Mainly two types of protocol:
- Long protocol (agonist) – Here down regulation of internal hormones of woman done with continuous daily subcutaneous injection of agonist (lupride or equivalent). This injection is started on 21st day of previous cycle and continued till day of HCG or till follicles reaches to the size of around 18-20 mm. There is another way of giving agonist injection. This is called depot protocol. Here instead of 20 or so daily injection single injection of Leuprolide or equivalent single injection is given.
- Short protocol (antagonist):
- Method no:1 :- Treatment is preceded with birth control pills in previous cycle. After stoppage of pills there will be withdrawal bleeding (menses). On Second day of cycle stimulation of ovary with gonadotropins started.
- Method no 2 (walking IVF) :- Here straight away woman is examined for her inclusion criteria which include absence of cyst in ovary and complete shedding of endometrium. From day 2 of menses stimulation started.
In both methods stimulation is given for 8 to 12 days. Once follicles reaches to the size of 13-14 mm inj cetrorelix or equivalent injections are also started subcutaneously to avoid premature LH surge and premature release of eggs. Cetrorelix are continued with gonadotropin stimulation till leading follicle is 18 to 21 mm of size.
1. Down regulation
Birth control pill is given for minimum 16 days and maximum 36 days to batch the cases and to smoothen hormones. Inj luprie depo 3.75 mg single dose is given on approximately one month prior to date of OPU or ET. After 10 days of inj lupride birth control pills are stopped. Within 7 days woman will get her period. From Day 2 or 3 of periods start stimulation.
Stimulation of ovary for getting or achieving more number of eggs are started with inj FSH, Recagon or HMG according to the need of the woman. This stimulation varies from person to person and how woman responds to medicines. Roughly it is between 7-12 days. Mostly it is 10 days. Throughout the stimulation period, you will need to visit us for ultrasound monitoring and sometimes blood hormone levels. This will help us know when there are enough follicles that are mature enough for us to go “egg pick-up”.
Simultaneously, an embryologist is present at the time of the procedure in the embryo culture laboratory, looking through the follicular fluid, finding the eggs, and scoring them according to their maturity and quality.
Inj HCG 5000 to 10000 iu is normally use for final maturation of Ovum. Rarely 2500 iu is used. Some time GnRH analogue is used when number of follicles are more and chances of development of OHSS (Hyperstimulation syndrome)
4. OPU (OVUM PICK UP)
Woman has to come fasting overnight or at least 6 hours. Pre-OPU on previous night she is advised to take mild purgative and antacids. Normally this procedure is done at 35 hrs+ of inj HCG sometimes it is delayed to 36 to 36.5 hrs. If done early chances of not getting mature egg develops and if done delayed than egg may get released into peritoneal cavity and we may not get any ovum. This procedure is done under anesthesia or deep sedation. Usually, the egg pick-up is performed through the wall of the vagina. A needle is placed through the wall of the vagina and into the ovary, where the follicles are emptied of their fluids and their eggs. Post operative recovery is usually quick and uneventful. OPU is carried out under sonographic control with special needle. The procedure lasts for about 15-30 min depends upon the number of follicles and position of ovaries.
After eggs are retrieved they are either put into petri dish with sperms for IVF or with micro injection pipette ICSI is done. All these procedure is done in a laboratory in an closed, clean environment. Incubators are use for this Heracell, cook mink and others. Approximately 18 hours of ICSI or IVF fertilization is checked. How many oocytes got fertilized that is being checked. Once this is confirmed on next day that means approximately 43-36 hours formation of embryos are checked. During this time there is tight control of all parameters are maintained in the lab like concentration of CO2, Trigas, PH of media, temperature inside( strictly 37 degree).
6. Collection of the sperm
We recommend that you ejaculate on the day prior to the trigger injection. Sperm collection can be done in one of our special, very private collection rooms. You may do this alone or together with your wife, whichever you prefer. If you anticipate that you will have any trouble providing a sample on the day of the egg pick-up procedure, we can arrange for you to collect ahead of time and have the sperm frozen.
7. Embryo transfer (ET)
Embryos are formed on day 3 of OPU. Once embryologist check the grade of embryos time of embryo transfer is decided. Two or three of the embryos will be chosen for the embryo transfer. Any remaining good quality embryos can be ‘frozen’ for future use if you wish. Here anesthesia is not necessary. You will be asked to drink moderate amount of water or any liquid and don’t go for urination for 2-4 hrs depend upon weather. Once bladder is filled partially you are ready for embryo transfer. Embryo transfer is done under USG guidance at precise point of endometrial cavity (uterine cavity) to get maximum implantation.
Watch video for embryo transfer.
8. How embryos are formed or steps of embryo formation:
Female gamete is called “Oocyte” or ovum or egg.
Male gamete is called “sperms”.
When with the help of IVF or ICSI (oocyte and sperms ) they get fertilized they converted into blastocyst in following order:
- Two PN embryo
- Two cell embryo (day-1)
- 4 cells embryo (day-2)
- 8 cells to morula ( more than 32 cells) (day-3-4)
- Blastocyst and hatching embryo—ready to implant in the uterus (endometrium)
The first week…
After the sperm enters the ovum, the sperm head\enlarges to form the male pronucleus. Within 24 hours, two small spheres, called the pronuclei, can be seen in the cytoplasm of the egg. These contain the genetic material from the mother and the father. When the two pronuclei fuse, joining the DNA from both parents together, fertilization is complete. As the pre-embryo grows, it undergoes cleavage, where the cell divides into smaller cells call blastomeres. After 2 days, the embryo will consist of 4-8 blastomeres. At this point it is impossible to tell which embryos are most likely to survive and develop normally. After about 3 days, when the embryo consists of 12-16 blastomeres, the cells begin to compact, forming a morula. This is the stage at which the embryo would normally enter the uterus, where it floats for a day or two before attaching to the lining of the uterus.
2 Cell Embryo
64 Cell Embryo
4 Cell Embryo
Complexion of Cells
2 PN Embryo
8 Cell Embryo
9. Blastocyst Transfer
A blastocyst transfer is the transfer of an embryo from the laboratory to the uterus at Day 5 of development, instead of Day 3. It is one way of selecting the embryo or embryos most likely to survive and implant, giving a better chance of pregnancy. To appreciate the difference two extra days can make to an embryo, first you need to understand a little about early embryo development.
During those two days, huge changes in the embryo’s appearance can be seen, as the cells begin to differentiate into those that will become the fetus, and those forming the amniotic sac and placenta. These changes are characterized by the formation of a cavity in the morula, to create a blastocyst.
After 4 or 5 days the embryo “hatches” out of the outer shell of the egg, the zona pellucida, and the blastocyst is able to attach to the endometrium. By Day 7, the embryo has completely implanted in the
Embryos that are cultured and grown for 5 -7 days (extended culture) into an incubator are called blastocyst. Here cells are multiplied to many and then there will be development of blastocoels (fluid filled cavity) is called blastocyst. The surface cells surrounding cavity are called trophectoderm and will convert into placenta. Centrally located cells are inner cell mass and convert into fetus. With high quality lab and infrastructure and use of special media for extended culture, we in our lab do routinely blastocyst culture. This embryo is having much more chances of implantation than day 2-3 embryo. Here we transfer fewer 1-2 blastocyst and thereby reduce the chances of multifetal pregnancy. This is the stage of embryo which is ready to hatch and ready to implant in the endometrium.
Why have a Blastocyst Transfer?
By watching the embryo develop to the blastocyst stage, the embryologists can have a better idea of which embryos are most likely to be healthy and continue to develop. One of the factors that decides an embryo’s fate is whether or not it has enough energy for the first week of development, hatching and implantation. Of course, there are still many stages of development that the embryo must pass through to create a successful pregnancy, but choosing the healthiest 5-day-old embryos and transferring them just before they would normally implant has given us the best success rates yet. Ultimately, our goal is to have such high success rates that only one blastocyst will need to be transferred in each cycle for a good chance at pregnancy.
Normally “spare” embryos are frozen at the 4-8 cell stage, and provide good results when transferred after thawing. We are now able to successfully freeze and thaw blastocysts.
10. Assisted Hatching
May be required if the egg has a very thick outer coat.
How is Assisted Hatching performed?
- The embryo is held with a specialized holding pipette.
- A very delicate, hollow needle is used to expel an acidic solution against the outer “shell” (zona pellucida) of the embryo.
- A small hole is made in the shell by digesting it with the acidic solution.
- The embryo is then washed and put back in culture in the incubator.
- The embryo transfer procedure is done shortly after the hatching procedure.
- Embryo transfer places the embryos in the woman’s uterus where they will hopefully implant and develop to result in a live birth.
- Sperm Freezing
- Embryo Freezing
B POSITIVE DAY : after 14 days of embryo transfer to confirm implantation and pregnancy.
Vitrification (freezing) & Thawing
This procedure is used to store embryo, ovum and sperm. Here gametes and embryos are stored at -196 degree celsius that means well below the freezing point of zero and well below antarctic and arctic region. So gametes and embryos are preserved in their live states. We can utilize this when we find comfortable at subsequent date or month or year. We can store this up to many years and many month. We use special jumbo cryo cans for that they are filled with liquid nitrogen. Once we decide the suitable environment and time for gamete or embryo to use, these are brought out and with special media they are brought to room temperature and they again start growing and developing and they are transferred in to woman’s womb, or gametes are utilize for IVF/ICSI(test tube baby procedure) purpose.
Success Rate of IVF / ICSI treatment:
Success rate is normally 30 % to 80 %. Success rate depends upon several factors like (a) Age of women (b) inherent sperm quality of husband or male partner (c) Receptivity of uterus of woman to embryo (d) inherent quality of ovum (egg)
Younger the woman better is the result and older the woman lesser is the result. Success rate can be as high as 80 % in a young woman of 27 to 30 years. So decision for IVF treatment should not be delayed. Start thinking and take decision as early as possible.
POSSIBLE COMPLICATION WHICH CAN OCCUR AT THE TIME OF IVF/ICSI/OPU:
Haemorrhage: Bleeding can occur from ovary or blood vessels while doing ovum pick up. Rarely it can be life threatening.
Bleeding from urine (haematuria): Due to an abnormal position of ovary and needle for ovum pick up has to traverse through urinary bladder.
OHSS (Ovarian hyper stimulation syndrome): It is due to woman’s hyper (more) response to the injections. Due to woman’s hyper response more number of follicles are produced and it leads to collection of fluid (water) in to the abdominal cavity, hyper concentration of blood, breathless ness, gas and acidity. Many a time this is unavoidable and this complication occurs due to individual’s body response to medicine. This is one of the most common complications.
How to avoid? If such complication occurs we will suggest you to freeze all embryos and embryo transfer should be avoided. Because if pregnancy happens the complication become more worse and we may have to terminate pregnancy.
How to treat? 1. Investigations are done in the form of blood and urine. 2. IV fluid and colloids are given, maximum fluid intake is suggested. 3. Cabergolin tab 0.5 mg two times are given. 4. In case of severe OHSS woman may require- removal of fluid from abdominal cavity, admission to ICCU, Inj Heparin and termination of pregnancy if it occurs.
INFECTION: Pelvic infection and pelvic abscess.
Expenses or Cost of IVF treatment:
Treatment cost varies with couple’s problem to conceive, age, weight and method of IVF technique used. Cost also varies with the molecule of medicine and injection use. Normally cost per cycle varies from 50,000/- INR to 150,000/- INR in first cycle. Cost decreases in second cycle.