik lainnya (M2: tC-tEB, M3: t2-tEB, DC, RC, MN dengan P> 0.05), serta dengan status kromosom embrio (euploid: 139.44 ± 133.12, aneuploid: 142.40 ± 111.30, p= 0.806). Penelitian ini menunjukkan bahwa jumlah salinan mtDNA di CGCs merupakan biomarker untuk memprediksi pencapaian blastokista dan fase awal perkembangan embrio, tetapi tidak status kromosom.

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This cohort study evaluates the association between the mtDNA copy number in cumulus granulosa cells (CGCs) with embryo morphokinetic parameters and chromosomal status. mtDNA copy number of 129 CGCs from 30 patients undergoing the IVF-IMSI program at Morula IVF Jakarta between July-October 2020 were analyzed using real-time PCR. Bivariate and multiple analyses were conducted to see its relationship with all parameters. There was a significant correlation between the mtDNA copy number and the blastocyst after adjusting the maternal age and sperm morphology (coefficient 0.832, p-value = 0.032, RR value 2.299). A significant link was observed between mtDNA copy number in CGCs and early embryo developmental phase M1 (t2-t8), with the equation of M1 is 5.702 - 0.271 mtDNA copy number of CGCs + 0.017 maternal age + 0.013 sperm motility -0.115 sperm morphology (p-value = 0.032). No correlation was found between the mtDNA copy number in CGCs with the other morphokinetic parameters (M2: tC-tEB, M3: t2-tEB, DC, RC, MN with p> 0.05), and the chromosomal status (euploid: 139.44 ± 133.12, aneuploid: 142.40 ± 111.30, p= 0.806). mtDNA copy number in CGCs can serve as a useful biomarker for blastocyst status and early embryo developmental phase but not for chromosomal status."

"Human milk oligosaccharides (HMO) adalah karbohidrat yang terdiri dari 3–10 monosakarida dan tidak dapat dicerna oleh manusia. Fungsi HMO adalah prebiotik untuk mikrobiota usus. Metabolit yang dihasilkan mikrobiota adalah asam lemak rantai pendek (short chain fatty acid/SCFA). Sintesis HMO ditentukan oleh enzim fukosiltransferase 2 (FUT2) dan fukosiltransferase 3 (FUT3), yang disandi gen FUT2 dan FUT3. Polimorfisme gen FUT2 menyebabkan perbedaan HMO pada ASI. Ibu dengan kadar 2’fukosillaktosa (2’FL) ≥ 50 mg/L disebut ibu sekretor. Proporsi ibu sekretor bervariasi, karena polimorfisme gen FUT2 berbeda antar ras. Proporsi sekretor di Eropa > 80%, namun belum ada data di Indonesia. Penelitian ini bertujuan menganalisis proporsi sekretor dan polimorfisme gen FUT2, serta profil SCFA berdasarkan pasangan genotipe ibu-bayi. Penelitian menggunakan desain potong lintang, dilakukan di RSIA Bunda selama bulan Desember 2021–Juli 2022. Subjek penelitian adalah ibu berusia minimal 18 tahun, menyusui eksklusif, dan sehat. Ibu dengan ras Kaukasia di atas 2 generasi dieksklusi. Bayi dari ibu yang memenuhi kriteria inklusi automatis menjadi subjek penelitian dan dieksklusi bila bayi pernah mendapat antibiotik. Pemeriksaan HMO dilakukan saat bayi berusia 2–5 minggu, sedangkan SCFA feses bayi saat usia 4 minggu. Sekuensing coding region FUT2 dilakukan pada ibu dan bayi. Sebanyak 120 pasangan ibu-bayi memenuhi kriteria inklusi dengan proporsi ibu fenotipe sekretor 65,8% dan genotipe sekretor 65,8%. Hubungan antara genotipe FUT2 dan kadar 2’FL bermakna. Penelitian ini menemukan varian baru c.851C>G yang bersifat merusak berdasarkan prediksi in silico. Berdasarkan genotipe FUT2, diusulkan nilai ambang baru 2’FL 425,9 mg/L dengan nilai sensitivitas 98,7% dan spesifisitas 100%. Tidak terdapat hubungan antara proporsi relatif asetat, propionat, butirat dan genotipe ibu, genotipe bayi, maupun pasangan genotipe ibu-bayi.

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Human milk oligosaccharides (HMO) are complex carbohydrates consisting of 3–10 monosaccharides which is undigestible to human. HMO acts as a prebiotic for gut microbiota, which produce short chain fatty acid (SCFA). The synthesis of HMO is determined by the activity of fucosyltransferase 2 (FUT2) and fucosyltransferase 3 (FUT3) enzymes, which are encoded by the FUT2 and FUT3 genes. Polymorphisms of the FUT2 gene result in different secretor status. Mothers with 2'-fucosyllactose (2'FL) level of ≥ 50 mg/L are referred to as secretor. The proportion of secretor varies worldwide due to FUT2 polymorphisms among races. The proportion of secretor in Europe is generally > 80%, but there is no data on secretor status in Indonesia. Thus, baseline data about secretor phenotype and genotype status in Indonesia is needed. This study aimed to analyze the proportion of secretor and FUT2 gene polymorphism in Indonesia, as well as the stool SCFA profile based on the mother-infant dyad genotype.

This was a cross-sectional study conducted at Bunda Mother and Child Hospital from December 2021 to July 2022. The study subjects were healthy mothers aged at least 18 years, exclusively breastfeeding. Mothers with Caucasion ancestor from two generations above were excluded. Infants from eligible mothers were automatically included as study subjects but excluded if they had history of antibiotic administration. Breastmilk samples were obtained at infant’s age 2–5 weeks old, while infant’s stool at 4 weeks old. Sequencing of the entire coding region of FUT2 was performed for mothers and infants.

A total of 120 mother-infant dyads met the eligibility criteria. The proportion of secretor mother was 65.8%. Secretor genotypes were found in 65.8% of mothers. There was a significant association between secretor genotype and 2’FL level. A novel variant was identified, c.851C>G, which showed deleterious effect based on in silico analysis. A new threshold value of 425.9 mg/L for 2'FL is proposed, with 98.7% sensitivity and 100% specificity. There was no significant relationship between the relative proportion of acetate, propionate, butyrate, and valerate among the mother-infant’s genotype dyads."

"Latar Belakang: DNA bebas dalam medium kultur embrio dan kemajuan pemodelan berbasis kecerdasan buatan berpotensi menjadi modalitas uji genetik yang non-invasif. Saat ini, tidak diketahui apakah DNA tersebut dilepaskan oleh sel embrio euploid atau aneuploid, sehingga melemahkan dasar keilmuan penggunaannya. Penelitian ini bertujuan untuk mengetahui sel sumber embrio pelepas DNA bebas dalam medium kultur, validasi potensi klinis penggunaan DNA bebas untuk skrining status ploidi embrio pasien Fertilisasi In-Vitro (FIV), dan konstruksi model pembelajaran mendalam menggunakan gambar embrio tersegmentasi untuk deteksi status ploidi embrio.Metode: Penelitian ini terbagi dalam dua desain penelitian yaitu eksperimental in-vitro menggunakan embrio hewan model dan observasi kohort menggunakan 28 sampel medium kultur embrio dari 21 pasien program FIV di Klinik Morula IVF Jakarta, periode September 2022–Januari 2023. Konstruksi model pembelajaran mendalam menggunakan gambar embrio pasien FIV yang menjalani program bayi tabung periode Januari 2021– Juni 2023. Deteksi sel embrio sumber pelepas DNA bebas dilakukan dengan memapar salah satu embrio (galur DDY atau C57BL) dengan reversin untuk memperoleh blastomer pembawa sel-sel aneuploid. Embrio kontrol dikultur bersamaan tanpa reversin sebagai pembawa sel-sel blastomer euploid. Agregasi membentuk embrio mosaik dilakukan antara embrio pembawa blastomer aneuploid (perlakuan) dan embrio pembawa blastomer euploid (kontrol). Polimorfisme gen GABRA2 antara galur DDY (alel wildtype) dan C57BL (alel delesi) mejadi alel target yang dikuantifikasi dengan metode qPCR. Empat jenis sampel dibuat sebagai berikut: medium kultur tanpa embrio, medium kultur embrio agregasi tanpa pemaparan reversin, rev-DDY, rev-C57BL. Embrio mosaik diwarnai dengan marka apoptosis untuk deteksi mekanisme pelepasan DNA bebas. Analisis status ploidi embrio menggunakan medium kultur embrio pasien FIV dilakukan dengan metode sekuensing. Konstruksi model pembelajaran mendalam menggunakan gambar embrio tersegmentasi yang dipotong urut selama 10 jam sebelum proses biopsi. Variabel yang diamati dalam penelitian adalah konsentrasi alel delesi dan wildtype gen GABRA2, jumlah sel terwarnai marka apoptosis, Pada sampel medium kultur embrio manusia, keberhasilan amplifikasi dan interpretasi hasil sekuensing, serta tingkat kesesuaian uji antara DNA bebas dengan biopsi trofoblas dianalisis. Kemampuan prediksi model berbasis kecerdasan buatan dinilai dengan akurasi dan loss. Analisis data penelitian dan konstruksi model pembelajaran mendalam menggunakan perangkat lunak SPPS versi 21, OpenEpi. pyhton.Hasil: Sebanyak 0,08 ng/reaksi alel wildtype ditemukan pada embrio mosaik dengan pemaparan reversin pada blastomer embrio DDY (rev-DDY, pembawa sel-sel aneuploid) dan 0,01 ng/reaksi alel delesi ditemukan pada embrio mosaik dengan pemaparan reversin pada blastomer embrio C57BL (rev-C57BL, pembawa sel-sel aneuploid). Median jumlah sel embrio terwarnai marka apoptosis antara ketiga group embrio (agregasi kontrol, rev- DDY dan rev-C57BL) tidak berbeda bermakna (nilai p = 0,95 untuk pewarnaan late apoptosis (propidium iodide) dan p = 0,42 untuk early apoptosis (Ann-V) menandakan adanya proses koreksi sel pada kedua group embrio mosaik selama masa perkembangan pra-implantasi. Keberhasilan amplifikasi DNA bebas medium kultur embrio manusia dalah 100%, dengan nilai interpretasi 92,8% (26/28). Nilai kesesuaian DNA bebas dengan biopsi trofoblas adalah rendah sebesar 65,4% (17/26) dengan kesesuaian kromosom seks adalah 61,5% (16/26). Sepuluh dari 11 embrio XY pada biopsi trofoblas terdeteksi XX pada DNA bebas. Seluruh model pembelajaran mendalam mengalami peningkatan akurasi menggunakan gambar embrio tersegmentasi dengan algoritma InceptionV3 mencapai akurasi tertinggi sebesar 0,67 dengan nilai loss sebesar 1,4.Kesimpulan: Sel embrio anueploid adalah sel sember pelepas DNA bebas medium kultur embrio pada embrio mosaik hewan coba mencit yang dilepaskan melalui mekanisme apoptosis. Embrio masik tersebut diperkirakan melakukan self-correction dengan mengeksklusi sel-sel aneploid untuk mempertahankan euploiditasnya. Rendahnya tingkat kesesuaian antara DNA bebas dengan biopsi trofoblas disebabbkan oleh adanya kontaminasi maternal yang ditandai dengan perubahaan koromosm seks yang signifikan. Penggunaan gambar blastosis tersegmentasi meningkatkan akurasi model prediksi pembelajaran mendalam.

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Background: Cell-free DNA and advanced artificial intelligence-based modeling uphold the potential of a non-invasive approach to determining embryo ploidy status. The specific embryonic cells (whether euploid or aneuploid) that release cell-free DNA are largely unknown, causing a weak scientific basis for its use. This study aimed to identify the source of embryonic cells releasing cell-free DNA in culture media, validate the clinical potential of using cell-free DNA to screen embryo ploidy status in an in-vitro fertilization (IVF) program and develop a deep learning model using segmented embryo images to detect embryo ploidy status.

Materials and Methods: This study employed two research designs including an in-vitro experimental study using animal model embryos and an observational cohort study using 28 samples of spent embryo culture media from 21 patients undergoing IVF program at Morula IVF Clinic Jakarta (September 2022 to January 2023). A deep learning model was constructed using images of embryos from IVF patients who participated in IVF program from January 2021 to June 2023. Detection of the source embryonic cells releasing cell-free DNA was achieved by exposing embryos (DDY or C57BL strains) to reversine to induce the formation of blastomeres carrying aneuploid cells. Control embryos were cultured simultaneously without reversine to serve as the source of euploid blastomeres. Mosaic embryo aggregation was performed by combining embryos carrying aneuploid blastomeres (treatment) with those carrying euploid blastomeres (control). The GABRA2 gene polymorphism between the DDY strain (wildtype allele) and the C57BL strain (deletion allele) was the target allele quantified using qPCR. Four types of samples were prepared: culture medium without embryos, culture medium of aggregated embryos without reversine exposure, rev-DDY, and rev-C57BL. Mosaic embryos were stained with an apoptosis marker to detect the mechanism of cell-free DNA release. The ploidy status of embryos using spent embryo culture media from IVF patients was determined using sequencing methods. The deep learning model was constructed using segmented images of embryos captured over 10 hours before the biopsy process. The variables observed in the study included the concentration of deletion and wildtype alleles of the GABRA2 gene, the number of cells stained with apoptosis markers, the success rate of amplification and interpretation of sequencing results from human spent embryo culture medium samples, and the concordance rate between cell-free DNA and trophectoderm biopsy analysis. The predictive ability of the artificial intelligence-based model was evaluated using accuracy and loss metrics. Data analysis and deep learning model construction were performed using SPSS version 21, OpenEpi, and Python.

Results: A total of 0.08 ng/reaction of the wildtype allele was detected in the culture media sample of mosaic embryos exposed to reversine in DDY embryo blastomeres (rev- DDY, carrying aneuploid cells), and 0.01 ng/reaction of the deletion allele was found in the sample exposed to reversine in C57BL embryo blastomeres (rev-C57BL, carrying aneuploid cells). The median number of embryonic cells stained with apoptosis markers among the three groups of embryos (control aggregation, rev-DDY, and rev-C57BL) did not differ significantly (p = 0.95 for late apoptosis staining with propidium iodide and p = 0.42 for early apoptosis with Annexin V), indicating the presence of cell correction processes in both groups of mosaic embryos during pre-implantation development. The success rate of cell-free DNA amplification in human spent embryo culture media was 100%, with an interpretability of 92.8% (26/28). The concordance between cell-free DNA and trophectoderm biopsy was low at 65.4% (17/26), with sex chromosome concordance at 61.5% (16/26). Ten out of eleven XY embryos from the trophectoderm biopsy were detected as XX in cell-free DNA analysis. All deep learning models showed improved accuracy using segmented embryo images with the InceptionV3 algorithm, achieving the highest accuracy of 0.67 with a loss of 1.4.

Conclusion: Aneuploid embryonic cells were identified as the source releasing cell-free DNA in culture media during embryo animal model experiments, releasing DNA through an apoptotic mechanism. These mosaic embryos were expected to activate embryonic cell correction mechanisms by excluding aneuploid cells to maintain their euploidy. The low concordance rate between cell-free DNA and trophectoderm biopsy was attributed to maternal contamination, as indicated by significant changes in sex chromosomes. The use of segmented blastocyst images improved the model's accuracy.

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