Embryo Life

Dr. Yasmin Magdi Abd-Elkreem

Embryo: The collection of cells (2n) that has developed from the fertilized oocyte (male n + female n), before all the major organs have developed.

In vitro

In vivo.Steps oh human embryo Pre-implantation development:1. Fertilization2. cleavage3. blastulation

Embryo Selection • Most Optimal selection profile would be through taking into

consideration:

1. Chromosomal integrity.

2. Appropriate genomic activation.

3. Metabolic activity.

4. Morphological changes and dynamics appropriate for the culture period Microscopic embryo assessment most used technique for being non-invasive, non-traumatic, simple, cost effectiveness and correlated with implantation and pregnancy rates techniques which help assess embryos without causing damage .

1. Chromosomal integrity• based on preimplantation genetic diagnosis (PGD), a procedure used to test

early human embryos for serious inherited genetic conditions and chromosomal abnormalities.

• Normal embryos were those in which each cell had two chromosomes of each kind.

Aneuploid :embryos could be monosomic or trisomic. Monosomic : embryos were those in which the same chromosome was missing

in each cell. Trisomic : embryos were those in which each cell had three chromosomes of the

same type instead of two. Haploid : Embryos in which all the cells had only a single chromosome of each

kind. Polyploid :embryos had three or more copies of each chromosome in each cell.

1. Chromosomal integrityPGD is recommended when:

1. Familial Single Gene Disorders2. Familial Sex-linked Disorders3. Familial Chromosomal Disorders4. Non-familial Chromosomal Disorders Associated with Advanced

Reproductive Age5. Non-familial Chromosomal Disorders Associated with Infertility

(recurrent implantation failure )6. Sex Selection 7. Select embryos with a genetic impairment seen in a parent

Two types of assessment techniques are common:a. chromosome “painting” (or FISH)b. genetic testing for specific disease loci (PCR or gene chips)

2. Appropriate genomic activation.

• Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA), and is informally termed gene regulation.

• With the emergence of new technologies such as Omics, the gene expression profiling of human oocytes, embryos, and hESCs has been performed and generated a flood of data related to the molecular signature of early embryo development.

3. Metabolic activity• Adenosine triphosphate (ATP) is the universal currency of energy

in biological systems (supplied by the Mitochondria).

• Too high and too low ATP production can be lethal to the cell, and any mitochondrial dysfunction may be critical to the embryo.

• spectrophotometric tests are used to measure metabolomic changes , in proteomic (proteins) profiling, in amino acid profiling, amino acid depletion and production , respiration-rate measurement.

• Pyruvate/Glucose uptake• Amino acids• Oxygen consumption (Respirometry)

ProductionGlucosePyruvate

Amino Acids

Other Sugars

Oxygen

Other Peptides & Factorsμl drop of defined culture medium

Modified from: Gardner and Leese(1993) Assessment of embryo metabolism and viability. In: Handbook of In Vitro Fertilization. EdsTrounson& Gardner CRC Press. pp195-211.

Lactate

Ammonium

Amino Acids

Enzymes, eg LDH

sHLA-G

HOXA10 regulator

PAFMetabolomics / Proteomics

3. Metabolic activityUptake

4. Morphological assessment• The best available method for embryo selection.• Microscopic embryo assessment most used technique • non-invasive, non-traumatic• Simple• cost effectiveness • correlated with implantation and pregnancy rates.

4. Morphological assessment

ObservationTiming (hours

post-insemination)

Expected stage of development

Fertilization check 17 ± 1 h Pronuclear stage

Syngamy check 23 ± 1 hUp to 50% in syngamy(up to 20% at the 2-cell

stage)

Early cleavage check

26 ± 1 h post-ICSI

28 ± 1 h post-IVF2-cell stage

Day 2 assessment 44 ± 1 h 4-cell stageDay 3 assessment 68 ± 1 h 8-cell stageDay 4 assessment 92 ± 2 h MorulaDay 5 assessment 116 ± 2 h Blastocyst

Standardized timing relative to insemination time

Report cell number/stage and grade (separately) – with time post-insemination

4. Morphological assessmentPronuclear Embryo Assessment • Evaluation 16-18 hours following oocyte insemination (IVF) or

ICSI.

• Pronucleus stadium/PN : the distribution pattern of the nucleoli (precursor bodies) can be determined through an expensive digitalized, image-generating method.

• Normal Fertilization: is assessed by two centrally positioned, juxtaposed PNs with clearly defined membranes and two polar bodies.

• There are different systems for 2PN scoring:

A] Zygote-Score System (Revised zygote scoring)B] Initial zygote scoring systemC] Ideal Features described by Tesarik and Greco

4. Morphological assessmentCleavage Embryo AssessmentThe embryo classification systems are based on the

evaluation of 1. The number of blastomeres. 2. The degree of fragmentation.3. The symmetry of the blastomeres.4. the presence of multinucleation.5. The compaction status.

4. Morphological assessment24-28 Hours : first cleavage, 2-cells stage

40-44 Hours: number of blastomeres should be 4-6, symmetric, fragmentation of less than 20%, and no multinucleated blastomeres.

64-68 Hours: number of blastomeres should be 8-12 , symmetric, fragmentation less than 20%, and no multinucleated blastomeres.

4. Morphological assessmentBlastocyst Assessment • Evaluation 104-110 hours after insemination or ICSI.

• Blastocyst cavity should be full, inner cell mass should be numerous and tightly packed, trophectoderm should be numerous and cohesive.

The Gardner and Schoolcraft scoring system

1. Degree of expansion2. ICM morphology3. TE morphology4. Cellular degeneration in blastocysts5. Cytoplasmic strings/bridges between ICM and TE 6. Other morphological features (vacuolation , More than one point of natural hatching)

4. Morphological assessment• For example:

Conclusions……….

• Embryo assessment is one of the most critical procedures that play a role in the success of IVF/ART

• Traditional embryo assessment is challenged by different factors, ie, subjectivity, low efficiency

• “Non-invasive” morphological assessment may provide valuable additional information to optimize embryo assessment and maximize the chances of IVF success

Thank You for your time…….

For contact: E-mail: Yas.magdi@hotmail.com