Expansion Grade and Morphokinetic Parameters Associated with Aneuploidy Status of Embryo Fifth Day: Tingkat Ekspansi dan Parameter Morfokinetik yang Diasosiasikan dengan Status Aneuploid pada Hari k Lima Sebuah Embrio

Objective: To determine the potential of examining embryo morphology, and morphokinetic parameters in predicting the chromosomal status of embryos. Methods: This study is a cross-sectional that requires patients undergoing IVF followed by chromosome examination with NGS that was conducted at the IVF Center at Pondok Indah Hospital and Morula IVF Center at Bunda Hospital from December 2021 to December 2022. Each embryo that reaches the blastocyst stage on day 5 or 6 will be washed and put into a PCR tube for a week; then, embryologists annotate them to determine morphological assessment and morphokinetic parameters using Time-Lapse Microscopy. The chi-square test was used to analyse bivariate variables. Results: One hundred twenty-four samples were collected on day 5 of patients undergoing the IVF procedure. 50.8% of the samples were aneuploid chromosomes, and 49.2% were euploid. The morphokinetic characteristics median was 3.86 fold. It was found that expansion grade, time to pro-nuclear fading, and time to the synchrony of the third cell cycle were significantly associated with euploid status (p = 0.000; 0.041 and 0.036). Conclusion: The expansion grade has been proven as the most influential component for accurately predicting the ploidy status of embryos. Keyword: blastocyst, embryo, euploid status, expansion grade, morphokinetics   Abstrak Tujuan: Untuk mengetahui potensi pemeriksaan morfologi embrio, dan parameter morfokinetik dalam memprediksi status kromosom embrio. Metode: Penelitian ini merupakan penelitian potong lintang yang memerlukan pasien yang sedang menjalani program bayi tabung yang dilanjutkan dengan pemeriksaan kromosom dengan NGS yang dilakukan di Pusat IVF RS Pondok Indah dan Pusat IVF Morula RS Bunda pada bulan Desember 2021 hingga Desember 2022. Setiap embrio yang mencapai tahap blastokista pada hari ke 5 atau 6 akan dicuci dan dimasukkan ke dalam tabung PCR selama seminggu; kemudian, ahli embriologi membuat anotasi untuk menentukan penilaian morfologi dan parameter morfokinetik menggunakan Mikroskop Time-Lapse. Uji chi-square digunakan untuk menganalisis variabel bivariat. Hasil: Seratus dua puluh empat sampel dikumpulkan pada hari ke 5 dari pasien yang menjalani prosedur IVF. 50,8% sampel adalah kromosom aneuploid, dan 49,2% adalah euploid. Median karakteristik morfokinetik sebesar 3,86 kali lipat. Ditemukan bahwa tingkat ekspansi, waktu menuju pemudaran pro-nuklir, dan waktu hingga sinkronisasi siklus sel ketiga berhubungan secara signifikan dengan status euploid (p = 0,000; 0,041 dan 0,036). Kesimpulan: Tingkat ekspansi telah terbukti sebagai komponen yang paling berpengaruh dalam memprediksi status ploidi embrio secara akurat. Kata kunci: blastosist, embrio, status euploid, tingkat ekspansi, morfokineti

Integrating imaging-based classification and transcriptomics for quality assessment of human oocytes according to their reproductive efficiency

PURPOSE: Utilising non-invasive imaging parameters to assess human oocyte fertilisation, development and implantation; and their influence on transcriptomic profiles. METHODS: A ranking tool was designed using imaging data from 957 metaphase II stage oocytes retrieved from 102 patients undergoing ART. Hoffman modulation contrast microscopy was conducted with an Olympus IX53 microscope. Images were acquired prior to ICSI and processed using ImageJ for optical density and grey-level co-occurrence matrices texture analysis. Single-cell RNA sequencing of twenty-three mature oocytes classified according to their competence was performed. RESULT(S): Overall fertilisation, blastulation and implantation rates were 73.0%, 62.6% and 50.8%, respectively. Three different algorithms were produced using binary logistic regression methods based on "optimal" quartiles, resulting in an accuracy of prediction of 76.6%, 67% and 80.7% for fertilisation, blastulation and implantation. Optical density, gradient, inverse difference moment (homogeneity) and entropy (structural complexity) were the parameters with highest predictive properties. The ranking tool showed high sensitivity (68.9-90.8%) but with limited specificity (26.5-62.5%) for outcome prediction. Furthermore, five differentially expressed genes were identified when comparing "good" versus "poor" competent oocytes. CONCLUSION(S): Imaging properties can be used as a tool to assess differences in the ooplasm and predict laboratory and clinical outcomes. Transcriptomic analysis suggested that oocytes with lower competence may have compromised cell cycle either by non-reparable DNA damage or insufficient ooplasmic maturation. Further development of algorithms based on image parameters is encouraged, with an increased balanced cohort and validated prospectively in multicentric studies

Cytoplasmic strings as potential markers of embryo development and clinical outcome.

Aims: The first study investigated the agreement between embryologists when assessing cytoplasmic strings (CS) in human blastocysts. The second study investigated the idea that embryos originating from fresh ejaculate sperm is comparable to those originating from frozen-thawed ejaculate sperm. The third study investigated and expanded on the association between the presence of CS, blastocyst quality and live birth rate. The fourth study used timelapse technology to investigate whether the presence of CS in human blastocysts was associated with a clinical pregnancy. Methods: In the first study, CS activity in one hundred blastocysts was assessed to measure inter- & intra-observer agreement. In the second study, timelapse was used to observe embryo development in those originating from fresh ejaculate sperm compared to those originating from frozen-thawed ejaculate sperm. In the third study, timelapse was used to investigate the association between the presence of CS and their characteristics, with blastocyst quality, development and live birth in one thousand day 5 human blastocysts. In the fourth study, timelapse was used to assess CS presence in blastocysts to investigate the link between those blastocysts that formed a fetal heart after transfer. Results: From the first study, a moderate level of inter and intra observer agreement was observed when five embryologist assessed day 5/6 blastocysts for the presence of CS and their vesicles. From the second study, it was found that no differences were detected in key developmental events between embryos originating from fresh ejaculate sperm compared to their frozen-thawed counterparts. The third study confirmed that CS presence in human blastocysts is associated with blastocyst quality. The fourth study, the presence of CS in human blastocysts was found to be associated with a fetal heart. Conclusions: The first study showed a moderate level of inter- and intra-observer agreement when the presence of CS and their vesicles was assessed. The second study found that there are no differences in the morphokinetic parameters of early embryo development when either fresh or frozen ejaculate sperm was used for ICSI insemination. The third study confirmed that CS presence in human blastocysts is associated with blastocyst quality. The fourth study found that the presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart

ENDOMET database – A means to identify novel diagnostic and prognostic tools for endometriosis

Endometriosis is a common benign hormone reliant inflammatory gynecological disease that affects fertile aged women and has a considerable economic impact on healthcare systems. Symptoms include intense menstrual pain, persistent pelvic pain, and infertility. It is defined by the existence of endometrium-like tissue developing in ectopic locations outside the uterine cavity and inflammation in the peritoneal cavity. Endometriosis presents with multifactorial etiology, and despite extensive research the etiology is still poorly understood. Diagnostic delay from the onset of the disease to when a conclusive diagnosis is reached is between 7–12 years. There is no known cure, although symptoms can be improved with hormonal medications (which often have multiple side effects and prevent pregnancy), or through surgery which carries its own risk. Current non-invasive tools for diagnosis are not sufficiently dependable, and a definite diagnosis is achieved through laparoscopy or laparotomy. This study was based on two prospective cohorts: The ENDOMET study, including 137 endometriosis patients scheduled for surgery and 62 healthy women, and PROENDO that included 138 endometriosis patients and 33 healthy women. Our long-term goal with the current study was to support the discovery of innovative new tools for efficient diagnosis of endometriosis as well as tools to further understand the etiology and pathogenesis of the disease. We set about achieving this goal by creating a database, EndometDB, based on a relational data model, implemented with PostgreSQL programming language. The database allows e.g., for the exploration of global genome-wide expression patterns in the peritoneum, endometrium, and in endometriosis lesions of endometriosis patients as well as in the peritoneum and endometrium of healthy control women of reproductive age. The data collected in the EndometDB was also used for the development and validation of a symptom and biomarker-based predictive model designed for risk evaluation and early prediction of endometriosis without invasive diagnostic methods. Using the data in the EndometDB we discovered that compared with the eutopic endometrium, the WNT- signaling pathway is one of the molecular pathways that undergo strong changes in endometriosis. We then evaluated the potential role for secreted frizzled-related protein 2 (SFRP-2, a WNT-signaling pathway modulator), in improving endometriosis lesion border detection. The SFRP-2 expression visualizes the lesion better than previously used markers and can be used to better define lesion size and that the surgical excision of the lesions is complete.ENDOMET tietokanta – Keino tunnistaa uusi diagnostinen ja ennustava työkalu endometrioosille Endometrioosi on yleinen hyvänlaatuinen, hormoneista riippuvainen tulehduksellinen lisääntymisikäisten naisten gynekologinen sairaus, joka kuormittaa terveydenhuoltojärjestelmää merkittävästi. Endometrioositaudin oireita ovat mm. voimakas kuukautiskipu, jatkuva lantion alueen kipu ja hedelmättömyys. Sairaus määritellään kohdun limakalvon kaltaisen kudoksen esiintymisenä kohdun ulkopuolella sekä siihen liittyvänä vatsakalvon tulehduksena. Endometrioosin etiologia on monitahoinen, ja laajasta tutkimuksesta huolimatta edelleen huonosti tunnettu. Kesto taudin puhkeamisesta lopullisen diagnoosin saamiseen on usein jopa 7–12 vuotta. Sairauteen ei tunneta parannuskeinoa, mutta oireita voidaan lievittää esimerkiksi hormonaalisilla lääkkeillä (joilla on usein monia sivuvaikutuksia ja jotka estävät raskauden) tai leikkauksella, johon liittyy omat tunnetut riskit. Nykyiset ei-invasiiviset diagnoosityökalut eivät ole riittävän luotettavia sairauden tunnistamiseen, ja varma endometrioosin diagnoosi saavutetaan laparoskopian tai laparotomian avulla. Tämä tutkimus perustui kahteen prospektiiviseen kohorttiin: ENDOMET-tutkimuk-seen, johon osallistui 137 endometrioosipotilasta ja 62 terveellistä naista, sekä PROENDO-tutkimukseen, johon osallistui 138 endometrioosipotilasta ja 33 terveellistä naista. Tässä tutkimuksessa pitkän aikavälin tavoitteemme oli löytää uusia työkalujen endometrioosin diagnosointiin, sekä ymmärtää endometrioosin etiologiaa ja patogeneesiä. Ensimmäisessä vaiheessa loimme EndometDB –tietokannan PostgreSQL-ohjelmointi-kielellä. Tämän osittain avoimeen käyttöön vapautetun tietokannan avulla voidaan tutkia genomin, esimerkiksi kaikkien tunnettujen geenien ilmentymistä peritoneumissa, endo-metriumissa ja endometrioosipotilaiden endometrioosileesioissa EndometDB-tietokantaan kerättyjä tietoja käytettiin oireiden ja biomarkkeripohjaisen ennustemallin kehittämiseen ja validointiin. Malli tuottaa riskinarvioinnin endometrioositaudin varhaiseen ennustamiseen ilman laparoskopiaa. Käyttäen EndometDB-tietokannan tietoja havaitsimme, että endo-metrioositautikudoksessa tapahtui voimakkaita geeni-ilmentymisen muutoksia erityisesti geeneissä, jotka liittyvät WNT-signalointireitin säätelyyn. Keskeisin löydös oli, että SFRP-2 proteiinin ilmentyminen oli huomattavasti koholla endometrioosikudoksessa ja SFRP-2 proteiinin immunohistokemiallinen värjäys erottaa endometrioosin tautikudoksen terveestä kudoksesta aiempia merkkiaineita paremmin. Löydetyllä menetelmällä voidaan siten selvittää tautikudoksen laajuus ja tarvittaessa osoittaa, että leikkauksella on kyetty poistamaan koko sairas kudos