1. Basics of embryo development in an IVF laboratory.
Dr. Samit Sekhar
Executive Director and Embryologist
Kiran Infertility Centre Pvt. Ltd
Embryology 101
Basics of embryo development in an IVF laboratory.

2. Embryology in Greek means "the unborn, embryo”) is the science of the development of an embryo from the fertilization of the ovum to the fetus stage.
In a routine ART (Assisted reproductive Technology) setting we create embryos by fusing sperm from the male partner with the egg of the female partner.

3. Treatment Option 1
In vitro fertilisation (IVF) commonly known as test tube baby as we all know is one of the most significant advances in the field of modern medicine.
The first IVF baby Louis Brown, born in England in 1978 was the result of more than a decade of research by Dr. Patrick Steptoe and Robert Edwards. They essentially changed the rules for how people can come into the world. Conception was now possible outside the body — in a petri dish. The technique has resulted in the births of millions of babies worldwide, many in multiple births.

4. Initially ivf was developed for women with tubal disease but now it is the treatment of choice for several other causes of infertility,since its introduction all the steps of ivf have been improved upon which has resulted in continuously rising success rates. IVF has also provided a platform for the development of other treatments including egg donation, Gestational surrogacy and preimplantation diagnosis

5. Steps of IVF
1. Ovulation Induction is as per the protocols based on several factors of the female partner and might be a Down regulated or Antagonist cycles. 2. Oocyte retrieval is performed under vaginal ultrasound guidance. After the vaginal ultrasound is placed in the vagina and the ovarian follicles are located a needle is directd through the back wall of the vagina and directed into the ovarian follicles. The fluid is aspirated and then examined by the embryologist to identify the microscopic egg - oocyte surrounded by granulosa cells (cumulus). During normal fertilisation the acrosome of the sperm releases enzymes which disperse the cumulus cells thereby allowing the sperm to penetrate and fertilise the oocyte. Once the eggs are retrieved they are placed in culture media with added nutrients and then placed in the incubator. The procedure is performed under a general Anesthesia and generally takes less than 10 to 15 minutes to complete. There are minimal complications(<1%)

6. Steps of IVF
3. Oocyte Insemination After the male partner produces the sperm sample by masturbation, the sperm concentration and motility are assessed. If found adequate the sperm preparation is done to collect the most motile sperm. A total of nearly 50,000 motile sperm is placed around the eggs in a culture dish which is then placed in the incubator Denudation is the process by which following oocyte insemination the surrounding cumulus is removed to check for signs of fertilisation using denuding pipettes. After a while the nuclei unite and the embryo will start to divide 4. Embryo Culture : The embryos are cultures in a petri dish inside specially designed incubators using nutritive media.
Oocyte Insemination
A fertilised Egg
Incubators
Petri Dish

7. 5. Embryo Transfer – usually performed 72 hours after the egg retrieval, generally 2 to 4 embryos are transferred into the uterine cavity under abdominal ultrasound guidance.
ET Catheter
The recommended number of embryos to transfer is determined by the women’s age, cause of infertility, previous pregnancy history and other factors including the acceptance of a twin pregnancy.
During transfer a full bladder creates an opportunity so that uterus can be clearly visualised and an echogenic catheter is easily seen as it is passed through the cervical canal and into the uterine cavity. The catheter is placed about 2cms away from the top of the cavity where the embryos are released.
Ultra Sound ET

8. ICSI - Intra Cytoplasmic Sperm Injection
ICSI - Intra cytoplasmic sperm injection is used in cases of male factor infertility or in cases where standard ivf results in <40% fertilised embryos from previous ivf cycles or there is no fertilisation at all in a previous ivf cycle. ICSI involves the injection of a single sperm directly into the oocyte. Fertilisation rates using icsi are between 70 to 80%. Males with severe oligospermia(<5 million/cc) should have a chromosomal karyotype performed since they are at a greater risk of chromosomal abnormality.
ICSI

9. Related procedure to IVF
1. FET (frozen embryo transfer) - extra embryos can be cryopreserved by standard freezing or by vitrification and can be transferred in the next cycle after spontaneous ovulation or creation of an artificial endometrium with estrogen and progesterone. The success rates using frozen embryos is slightly lower than fresh embryos. The main advantage of freezing embryos is that ovulation induction need not be given to the donor again. Studies have shown that there are no increased risk of congenital anomalies in infants born following the use of frozen / thawed embryos. 2. Epididymal Sperm Aspiration In some cases of Azoospermia, sperm is produced but are not present in the ejaculate as a result of Obstruction because of infection, congenital conditions or a previous sterilisation. In these cases aspiration of epididymal sperm or testicular sperm by a specialist is carried out and the resultant sperm is used for ICSI(image) The sperm aspiration maybe carried out on the day of oocyte recovery or prior to ivf cycle and the samples maybe frozen.

10. Related procedure to IVF
3. Assisted Hatching- is a procedure in which the zona pellucida, the outer membrane surrounding the embryo is thinned by the addition of a dilute acidic solution and mechanically broken down based on the theory that implantation failure maybe the reslt of failure of the embryo to hatch out from within the zona pellucida,commonlyused for women over 35 years of age or after several unsuccessful transfers. Hatching may be done using chemicals or by laser. 4. Blastocyst Culture- The blastocyst stage of embryonic development occurs just prior to implantation. The blastocyst is an embryo made up of 80 to 100 cells and reaches this stage 5 to 6 days after egg retrieval. Usually after day 3 the embryos need to be transferred into a special “ Blastocyst medium” for them to grow to Blastocyst stage.generally 40 to 50% of embryos develop till Blastocyst stage, the Advantages of Blastocyst Allows for selection of best embryos. Reduces the chances of twins or triplets. Disadvantage - Monozygotic twinning(3%)
Blastocyst Embryo

11. Related procedure to IVF
5. PIGD has turned out to be a big boon for couples who were at a risk of having a child born with a genetic or a chromosomal disorder,the only options earlier were chorionic villous sampling or amniocentesis. These are invasive and not the first choice anymore. In PIGD a single blastomere is removed from the embryo and tested prior to transfer. It was carried out first in 1990 for a couple who were at risk of having a child born with cystic fibrosis. Commonly trisomy 13/18 and 21 and common genetic disorders are tested however with the availability of more and more genetic probes the increase in demand will only grow further. 6. Oocyte freezing is another emerging technology.the oocyte is more sensitive to the cryopreservation process than the embryo because of the high water content in the egg which predisposes it to ice crystal damage Indications-women undergoing cancer treatment to preserve their fertility,for younger women who are career minded and want to preserve their fertility for future. Couples undergoing ivf also sometimes prefer to freeze eggs instead of embryos because once their family is complete is would be emotionally easier to discard frozen eggs instead of embryos.

12. Oocyte scoring and embryo grading
Oocyte scoring The factors that are included in the evaluation of oocyte quality were oocyte cytoplasmic dysmorphisms, extracytoplasmic dysmorphisms and the oocyte–corona–cumulus complex. Assessing fertilization and zygotes (Day 1) Assessment of fertilization is straightforward, as a fertilized oocyte should have two pronuclei and two polar bodies. Cleavage-stage embryo scoring (Day 2 and 3) A. Top quality B. Good quality (not for elective single embryo transfer) C. Impaired embryo quality D. Do not recommend to transfer
Fertilized Oocyte
Oocyte Scoring

13. Embryo quality evaluation is of prime importance in order to sustain a successful IVF / Surrogacy program. Standard assessment by an embryologist relies on · Cell numbers · Fragmentation · Blastomere size · Cytoplasmic anomalies · Compaction Cleavage stage embryos range from the 2-cell stage on day 1 to the compacted morula composed of 8–16 cells day 3 and 4. The number of blastomeres on day 2 or 3 is used as the primary criteria for embryo assessment. Good quality embryos (A grade) must exhibit appropriate division and synchrony of division. In normal embryos, cell division occurs every 18–20 hours. Embryos dividing either too slow or too fast may have metabolic and/or chromosomal defects. It is also pertinent to note that the environment in the specific laboratory, such as culture media and temperature,PH etc influences the quality and grading of embryos.

14. Stages of Embryo Development
2 Cells
3 Cells
4 Cells
5 Cells
6 Cells
7 Cells
8 Cells
9 Cells

15. Each blastomere should have a single nucleus
Each blastomere should have a single nucleus. Multinucleation has been described to be associated with genetic disorders of the embryo and has been associated with an increased abortion rate Multinucleation can be evaluated on Days 1, 2 and 3 of development. Grade-A embryo characteristics: blastomeres of equal size, little or no fragmentation, and a zona pellucida that is not extremely thick or dark in appearance. Grade-B embryo characteristics: blastomeres of equal size, minor cyotplasmic fragmentation covering < 10% of the embryo surface. Grade-C embryo characteristics: blastomeres of distinctly unequal size and moderate to significant cytoplasmic fragmentation covering >10% of the embryo surface.

16. A clear homogeneous cytoplasm is a sign of normal cleavage stage embryo. The presence of multiple vacuoles is a sign of a poor quality embryos Compaction- After the embryo reaches the 8-cell stage, the blastomeres begin to show an increase in cell to cell adherence and this is known as compaction and the process continues until the boundaries between the cells are barely detectable . If some of the blastomeres are excluded from this compaction process, the embryo may have a reduced potential for becoming a normal blastocyst
Compaction

17. Blastocyst transfer Day 5 or day 6 embryos are called blastocyst stage embryos. The blastocyst stage is the stage of development that an embryo must reach before it can hatch and implant in the uterus Blastocyst embryos consists of a blastocoel which causes the embryo to start to expand and increase in overall size. Blastocyst embryos have two distinct parts, the inner cell mass (these cells develop and become the fetus), and the trophectoderm cells (these cells will develop into the placenta). The blastocyst is given a grade based upon the three main components of the blastocyst embryo.

18. Expansion Grade
Early Blastocyst: the blastocoel filling more than ½ the volume of the embryo, but not expansion in overall size compared to earlier stages.
Blastocyst: the blastocoel filling more than ½ the volume of the embryo, with slight expansion in overall size and notable thinning of the zona.
Full Blastocyst: a blastocoel filling more than 50% of the embryo volume and overall size fully enlarged with a very thin zona.
Hatching Blastocyst: The trophectoderm has started to herniate through the zona.
Fully Hatched Blastocyst: Free blastocyst fully removed from the zona.

19. Inner Cell Mass A Tightly packed compacted cells
Inner Cell Mass A Tightly packed compacted cells. B Large, loose cells C No ICM distinguishable D Cells of the inner cell mass appear degenerative Trophectoderm Grade A Many healthy cells forming a cohesive epithelium B Few but healthy cells, large in size C Poor, unevenly distributed cells. Many appear as few cells squeezed to the side D Cells of the trophectoderm appear degenerative embryo surface.
Hatching Blastocyst

20. Embryo freezing Step 1. consent Step 2
Embryo freezing Step 1. consent Step 2. Screening for both people who provided the eggs and sperm used to create the embryos for infectious diseases such as HIV, Hepatitis B & C. Step 3. During treatment, the unused embryos are frozen and then stored in tanks of liquid nitrogen. A liquid called a cryoprotectant is added to protect the embryos during freezing. In the standard freezing method, embryos are slowly frozen down to -196 degrees celcius. ‘vitrification’. is a fast freeze process – the embryo undergoes instantaneous ‘glass-like’ solidification without the damaging formation of ice crystals (which can occur with the standard method of freeze)

21. How long can the embryos be stored?
The normal maximum period that frozen embryos can be stored is ten years. This may be extended depending on the medical circumstances of the woman undergoing treatment, her partner and/or a donor.