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
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Purpose
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the way for direct assessment of embryos through the mass spectrometry-based analysis of blastocoele fluid, which is withdrawn from the blastocoele cavity prior to cryostorage of blastocysts.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.
| Condition |
|---|
|
Blastocoele Fluid |
| Study Type: | Observational |
| Study Design: | Observational Model: Case-Only Time Perspective: Retrospective |
| Official Title: | 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 |
- standardize the method of aspiration [ Time Frame: 2 months ] [ Designated as safety issue: Yes ]
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.
- metabolite detection [ Time Frame: 6 months ] [ Designated as safety issue: Yes ]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; ii) glucose-6-phosphate; iii) lactate; iv) NAD+ and v) NADH and vi) NADPH; vii) 6-phosphogluconic acid; viii) glutamic acid and ix) α-ketoglutarate.
- analysis of the correlation between blastocyst morphology and metabolic profile in the blastocoele fluid [ Time Frame: 1 year ] [ Designated as safety issue: Yes ]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
Biospecimen Retention: Samples Without DNA
blastocoele fluid
| Estimated Enrollment: | 100 |
| Study Start Date: | September 2011 |
| Estimated Study Completion Date: | January 2013 |
| Estimated Primary Completion Date: | January 2012 (Final data collection date for primary outcome measure) |
| Groups/Cohorts |
|---|
|
Hyper1
Only patients who achieved hyperstimulation pathology after external administration of gonadotrophin during IVF treatment
|
Eligibility| Ages Eligible for Study: | 18 Years to 45 Years |
| Genders Eligible for Study: | Female |
| Accepts Healthy Volunteers: | No |
| Sampling Method: | Probability Sample |
All the patients that are unable to be subjected to embryo transfer due to her ovarian hyperstimulation. In those cases the embryos will be vitrified and during this procedure we collapse the blastocyst sucking the fluid.
Inclusion Criteria:
- Ovarian hyperstimulation disease
- Blastocyst formation
Contacts and Locations| Contact: Simone Palini, biology | +39 339 4572101 | simonepalini@yahoo.it |
| Contact: Silvia De Stefani, Biotecnology | +39 320 1111937 | silvia.destefani83@libero.it |
| Italy | |
| Cervesi Hospital | Not yet recruiting |
| Cattolica, Rimini, Italy, 47841 | |
| Contact: Silvia De Stefani, Biotecnology +39 320 1111937 silvia.destefani83@libero.it | |
| Contact: Simone Palini, biology +39 339 4572101 simonepalini@yahoo.it | |
| Study Director: | Simone Palini, biology | Cervesi Hospital, Cattolica, Italy |
More Information
No publications provided
| Responsible Party: | Palini Simone, Embryologist, Cervesi Hospital, Cattolica, Italy |
| ClinicalTrials.gov Identifier: | NCT01427413 History of Changes |
| Other Study ID Numbers: | SS4e |
| Study First Received: | August 29, 2011 |
| Last Updated: | August 31, 2011 |
| Health Authority: | Italy: Comitato Etico di Area Vasta Romagna e I.R.S.T. |
Keywords provided by Cervesi Hospital, Cattolica, Italy:
|
blastocoele fluid mass spectrometry metabolomics |
ClinicalTrials.gov processed this record on June 18, 2013