Identification and Standardization of a Method That Would Allow the Study of the Metabolic Profile of Blastocoele Lays the Foundation to Assess Blastocyst Metabolomic Profile and Its Relation With Embryo Morphology and Embryo Implantation

Study Overview
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Brief Summary
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While the number of assisted reproduction cycles increases worldwide, the introduction of actual technological improvements in the ability to quickly and non-invasively identify the best embryos for transfer still represents a critical goal for reproductive medicine. Indeed, embryo assessment is currently performed through the analysis of morphology and cleavage rate. Recent studies have sought to identify a correlation between qualitative-quantitative profiles of small molecules of metabolic interest and the outcome of embryo transfer. Some of these molecules seem to be best suited for this purpose, including glucose, lactate, pyruvate or amino acid levels. Approaches relying on both optical and non-optical spectroscopy have been proposed to non-invasively monitor the embryo culture media. However, the non-invasive approach only offers an indirect strategy to monitor embryos and a turn-around solution to bypass the limits of detection of these analytical techniques. In this paper the investigators pave the way for direct assessment of embryos through the mass spectrometry-based analysis of blastocoele fluid, which is withdrawn from the blastocoele cavity prior to cryostorage of blastocysts. The investigators show how it is possible to detect most of the already documented metabolites of interest right at the very heart of the blastocyst, without disrupting the workflow of a classic laboratory pipeline.

Official Title
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Identification and Standardization of a Method That Would Allow the Study of the Metabolic Profile of Blastocoele Lays the Foundation to Assess Blastocyst Metabolomic Profile and Its Relation With Embryo Morphology and Embryo Implantation

Conditions
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Blastocoele Fluid

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Ovarian hyperstimulation disease Blastocyst formation

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 45 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Hyper1<br>Only patients who achieved hyperstimulation pathology after external administration of gonadotrophin during IVF treatment | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| standardize the method of aspiration | The method for blastocyst micropuncturing and aspiration of blastocoel fluid is according also to the last literature about blastocyst vitrification. In brief, expanded day 5 blastocysts were removed from culture and transferred to a 30 nl droplet of pre-warmed Hepes buffer. An injection pipette was introduced avoiding contaminations through the trophectoderm, and blastocoel fluid was aspirated until the blastocyst had fully collapsed around the pipette. The retrieved fluids were expelled into new purified water drops and frozen at -80°C alongside 0.5 nl control droplets of purified water. | 2 months | 
| metabolite detection | metabolite detection through rapid resolution reversed phase (RR-RP) high performance liquid chromatography (HPLC)-mass spectrometry (MS). From sample volumes as low as 0.5 nl we could detect and quantify against external standards a group of metabolites, whose roles in blastocyst development and embryo metabolism have long been postulated. The list included i) ATP adenosine triphosphate ; ii) glucose-6-phosphate; iii) lactate; iv) NAD+ nicotinamide-adenine dinucleotide+ and v) NADH and vi) NADPH nicotinamide-adenine dinucleotide phosphate; vii) 6-phosphogluconic acid; viii) glutamic acid and ix) α-ketoglutarate. | 6 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| analysis of the correlation between blastocyst morphology and metabolic profile in the blastocoele fluid | blastocyst classify according to morphological criteria in the book Atlas of Human Blastocyst by L. Veeck and associate, to each morphological class a characteristic metabolic profile, in order to scientifically validate the observational and objective criteria up to now used in our lab | 1 year | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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blastocoele fluid, mass spectrometry, metabolomics