INTRA CYTOPLASMIC SPERM INJECTION :
Definition: It is a latest of several microfertilization techniques that have been utilized predominantly to overcome severe male infertility. Procedure in which a single sperm is injected directly into a single egg. The fertilized embryo is then transferred into the uterine cavity.
History: The procedure was first performed in 1987, though it only went to the pronuclear stage. The first activated embryo by ICSI was produced in 1990.First successful birth by ICSI took place on jan14,1992 after an april 1991 conception.
Why it is done:
Male infertility– Low sperm count, Poor sperm motility, Abnormal morphology, Underwent vasectomy sperms have been collected from the testicles or epididymis)
Female infertility – blocked fallopian tubes, Several failed IUI’s, Poor ovarian reserve, Unexplained infertility.
ICSI is used to treat severe male infertility, as when little or no sperm are ejaculated in the semen. Icsi is also used for couples who are planning to test the embryo for genetic problems. Icsi uses only one sperm for each egg, so there is no chance the genetic test can be contaminated by other sperm.
SUCCESS RATE: Successful rate on ICSI is more, as we fertilise the egg and transfer at P2N stage. Here are the 2011 live birth ICSI success rates from couples diagnosed with male factor infertility (statistics are based on the mother’s age).
These success rates are based on live birth rates per fresh embryo transfer.
35 and younger: 48.3 percent gave birth
35 to 37: 42 percent
38 to 40: 29.8 percent
41 to 42: 20.4 percent
- Stimulation of multiple follicles
- Retrieval of mature oocytes under ultrasound guidance
- Retrieving sperm from husband & sperm preparation
- Oocyte maturity assessment & Grading of embryos
- Transfer of embryos into uterus & freezing the remaining embryos
- Pregnancy test should be done after 2 weeks
STIMULATION OF FOLLICLES and Retrieval: Gonadotropins are started from day 3, and dosage is altered depending on the growth of the follicles. The follicle normally grow at a rate of 2-3mm/day in a stimulated cycle. HCG is administrated to mature the follicles. After 36 hours of administration of HCG, under general anesthesia transvaginal ultrasound guided oocyte retrieval done.
RETRIEVING SPERM FROM HUSBAND: If there is sperm in the semen, sperm sample can be produced by normal ejaculation. If there so no sperm in the semen, extract sperm. Epididymis- Percutaneous epididymal sperm aspiration (PESA).Testicle- Testicular sperm aspiration (TESA)
FERTILIZATION OF EGGS: Using micromanipulator sperm is injected into cytoplasm of the egg. Eggs are checked after 16-18 hours for evidence of fertilization. By 48 hours embryos must be atleast 2 cells preferably 4 cell stage. By 72 hours embryos must be atleast 6 cells preferably 8 cell stage. Egg now becomes the embryo and incubated for 3-5 days and transfer. Until day 3 embryos remain in cleavage medium (pronuclear to 8cell stage), and they are changed over to blastocyst cleavage medium
Quality of embryo:
Usually, determinations of “quality” are not made until at least 48 hours after the egg retrieval. Assessing embryos on the first day after egg retrieval at the zygote, or 1-cell embryo stage. They use morphologic parameters of the pronuclei on day one as part of the overall embryo quality assessment, and also as part of their selection criteria for the best embryos to transfer. This assessment on day one is an interesting tool, but it has not been shown to be consistently valid in different laboratories.
By 48 hours (“day 2”), the embryos must be to at least 2 cells – or they have “arrested”. We prefer that at least some of them are at the 3 or 4 cell stage by then.
By 72 hours (“day 3”), we like to see at least 6 cells – and preferably some at about 8 cells. We have seen babies that came from 4 cell embryos on day 3, but chances for pregnancy increase significantly with increasing cell number.
Embryos with higher cell numbers, regular appearing cells and little or no fragmentation have a higher overall chance of implanting than do other embryos with less cells, more irregularity and significant fragmentation.
Embryo quality is mainly evaluated by microscopy at certain time points using a morphological scoring system. This has shown to significantly improve pregnancy rates.
Time-lapse microscopy is an expansion of microscopy wherein the morphology of embryos is studied over time.
GRADING OF EMBRYO:
Embryo picture of a “perfect” looking 8-cell embryo (day 3 embryo)
There is no embryo fragmentation and the cells are very even, regular, and similarly sized
We call this is a “high quality” day 3 embryo
Embryo grading: 8 cell, grade 4
Embryo quality assessment and grading done on day 3 of development:
Cell number: Embryos should be at 2 to 4 cells at 48 hours after egg retrieval and preferably about 7 to 10 cells by 72 hours. The cells in an embryo are also referred to as “blastomeres”.
Cell regularity – degree of regularity of size of blastomeres
It is generally best if the size of the individual cells (referred to as blastomeres) in the embryos are similar in size. If they are not, it is better if they are close to the same size, as compared to very different in size.
Degree of fragmentation
Fragmentation, also called blebbing, is a process where portions of the embryo’s cells have broken off and are now separate from the nucleated portion of the cell. It is preferable to have little or no fragmentation. However, fragmentation in human embryos is quite common and many beautiful babies have resulted from implantation of embryos with fragments. Embryos with more than 25% fragmentation have a low implantation potential.
Presence of multinucleation
An embryo is multinucleated if more than one nucleus can be seen in any individual cell on either day 2 or day 3. After day 3 it is extremely difficult to identify the presence of multinucleation. The great majority of multinucleated embryos have been shown to be chromosomally abnormal.
However, sometimes multinucleated embryos implant and lead to a healthy pregnancy and birth of a normal baby. These embryos are generally only transferred to the uterus if they are the only ones available. Additional factors involved with embryo grading and selection for transfer.Other aspects of the microscopic appearance of the embryos are also noted including the presence of vacuoles, granularity, thickness of the outer shell (or zona pellucida), etc.
- A slightly fragmented 8-cell embryo
- Assisted hatching is being performed
- The holding pipette is on the far left
- A sperm is clearly visible at 7 o’clock (this sperm lost the race)
- This embryo is high quality because it has an optimal cell number, the cells are fairly regular in size, and there is minimal fragmentation
Embryo grading: 8 cell, grade 3
This 5-cell embryo is moderately fragmented and has unevenly sized (irregular) cells
This embryo is expected to have a significantly lower chance of implanting because it is somewhat “slow” (more than 5 cells is preferred on day 3) and because of the significant fragmentation and irregularity of the cells
Embryo grading: 5 cell, grade 2
This embryo is severely fragmented and has unevenly sized cells
It is a poor quality day 3 embryo
This embryo probably does not have much chance to implant and make a viable pregnancy.
Embryo grading: 6 cell, grade 1
Photo of a severely fragmented and very poor quality day 3 embryo
This embryo is essentially non-viable and is degenerating
It will not be able to continue normal development and implant
Embryo grading: 2 cell, grade 0
Multinucleated 2 cell embryo
- Multiple nuclei (circular structures that look like moon craters) seen in both cells
This embryo does not have long term viability potential
- Usually, determinations of “quality” are not made until at least 48 hours after the egg retrieval. By carefully assessing embryos on the first day after egg retrieval at the zygote, or 1-cell embryo stage. They use morphologic parameters of the pro nuclei on day one as part of the overall embryo quality assessment, and also as part of their selection criteria for the best embryos to transfer. This assessment on day one is an interesting tool, but it has not been shown to be consistently valid in different laboratories.
- By 48 hours (“day 2”), the embryos must be to at least 2 cells – or they have “arrested”. We prefer that at least some of them are at the 3 or 4 cell stage by then.
- By 72 hours (“day 3”), we like to see at least 6 cells – and preferably some at about 8 cells. We have seen babies that came from 4 cell embryos on day 3, but chances for pregnancy increase significantly with increasing cell number.
- Embryos with higher cell numbers, regular appearing cells and little or no fragmentation have a higher overall chance of implanting than do other embryos with less cells, more irregularity and significant fragmentation.
Embryo is transferred usually after 3 days after egg collection under ultrasound guidance (with full bladder) using special catheter.2-3 embryos are transferred into uterine cavity.Remaining embryos will be frozen. There is now good evidence that luteal support improves outcome. Luteal support can be provided by either hCG or parenteral or vaginal progesterone. Vaginal progesterone should be stop after 14 days after embryo transfer, when parenteral progesterone is used it suppress corpus luteum profoundly ,so it should be continued until end of first trimester of pregnancy.Administration of hCG should be avoided if there is any risk of OHSS.
Check for pregnancy: After 14 days of embryo transfer, you need to check for pregnancy. By b-Hcg in blood. When the cycle is not succesful,you can use the remaining frozen embryos.
ADVANTAGES OF ICSI:
Revolutionized treatment with a few or one sperm to father genetically related children.
Icsi is an extremely useful where only few eggs are available.
Disadvantages of Icsi:
Risk of multiple birth.
Ovarian hyper stimulation syndrome: OHSS has been characterized by the presence of multiple luteinized cysts within the ovaries leading to ovarian enlargement and secondary complications, but that definition includes almost all women undergoing ovarian stimulation. The central feature of clinically significant OHSS is the development of vascular hyper permeability and the resulting shift of fluids into the third space. As hCG causes the ovary to undergo extensive luteinization, large amounts of estrogens, progesterone, and local cytokines are released. It is almost certain that vascular endothelial growth factor (VEGF) is a key substance that induces vascular hyperpermeability, making local capillaries “leaky”, leading to a shift of fluids from the intravascular system to the abdominal and pleural cavity. Supraphysiologic production of VEGF from many follicles under the prolonged effect of hCG appears to be the specific key process underlying OHSS. Thus, while the woman accumulates fluid in the third space, primarily in the form of ascites, she actually becomes hypovolemic and is at risk for respiratory, circulatory (such as arterial thromboembolism since blood is now thicker), and renal problems. Women who are pregnant sustain the ovarian luteinization process through the production of HCG. Avoiding OHSS typically requires interrupting the pathological sequence, such as avoiding the use of HCG. One alternative is to use a GnRH agonist instead of HCG. Symptoms generally resolve in 1 to 2 weeks, but will be more severe and persist longer if pregnancy occurs. This is due to human chorionic gonadotropin (HCG) from the pregnancy acting on the corpus luteum in the ovaries in sustaining the pregnancy before the placenta has fully developed. Typically, even in severe OHSS with a developing pregnancy, the duration does not exceed the first trimester.