Non-invasive pre-implantation genetic testing's reliability for aneuploidy using Cell-free DNA in embryo culture media.

OBJECTIVE: The presence of embryonic cell-free DNA (cfDNA) in spent embryo culture media (SECM) may offer valuable advantages for non-invasive testing of embryo ploidy or genetic characteristics compared to trophectoderm (TE) biopsy. This study aimed to assess the diagnostic potential of SECM cfDNA as a non-invasive sample for chromosomal copy number testing in blastocysts within the clinical setting of in-vitro fertilization. METHOD: This prospective observational study collected 28 SECM cfDNA samples matched with TE biopsy samples from 21 infertile couples who underwent IVF-PGT-A cycles. SECM samples were obtained from blastocysts that were cultured for approximately 5/6 days in an uninterrupted time-lapse incubator. Both sets of samples were collected during the biopsy procedure. The Variseq Illumina platform was utilized for ploidy measurement. The study evaluated the informativity and interpretability of SECM cfDNA, concordance of general ploidy status, and sex chromosome agreement between the two sample types. RESULTS: SECM cfDNA had a high informativity rate (100 %) after double amplification procedure, with a result interpretability of 93 %. Two out of the 28 SECM cfDNA samples were uninterpretable and regarded as overall noise samples. The diagnostic potential of SECM cfDNA, when compared to TE biopsy the standard reference, was relatively low at 50 %. Maternal DNA contamination remains the major obstacle that hinders the widespread clinical adoption of SECM cfDNA in the routine practice of pre-implantation genetic testing for aneuploidy within IVF settings. CONCLUSION: A significant modification must be implemented in the IVF laboratory to minimize DNA contamination and this necessitates suggesting adjustments to oocyte denudation, embryo culture media preparation, and sample collection procedures.

Vitrification alters growth differentiation factor 9 and follicle-stimulating hormone receptor expression in human cumulus-mural granulosa cells.

Objective: Ovarian tissue vitrification is widely utilized for fertility preservation in prepubertal and adolescent female patients with cancer. The current literature includes reports of successful pregnancy and live birth following autografting. However, the effects of the vitrification process on cumulus-mural granulosa cells (C-mGCs)-somatic cells in ovarian tissue crucial for oocyte maturation and early embryonic development-remain unclear. This study was conducted to explore the impact of vitrification on the cellular function of C-mGCs by quantifying the expression of growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15), follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), connexin 37, survivin, and caspase 3. Methods: Mature and immature C-mGCs were obtained from 38 women with polycystic ovary syndrome who participated in an in vitro fertilization program. The C-mGCs were then divided into two groups: fresh and vitrified. The expression levels of target genes were assessed using real-time quantitative polymerase chain reaction. Results: After vitrification, GDF-9 expression was significantly decreased among both mature and immature C-mGCs, with 0.2- and 0.1-fold changes, respectively (p<0.01). Similarly, FSHR expression in the mature and immature groups was reduced by 0.1- and 0.02-fold, respectively, following vitrification (p<0.01). The expression levels of the other genes, including BMP-15, LHR, connexin 37, survivin, and caspase 3, remained similar across the examined groups (p>0.05). Conclusion: Vitrification may compromise oocyte maturation through reduced GDF-9 and FSHR expression in C-mGCs after warming.

Reference range of placental growth factors at 11-14 weeks' gestation in Indonesia.

OBJECTIVES: To develop a reference chart for placental growth factor (PLGF) value during 11-14 weeks' gestation in Indonesian population. METHODS: This was an observational study observing women in their first trimester. Maternal characteristics, biophysical tests, and serum PLGF levels were collected during the visit. PLGF Multiple of Median (MoM) was adjusted for maternal characteristics including age, parity, smoking habits, diabetes mellitus, weight, height, body mass index, gestational age, and crown-rump length (CRL) utilizing the linear regression analysis. Plot distributions of PLGF level and PLGF MoM adjusted to CRL were developed using logistic regression technique. RESULTS: Out of 2.062 consecutive women undergoing 11-14 weeks' gestation ultrasound screening, the median of PLGF level and PLGF MoM were 50.38 pg/ml (1.09-265.20 pg/ml) and 1.00 (0.02-4.80), respectively. In the multivariate analysis, PLGF MoM was not significantly influenced by maternal factors such as age, parity, smoking habit, diabetes mellitus, height, weight, and BMI. The adjusted PLGF MoM reference chart according to the CRL was developed using quadratic linear regression. CONCLUSION: PLGF levels at 11-14 weeks' gestation were notably influenced by CRL but not by maternal characteristics. The usefulness of this parameter in combining with other established markers as a screening tool for the Indonesian population basis requires further investigation.

Embryo ploidy status classification through computer-assisted morphology assessment.

BACKGROUND: Preimplantation genetic testing for aneuploidy has been proven to be effective in determining the embryo's chromosomal or ploidy status. The test requires a biopsy of embryonic cells on day 3, 5, or 6 from which complete information on the chromosomes would be obtained. The main drawbacks of preimplantation genetic testing for aneuploidy include its relatively invasive approach and the lack of research studies on the long-term effects of preimplantation genetic testing for aneuploidy. OBJECTIVE: Computer-assisted predictive modeling through machine learning and deep learning algorithms has been proposed to minimize the use of invasive preimplantation genetic testing for aneuploidy. The capability to predict morphologic characteristics of embryo ploidy status creates a meaningful support system for decision-making before further treatment. STUDY DESIGN: Image processing is a component in developing a predictive model specialized in image classification through which a model is able to differentiate images based on unique features. Image processing is obtained through image augmentation to capture segmented embryos and perform feature extraction. Furthermore, multiple machine learning and deep learning algorithms were used to create prediction-based modeling, and all of the prediction models undergo similar model performance assessments to determine the best model prediction algorithm. RESULTS: An efficient artificial intelligence model that can predict embryo ploidy status was developed using image processing through a histogram of oriented gradient and then followed by principal component analysis. The gradient boosting algorithm showed an advantage against other algorithms and yielded an accuracy of 0.74, an aneuploid precision of 0.83, and an aneuploid predictive value (recall) of 0.84. CONCLUSION: This research study proved that machine-assisted technology perceives the embryo differently than human observation and determined that further research on in vitro fertilization is needed. The study finding serves as a basis for developing a better computer-assisted prediction model.

The origin and possible mechanism of embryonic cell-free DNA release in spent embryo culture media: a review.

The presence of cell-free DNA in spent embryo culture media (SECM) has unveiled its possible utilization for embryonic ploidy determination, opening new frontiers for the development of a non-invasive pre-implantation genetic screening technique. While a growing number of studies have shown a high concordance between genetic screening using cell-free DNA (cfDNA) and trophectoderm (TE), the mechanism pertaining to the release of cfDNA in SECM is largely unknown. This review aims to evaluate research evidence on the origin and possible mechanisms for the liberations of embryonic DNA in SECM, including findings on the self-correction abilities of embryos which might contribute to the presence of cfDNA. Several databases including EMBASE, PUBMED, and SCOPUS were used to retrieve original articles, reviews, and opinion papers. The keywords used for the search were related to the origins and release mechanism of cfDNA. cfDNA in SECM originates from embryonic cells and, at some levels, non-embryonic cells such as maternal DNA and exogenous foreign DNA. The apoptotic pathway has been demonstrated to eliminate aneuploid cells in developing mosaic embryos which might culminate to the release of cfDNA in SECM. Nonetheless, there is a recognized need for exploring other pathways such as cross-talk molecules called extracellular vesicles (EVs) made of small, round bi-layer membranes. During in vitro development, embryos physiologically and actively expel EVs containing not only protein and microRNA but also embryonic DNA, hence, potentially releasing cfDNA of embryonic origin into SECM through EVs.

Genetic algorithm-assisted machine learning for clinical pregnancy prediction in in vitro fertilization.

BACKGROUND: A clinical pregnancy prediction model was developed by implementing machine learning technology that uses a combination of static images and medical data to calculate the outcome of an in vitro fertilization cycle. OBJECTIVE: To provide a system that can accurately and sufficiently assist with decision making that is critical to in vitro fertilization cycles, primarily embryo selection. STUDY DESIGN: Historical medical data, which consist of clinical information and a complete transferred embryo image dataset, of 697 patients who underwent unique in vitro fertilization were collected. Various techniques of machine learning were used, namely decision tree, random forest, and gradient boosting; each technique used the same data configuration for performance comparison and was subsequently optimized using genetic algorithm. RESULTS: A prediction model with a peak accuracy of approximately 65% was achieved. Significant differences in the performances of the 3 selected algorithms were apparent. Nonetheless, additional metric measurements, such as receiver operating characteristic, area under the receiver operating characteristic curve score, accuracy, and loss, suggested that the gradient boosting model performed the best in predicting clinical pregnancy. CONCLUSION: This study served as a stepping stone toward the application of in vitro fertilization prediction models that use machine learning techniques. However, additional validation steps are required to boost the model's performance for its implementation in the clinical setting.

A Homogeneous Ensemble of Robust Pre-defined Neural Network Enables Automated Annotation of Human Embryo Morphokinetics.

Background: The purpose of the current study was to reduce the risk of human bias in assessing embryos by automatically annotating embryonic development based on their morphological changes at specified time-points with convolutional neural network (CNN) and artificial intelligence (AI). Methods: Time-lapse videos of embryo development were manually annotated by the embryologist and extracted for use as a supervised dataset, where the data were split into 14 unique classifications based on morphological differences. A compilation of homogeneous pre-trained CNN models obtained via TensorFlow Hub was tested with various hyperparameters on a controlled environment using transfer learning to create a new model. Subsequently, the performances of the AI models in correctly annotating embryo morphologies within the 14 designated classifications were compared with a collection of AI models with different built-in configurations so as to derive a model with the highest accuracy. Results: Eventually, an AI model with a specific configuration and an accuracy score of 67.68% was obtained, capable of predicting the embryo developmental stages (t1, t2, t3, t4, t5, t6, t7, t8, t9+, tCompaction, tM, tSB, tB, tEB). Conclusion: Currently, the technology and research of artificial intelligence and machine learning in the medical field have significantly and continuingly progressed in an effort to develop computer-assisted technology which could potentially increase the efficiency and accuracy of medical personnel's performance. Nonetheless, building AI models with larger data is required to properly increase AI model reliability.

Birth of spinal muscular atrophy unaffected baby from genetically at-risk parents following a pre-implantation genetic screening: A case report.

Background: Spinal muscular atrophy (SMA) is characterized by the homozygous deletion of the survival motor neuron-1 gene. Pre-implantation genetic testing for monogenic diseases through in-vitrofertilization program was developed to provide a reliable genetic diagnostic method for SMA. Case presentation: The couple who was confirmed as carriers of SMA visited the Morula IVF Clinic, Jakarta, Indenesia seeking for an in-vitro fertilization expert opinion in relation to the pre-implantation genetic testing for SMA. Utilizing polymerase chain reaction-restriction fragment length polymorphism, we have successfully screened for unaffected embryos that were characterized by a normal presence of the survival motor neuron-1 exon 7-8 and survival motor neuron-2 exon 7-8. The frozen embryo was subsequently transferred and a healthy unaffected female baby was born with undetected deletion of the survival motor neuron-1 gene. Conclusion: This successful embryo pre-implantation screening case could potentially accommodate the demands of genetically at-risk couples who are apprehensive about conceiving a child who might inherit monogenic disorders such as SMA.

Potential Use of Immature Oocyte to Improve Fertility Preservation Outcome: A Narrative Review.

Fertility preservation through gamete vitrification has become one of the critical strategies to secure a childbearing potential in patients who are diagnosed with cancer or risks of infertility. Preserving the gametes would prevent the deleterious effects of cancer drugs or radiotherapy exposure on the quality of the gametes. Furthermore, in vitro fertilisation of vitrified mature human oocytes has lately demonstrated promising results that are reflected in the increased survival rate of thawed oocytes and the resultant clinical pregnancy rate. However, limitations in the cryopreservation of mature oocytes of cancer patients persist. Ovarian stimulation protocols which comprise administering gonadotrophin-releasing hormones could aggravate cancer or delay essential cancer therapy. Considering such circumstances, vitrification of immature oocytes would become a rational option. While the vitrification procedure of mature oocytes has been established, the vitrification of immature oocytes remains controversial due to a low post-thaw in vitro maturation and fertilisation rate. Apparent cryoinjuries to the immature oocytes post thawing or warming have been observed in both human and animal model oocytes. An alternative strategy was therefore proposed to improve the effectiveness of utilising immature oocytes for fertility preservation by conducting the in vitro oocyte maturation process first before vitrification. This method has prevailed, especially in oncofertility patients. Although the success rate of the clinical outcomes remains low, this approach, in conjugation with proper counselling, might provide oncofertility patients with an opportunity to preserve their reproductive potential.

Role of three-dimensional Doppler ultrasonography and leukemia inhibitory factor from endometrial secretion in predicting endometrial receptivity in IVF treatment: a pilot study.

PURPOSE: This pilot study aimed to evaluate the potential synergistic role of three-dimensional power Doppler angiography ultrasound and the expression of Leukemia Inhibitory Factor (LIF) protein in predicting the endometrial receptivity of fresh In-Vitro Fertilization (IVF) cycles. MATERIALS AND METHODS: This prognostic cohort study involved 29 good prognosis women who underwent fresh IVF cycles with fresh blastocysts transfer. Serial measurements of sub-endometrial parameters including vascularity index (VI), flow index (FI), and vascularization flow index (VFI) were conducted consecutively via power Doppler angiography on the day of oocyte maturation trigger, oocyte retrieval, and blastocyst transfer. Aspiration of endometrial secretion was performed on the day of embryo transfer. RESULTS: The mean index of VI and VFI on the trigger and oocyte retrieval day and also LIF protein concentration at the window of implantation were significantly higher in clinically pregnant women than that of the non-pregnant women (p < 0.05). The area under the curve (AUC) of VI and VFI was shown to have a powerful predictive value to forecast receptive endometrium on either trigger day (0.788 and 0.813, respectively) or oocyte retrieval day (0.813 and 0.818). Likewise, LIF concentration on the day of embryo transfer was adequate to become a predictor for endometrial receptivity (AUC 0.874). A combination of the VI and VFI on the trigger day and LIF concentration at specific cut-off values (VI > 5.381, VFI > 1.483, LIF 703.5 pg/mL) produced an algorithm with high AUC (0.881) and high specificity (94.4%) for an adequate prediction of non-receptive endometrium. CONCLUSION: VI and VFI index assessed on maturation trigger day and the expression of LIF protein concentration at the window of implantation provided sufficient information to predict endometrial receptivity. A large randomized control trial is needed to validate these findings.

Review of computer vision application in in vitro fertilization: the application of deep learning-based computer vision technology in the world of IVF.

In vitro fertilization has been regarded as a forefront solution in treating infertility for over four decades, yet its effectiveness has remained relatively low. This could be attributed to the lack of advancements for the method of observing and selecting the most viable embryos for implantation. The conventional morphological assessment of embryos exhibits inevitable drawbacks which include time- and effort-consuming, and imminent risks of bias associated with subjective assessments performed by individual embryologists. A combination of these disadvantages, undeterred by the introduction of the time-lapse incubator technology, has been considered as a prominent contributor to the less preferable success rate of IVF cycles. Nonetheless, a recent surge of AI-based solutions for tasks automation in IVF has been observed. An AI-powered assistant could improve the efficiency of performing certain tasks in addition to offering accurate algorithms that behave as baselines to minimize the subjectivity of the decision-making process. Through a comprehensive review, we have discovered multiple approaches of implementing deep learning technology, each with varying degrees of success, for constructing the automated systems in IVF which could evaluate and even annotate the developmental stages of an embryo.

Morphokinetics of embryos after IMSI versus ICSI in couples with sub-optimal sperm quality: A time-lapse study.

An investigation was conducted to determine the influence of two sperm selection modalities, IMSI and ICSI, on the morphokinetics, dynamic development and ploidy status of embryos derived from males with sub-optimal sperm profiles during IVF program. A total of 209 PGTA-tested top-quality blastocysts (IMSI = 129, ICSI = 80) from 84 couples (IMSI = 51, ICSI = 33) were assessed retrospectively. This study found that both IMSI and ICSI yielded comparable embryo morphokinetics, except for the T7, TEB and CC3 parameters (p < 0.05). A significant lower incidence of multinucleation was observed in the IMSI group when compared to the ICSI group (48.8% vs. 71.3%, p = 0.002), while other parameters of embryo development such as direct cleavage, distorted cytoplasmic movement, reverse cleavage and vacuole(s) appearance did not differ (p > 0.05). No differences were noticed in the proportion of generating chromosomally euploid embryos (44.2% vs. 51.3%, p = 0.394, respectively, for IMSI and ICSI). The implementation of IMSI or ICSI in couples with sub-optimal sperm profiles resulted in embryos with comparatively similar morphokinetics. Furthermore, the incidence of multinucleation at the two- to four-cell stage was lower following the practice of IMSI, although the method did not improve the proportion of gaining euploid embryos.

Robotic myomectomy for a non-pregnant reproductive age woman with severe acute urinary retention: A case report.

Urinary retention in non-pregnant reproductive age women is a very rare condition. Hereby, we reported a rare case of acute urinary retention in a non-pregnant reproductive-age woman with hydronephrosis and hydroureter due to a large fibroid. The fibroid had resulted in constant pressure to the urethral sphincter, which causes urinary retention. Robotic myomectomy was performed after insertion of ureteric catheters. To our knowledge, this is the first case report of a robotic surgery being utilised to manage acute urinary retention in a non-pregnant individual due to large fibroid.

In vitro Activation of Mouse Oocytes through Intracellular Ca2+ Regulation.

BACKGROUND: Ca2+ signaling pathway is suggested to play an essential role in mediating oocyte maturation. AIMS: The aim of this study was to evaluate intracellular Ca2+ of resistant immature oocytes that failed to resume meiosis following subsequent in vitro culture reach metaphase II after calcium ionophore A23187 activation. SETTINGS AND DESIGN: This in vitro analytical experimental study was conducted at Animal Science Laboratory of Indonesian Medical Education and Research Institute (IMERI), Human Reproductive Infertility and Family Planning of IMERI, and Electrophysiology Imaging of Terpadu Laboratory, Faculty of Medicine, University of Indonesia. METHODS: A total of 308 oocytes classed as resistant immature following in vitro culture were randomly allocated to control (n = 113) and treatment groups (n = 195). The oocyte activation group was exposed to A23187 solution for 15 min and then washed extensively. Maturation was evaluated by observing the first polar body extrusion 20‒24 h after A23187 exposure. Ca2+ imaging was conducted using a confocal laser scanning microscope to identify the dynamic of Ca2+ response. STATISTICAL ANALYSIS: SPSS 20, Chi-square, and Mann-Whitney U-test were used in this study. RESULTS: Activation of resistant immature oocytes with A23187 significantly increased the number of oocyte maturation compared with the control group (P < 0.001). Furthermore, fluorescent intensity measurements exhibited a significant increase in the germinal vesicle stage when activated (P = 0.005), as well as the metaphase I stage, even though differences were not significant (P = 0.146). CONCLUSION: Artificial activation of resistant immature oocyte using chemical A23187/calcimycin was adequate to initiate meiosis progress.

Embryo Banking with Mild Ovarian Stimulation for IVF: An Alternative Strategy for Poor Prognosis Patients.

BACKGROUND: Management of Poor Ovarian Reserve (POR) in in vitro fertilization remains a difficult challenge. The purpose of this retrospective cohort study was to compare the effectiveness of embryo banking strategy over a cohort of several mild stimulation cycles (Embryo Banking Strategy for Poor Prognosis/Embargo) to conventional full-dose antagonist protocol for IVF. METHODS: Subjects identified as having poor ovarian response (POR) based on the Bologna criteria were recruited. In total, there were 113 subjects included in the analysis. Fifty-three subjects underwent embryo banking procedure (Embargo) protocol, and sixty subjects underwent the conventional full-dose antagonist protocol for IVF. The Chi-square test was used to compare the clinical pregnancy rate, miscarriage rate as well as live birth rate, while the Mann-Whitney U test was utilized to analyze the cost per clinical pregnancy between the two groups. A p<0.05 was considered statistically significant. RESULTS: The two studied groups showed similar outcomes regarding clinical pregnancy rate, miscarriage rate, as well as live birth rate (p=0.966, p=0.310, and p= 0.469, respectively). Cost analysis of subjects who underwent mild ovarian stimulation followed by Embargo revealed the high cost of the protocol compared to conventional full-dose antagonist protocol ($10.507±6.181 vs. $9.533±2.530, p=0.002). CONCLUSION: The clinical outcomes of both protocols were comparable. Embargo procedure was not efficient in improving the overall clinical outcomes in patients who were expected poor ovarian responders as the protocol costed more comparing with conventional full-dose antagonist protocol. A larger prospective randomized control trial is needed to evaluate this finding.