The Process of Fertilization and Embryo Development up to Implantation
Aspirated oocytes have three main different morphologies:
Prophase I - the germinal vesicle (GV) is present Metaphase I - the first polar body has not been extruded and there is no GV
Metaphase II - The first polar body is present
In-Vitro Fertilization (IVF)
Insemination of oocytes
Oocytes are inseminated between 3 and 20 hours after recovery.
There are various methods of preparing the spermatozoa for IVF insemination. The normal "swim-up" technique involves mixing a portion of the ejaculate with culture medium, centrifuging it, discarding the supernatant containing the seminal plasma, and overlaying fresh culture medium onto the pellet containing the spermatozoa.
During subsequent incubation of the pellet for 30-60 minutes the motile sperm migrate into the overlay medium. The medium containing these spermatozoa is then collected by aspiration with a pipette, and used for insemination. The purpose of this procedure is to:
- remove the seminal plasma, which inhibits fertilization
- concentrate the motile sperm
- remove contaminating cells and debris
Usually, motile sperm are added to oocytes at a concentration of 100,000 sperm/ml.
This stage takes place immediately after fertilization before the gametes have fused, and is visible for a short time at 18-22 hours after fertilization. The zygote shows two polar bodies, male and female pronuclei, prior to their fusion. These two pronuclei contain the DNA from each parent that will unite to form a new individual. The zona pellucida is still present.
One-cell embryo, the zygote, with the two pronuclei fused to one genetic substance
Two-cell embryo (day 1-2), usually starts at about 26 hours after sperm and eggs unite.
Four-cell embryo (day 2, approximately 48 hours after insemination). During each mitotic division the embryo does not increase in size, and divides the existing cytoplasm.
Day 3: Human embryo at the 8-cell stage (approximately 72 hours after insemination).
Six cells are visible in this focal plane
Several parameters are needed to score the quality of the embryo:
Rrate of development
Percentage of fragmentation
The following score (on embryo day 3) was suggested by Michael J. Tucker of the FIBiol HCLD Georgia Reproductive Specialists, Atlanta, USA, and appears on: http://www.ivf.com/morphology.html
1) Grade I; 2) Grade II; 3) Grade III; 4) Grade IV; 5) Grade V
Morula (scientific word used to describe a ball of cells, about 96 hours after insemination). Still the overall size of the embryo continues to be almost the same as the one-cell embryo. This term is applicable until a cavity begins to form inside the spherically-shaped clump of cells.
Once the morula develops as an inner cavity, the embryo has achieved a new developmental milestone and is termed a blastocyst (day 5). The cells lining the embryonic pole (trophoblast) will bring water, minerals, and amino acids from the nutrient-rich environment outside the embryo into the blastocyst cavity where they can reach the cells of the inner-cell mass.
Scoring for quality of the blastocyst: Again, one of the scoring systems is a modification from Shady Grove Fertility Center, Rockville, and Georgia Reproductive Specialists.
Give each blastocyst an overall numerical score based on rate of development and degree of expansion, i.e.,
GOOD 1 Fully expanded or hatching: day-5
ADEQUATE 2 Fully expanded or hatching: day-6
Moderate expansion: day-5
MEDIOCRE 3 Moderate expansion: day-6
Early cavitation: day-5
POOR 4 Morula: day-5
Early cavitation: day-6
Two alphabetic scores for grading (to grade) are added to this, i.e.,
1st) Inner cell mass
GOOD A high cell number with good cell/cell adhesion
MEDIOCRE B lower cell number with poorer cell/cell attachment
POOR C no cells apparent (ICM) sparse granular cells (trophectoderm)
Example 1: A good-quality, well-expanded blastocyst on day 6 with good cell count in the ICM (>12 to 15), and good integrity of the trophectoderm would be scored as 2AA
Example 2: A fully-expanded blastocyst on day 5 with a nice trophectoderm but nonexistent ICM would be scored as 1CA
Another scoring system was that of a blastocyst as suggested by Gardner et al. Fertility and Sterility 73: 1155-1158, 2000.
High-quality human blastocyst. The cells which will become the fetus are in the area marked "ICM" (inner cell mass), the blastocele cavity in the center - "C", the trophectoderm cells that will form the placenta surround the cavity - one is marked "T"
Hatched blastocyst. The hatching process, by which the blastocyst emerges from the zona pellucida before implantation, is a phenomenon seen with embryos developing outside the body. Under natural conditions inside the body, the zona pellucida is believed to degenerate and disappear after the embryo reaches the uterine cavity and prepares to implant.
The process of implantation from Norwitz et al, NEJM, 345:1400, 2001
This occurs 6 to 7 days after conception and includes three stages:
Stage 1: The initial adhesion of the blastocyst to the uterine wall, called apposition
Stage 2: Stable adhesion, increased physical interaction between the blastocyst and the uterine epithelium. Penetration of the uterine epithelium, with orientation of the embryonic pole towards the uterine epithelium (Norwitz et al., NEJM,345:1400, 2001)
By day 10 the blastocyst is completely embedded in the stromal tissue covered by regrown endometrium (Norwitz et al., NEJM, 345:1400, 2001)