Nuevas estrategias para estimar la calidad embrionaria y el éxito de las embrio-transferencias mediante la evaluación no invasiva y selección automática en sistemas de time-lapse

La introducción de la tecnología de lapso de tiempo en la práctica clínica de fecundación in vitro permitió la supervisión ininterrumpida de los embriones durante todo el período de cultivo. Inicialmente, el objetivo principal era lograr un mejor desarrollo embrionario. Sin embargo, esta tecnología también introdujo el novedoso concepto de la morfocinética, parámetros relacionados con la dinámica de las células embrionarias. La gran cantidad de datos obtenidos permitió definir los rangos óptimos de la duración del ciclo celular en diferentes etapas del desarrollo del embrión, lo cual añadió información complementaria para asistir en la evaluación del embrión antes de la transferencia. Los marcadores cinéticos se convirtieron en parte de la estrategia de evaluación embrionaria con un gran potencial en aumentar las probabilidades de éxito clínico. Sin embargo, la anotación de estos parámetros aún depende de la subjetividad y experiencia de los profesionales que realizan las anotaciones. El uso de algoritmos de aprendizaje profundo para analizar eventos de desarrollo automáticamente es un paso hacia la implementación de Inteligencia Artificial en la evaluación de embriones, que se está convirtiendo en una tendencia importante en el futuro. El objetivo principal de la presente tesis es la optimización de la selección de embriones a través de protocolos no invasivos, con el objetivo de estandarizar la transferencia de un solo embrión en la práctica clínica. Se presta especial atención a la validación de una herramienta desarrollada mediante técnicas de Inteligencia Artificial para la automatización de la anotación de parámetros morfocinéticos y la evaluación de la metabolómica embrionaria mediante el análisis del estrés oxidativo en los medios de cultivo. Para ello, se establecieron tres objetivos específicos principales: Objetivo específico I. Caracterización de la sensibilidad y precisión de detección de los eventos de desarrollo del embrión humano preimplantacional mediante un software automatizado. Objetivo específico II. Comparación de la predicción de los resultados clínicos al aplicar las anotaciones morfocinéticas manuales y automatizadas de embriones con implantación conocida. Objetivo específico III. Análisis no invasivo del estado oxidativo del medio de cultivo de los embriones en combinación con la morfocinética de sistemas de lapso de tiempo. Gracias a la consecución de objetivos específicos, podemos concluir que el software de evaluación de embriones puede ser una alternativa automatizada y objetiva de anotar los parámetros morfocinéticos. Las anotaciones automatizadas aliviarían la carga de trabajo de los embriólogos, especialmente durante los eventos tempranos, los cuales se anotaron con gran precisión. Por otro lado, los eventos tardíos, pese a ser más variables, mostraron una mayor relevancia clínica en la predicción de resultados cuando se utilizaron algoritmos de selección publicados. Los eventos no anotados siempre pueden añadirse manualmente, lo cual aumenta la precisión de los datos. El ensayo de termoquimioluminiscencia (TCL) se validó con éxito como una herramienta no invasiva para realizar el análisis del estrés oxidativo de los medios de cultivo del embrión. La combinación de los parámetros oxidativos de TCL con los criterios morfocinéticos de lapso de tiempo presentaron un mayor poder discriminatorio que la evaluación morfológica en la identificación de embriones de alta calidad, sentando las bases para un método de selección de embriones más objetivo y no invasivo para reducir las transferencias múltiples de embriones.The introduction of time-lapse imaging to clinical in vitro fertilization practice enabled the undisturbed monitoring of embryos throughout the entire culture period. Initially, the main objective was to achieve a better embryo development. However, this technology also provided an insight into the novel concept of morphokinetics, parameters regarding embryo cell dynamics. The vast amount of data obtained defined the optimal ranges in the cell-cycle lengths at different stages of embryo development, which added valuable information to embryo assessment prior to transfer. Kinetic markers became part of embryo evaluation strategies with the potential to increase the chances of clinical success. However, the annotation of these parameters still depend on the subjectivity and experience of the professionals performing the annotations. The use of deep learning algorithms to analyze developmental events automatically is a step towards implementation of Artificial Intelligence into embryo assessment, which is becoming a significant trend in the future. The present thesis major research target is the optimization of embryo selection though non-invasive protocols, aiming for the standardization of single embryo transfer as gold standard. Special focus being given to the validation of an Artificial Intelligence developed tool for the automatization of the morphokinetic parameter annotation and assessment of embryo metabolomics through the analysis of oxidative stress in the spent culture media. For this, three main specific objectives were stablished: Specific objective I. Characterization of the detection sensitivity & accuracy when performing the annotation of the human preimplantation embryo developmental events with an automated software. Specific objective II. Clinical result prediction comparison when applying the manual and automated morphokinetic annotations of known implantation data embryos. Specific objective III. Non-invasive oxidative status analysis of the spent embryo culture medium in combination with Time-Lapse morphokinetics. Thanks to our specific objective attainment, we can conclude the embryo assessment software can be an automated and objective alternative of annotating the morphokinetic parameters. Automated annotations would ease the embryologists’ workload, especially during early events, which can be annotated with high accuracy. On the other hand, the more variable later events showed more clinical relevance in outcome prediction when using published embryo selection algorithms. Non-annotated events can still be annotated manually, increasing the accuracy of the data. The thermochemiluminescence (TCL) assay was successfully validated as a non-invasive tool to perform the analysis of the oxidative stress of the spent culture media of the embryo. The combination of the TCL oxidative parameters with the time-lapse morphokinetic criteria presented a greater discriminatory power than morphological assessment in the identification of high-quality embryos, providing the foundation for a more objective and non-invasive embryo selection method to reduce multiple embryo transfers

A longitudinal study of the experiences and psychological well-being of Indian surrogates

Study question: What is the psychological well-being of Indian surrogates during and after the surrogacy pregnancy? Summary answer: Surrogates were similar to a matched group of expectant mothers on anxiety and stress. However, they scored higher on depression during and after pregnancy. What is known already: The recent ban on trans-national commercial surrogacy in India has led to urgent policy discussions regarding surrogacy. Whilst previous studies have reported the motivations and experiences of Indian surrogates no studies have systematically examined the psychological well-being of Indian surrogates, especially from a longitudinal perspective. Previous research has shown that Indian surrogates are motivated by financial payment and may face criticism from their family and community due to negative social stigma attached to surrogacy. Indian surrogates often recruited by agencies and mainly live together in a “surrogacy house.” Study design, size, duration: A longitudinal study was conducted comparing surrogates to a matched group of expectant mothers over two time points: (a) during pregnancy (Phase1: 50 surrogates, 70 expectant mothers) and (b) 4–6 months after delivery (Phase 2: 45 surrogates, 49 expectant mothers). The Surrogates were recruited from a fertility clinic in Mumbai and the matched comparison group was recruited from four public hospitals in Mumbai and Delhi. Data collection was completed over 2 years. Participants/materials, setting, methods: Surrogates and expectant mothers were aged between 23 and 36 years. All participants were from a low socio-economic background and had left school before 12–13 years of age. In-depth faceto-face semi-structured interviews and a psychological questionnaire assessing anxiety, stress and depression were administered in Hindi to both groups. Interviews took place in a private setting. Audio recordings of surrogate interviews were later translated and transcribed into English. Main results and the role of chance: Stress and anxiety levels did not significantly differ between the two groups for both phases of the study. For depression, surrogates were found to be significantly more depressed than expectant mothers at phase 1 (p = 0.012) and phase 2 (p = 0.017). Within the surrogacy group, stress and depression did not change during and after pregnancy. However, a non-significant trend was found showing that anxiety decreased after delivery (p = 0.086). No participants reported being coerced into surrogacy, however nearly all kept it a secret from their wider family and community and hence did not face criticism. Surrogates lived at the surrogate house for different durations. During pregnancy, 66% (N = 33/50) reported their experiences of the surrogate house as positive, 24% (N = 12/50) as negative and 10% (N = 5/50) as neutral. After delivery, most surrogates (66%, N = 30/45) reported their experiences of surrogacy to be positive, with the remainder viewing it as neutral (28%) or negative (4%). In addition, most (66%, N = 30/45) reported that they had felt “socially supported and loved” during the surrogacy arrangement by friends in the surrogate hostel, clinic staff or family. Most surrogates did not meet the intending parents (49%, N = 22/45) or the resultant child (75%, N = 34/45). Limitations, reasons for caution: Since the surrogates were recruited from only one clinic, the findings may not be representative of all Indian surrogates. Some were lost to follow-up which may have produced sampling bias. Wider implications of the findings: This is the first study to examine the psychological well-being of surrogates in India. This research is of relevance to current policy discussions in India regarding legislation on surrogacy. Moreover, the findings are of relevance to clinicians, counselors and other professionals involved in surrogacy. Trial registration number: N/A

Assessment of Pre-Eclampsia Risk in Early Pregnancy

My thesis aims to improve pre-eclampsia prevention and outcomes in Australian women by providing evidence that can be used to enhance pre-eclampsia risk assessment in broad antenatal settings. I conducted the thesis in three stages. In stage 1, a systematic review identified major limitations in the existing evidence for current guideline approaches and ‘simple’ risk models incorporating maternal characteristics to identify women at increased risk of pre-eclampsia in early pregnancy. While the review demonstrated models including specialised tests such as uterine artery Doppler and serum biomarkers improved sensitivity to predict pre-eclampsia compared to simple models, there was insufficient evidence to compare the performance of simple models versus clinical guideline decision rules; and few simple models had been externally validated. To address these gaps, I conducted studies with the aim to validate current guideline approaches and published simple models; and to develop and validate a simple risk model for predicting pre-eclampsia in settings where specialised tests are not available. Studies were undertaken using the Perinatal Antiplatelet Review of International Studies (PARIS) clinical trials dataset (Stage 2); and the Western Sydney Local Health District’s ObstetriX database (Stage 3). The findings support the use of current guideline approaches to offer a simple and specific approach for predicting pre-eclampsia. Analysis of the PARIS dataset demonstrated the methodological challenges for developing and validating simple risk models to improve on guideline approaches. The PARIS model supported the value of including maternal mean arterial pressure (MAP) in simple models for pre-eclampsia prediction. Analysis of Western Sydney data demonstrated ethnic variation in the risk of preeclampsia independent of established clinical risk factors. These findings support the inclusion of ethnicity for pre-eclampsia risk assessment in Australia’s diverse multiethnic population. Further research is needed to understand the underlying causes for the reduced risk of preeclampsia observed for immigrant women. The Western Sydney (WS) risk model achieved modest performance for prediction of pre-eclampsia in nulliparous women. The model did not outperform the current antenatal guidelines; but has the advantage of providing individualised risk estimates to better inform patient counselling and guide decisions for further testing and prophylaxis. Further research is needed to assess the impact of adding MAP to the WS model. These findings can inform the development of Australian antenatal guidelines for the preeclampsia risk assessment and contribute towards the development of an improved nation-wide pre-eclampsia preventive strategy

Screening of fetal trisomies 21, 18 and 13 by noninvasive prenatal testing. Rapid assessment of other health technologies using the HTA Core Model for Rapid Relative Effectiveness Assessment.

The aim of this collaborative assessment is to evaluate the relative effectiveness and safety of noninvasive prenatal testing (NIPT) for the screening of fetal trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13) in pregnant women of at least 8–9 weeks’ gestation. Five screening pathways are considered for the purpose of NIPT assessment: 1. NIPT as a primary screening test (total replacement of first trimester combined testing (FCT)) 2. NIPT as part of FCT (replacement of serum testing) 3. NIPT as an add-on to FCT for the high-risk population 4. NIPT as an add-on to FCT for the high- and intermediate-risk population 5. NIPT as a replacement for invasive testing The comparator, chosen by application of EUnetHTA criteria, is first-trimester serum screening (pregnancy-associated plasma protein A (PAPP-A) and β subunit of human chorionic gonadotropin (β-hCG)) and/or an ultrasound scan to measure fetal nuchal translucency (NT) or fetal crown-rump length (CRL) and maternal age. Fetal karyotyping or birth outcomes determined through clinical examination or follow-up of the newborn are considered the reference standards. The effectiveness of the screening processes is evaluated in terms of secondary outcomes (sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV)) but also in terms of primary outcomes, reducing unecessary invasive tests, assessing the impact on children born with undiagnosed T13, T18 and T21, natural miscarriages or stillbirths, and miscarriages related to invasive testing (amniocentesis or chorionic villus sampling (CVS)). False positive (FP) rates, false negative (FN) rates and test failures were chosen as critical safety issues. The increase in the number of children born with other major prenatally undetected chromosomal conditions/ anomalies (not targeted by prenatal aneuploidy screening) and the increase in elective pregnancy termination for other chromosomal anomalies with uncertain significance were considered important safety issues. Several organisational, ethical and social outcomes were also considered of relevance. Randomised controlled clinical trials, nonrandomised controlled clinical trials and diagnostic test accuracy (DTA) studies on the index test, the comparator and the reference standard (crosssectional studies) are included for the effectiveness and safety domain. In addition, registries are included for the safety domain and qualitative studies and consensus documents are included for the organisational, ethical and social domains

Prostaglandin E2 and prostaglandin F2 alpha as endometrial receptivity biomarkers in successful embryo implantation

Failure in the adhesion of the human blastocyst to the endometrium has been described as an important cause of infertility. Establishing the period of the so-called window of implantation and understanding the molecular mechanisms associated with embryo implantation has clinical and scientific implications. While over the last decades histological evaluation has been used to determine the phase of the menstrual cycle of the endometrium, the poor information obtained has made the case of using new technologies to identify specific markers, understand and characterize the receptive stage. This doctoral thesis investigates the existence, function and clinical impact of two specific lipids, the prostaglandins E(2) and F(2α), which are abundant in human endometrial fluid (EF) during the window of implantation in natural, IVF, and ovum recipient cycles, which is abrogated with the insertion of an IUD. Developments in endometrial receptivity diagnosis using lipidomics demonstrate the correlation between those prostaglandins (PGs) and the receptive stage of the endometrium. The mechanisms that influence the production of these individual PGs in the endometrium were studied with a clinical approach that sheds light on the sequence of events that leads to the development of endometrial receptivity. Our results indicate that PG synthases required for the production of PGE2 and PGF2α are located in the endometrial epithelium and EF for the regulation of PGs concentrations during the window of implantation. Most of the accumulated evidence, using an in vitro model of embryonic adhesion, indicates that inhibition of PGE2 and PGF2α or the PG receptors EP2 and FP prevents embryo adhesion, which can be reversed by adding back these molecules or by using EP2 and FP agonists. Finally, our pilot study demonstrates that PGE2 and PGF2α concentrations in EF aspirated 24 hours prior to embryo transfer showed to be predictive of a successful pregnancy outcome. In summary, our findings indicate that embryo implantation is associated with an active crosstalk of PGE2 and PGF2α via EP2 and FP receptors, respectively, that might serve to nurse the blastocyst at the time of embryo implantation. Likewise the levels of these PGs in EF could potentially serve as non-invasive biomarkers to define the receptive phase of the endometrium and, therefore, have a significant impact in clinical translation