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IVF Step-by-Step

The Embryology Laboratory at the Strong Fertility Center is a state-of-the-art facility accredited by the New York State Department of Health. We offer a wide variety of laboratory techniques to help patients achieve success with assisted reproduction. These include in-vitro fertilization (IVF) with standard insemination, intracytoplasmic sperm injection (ICSI), assisted hatching (AHA), embryo cryopreservation, blastocyst culture, TESE and MESA for male factor, and embryo biopsy for pre-implantation genetic diagnosis.

Step-by-Step Descriptions of the IVF Procedures

Step 1: Control Ovarian Hyperstimulation (COH)

COH is done using different protocols. The most common one is a GnRH Antagonist protocol where the secretion of gonadotropin hormones is suppressed in order to prevent premature ovulation. Once optimal suppression is achieved, the next step is the recruitment of multiple follicles by daily injections of gonadotropins. Ultrasound imaging and hormone assessments are used to monitor follicular development. When the lead follicles have reached the appropriate size, the final maturation of eggs is completed by HCG administration. Egg retrieval is scheduled 34 - 36 hours after HCG injection.

Step 2: Egg Retrieval



Egg retrieval is performed in a surgical suite under intravenous sedation. Ovarian follicles are aspirated using a needle guided by trans-vaginal ultrasonography. Follicular fluids are scanned by the embryologist to locate all available eggs. The eggs are placed in a special media and cultured in an incubator until insemination.

Step 3: Fertilization and Embryo Culture



  • If sperm parameters are normal, approximately 50,000 to 100,000 motile sperm are transferred to the dish containing the eggs. This is called standard insemination.
  • The ICSI technique is used to fertilize mature eggs if sperm parameters are abnormal. This procedure is performed under a high-powered microscope. The embryologist picks up a single spermatozoa using a fine glass micro needle, and injects it directly into the egg cytoplasm. ICSI increases the chance that fertilization will occur if the semen sample has a low sperm count and/or motility, poor morphology, or poor progression. If there are no sperm in the ejaculate, sperm may be obtained via a surgical procedure. ICSI is always used to achieve fertilization if the sperm is surgically retrieved.
  • Fertilization is assessed 16 - 18 hours after insemination or ICSI. The fertilized eggs are called zygotes and are cultured in a specially formulated culture medium that supports their growth. They will be assessed on the second and third day after retrieval. If sufficient numbers of embryos exhibit good growth and development, they may be selected to grow to the blastocyst stage in a specially designed culture medium. Blastocyst culture has several advantages. Embryos at this stage have a higher potential for implantation, therefore fewer embryos can be transferred on day 5 to reduce the chance of multiple pregnancies. Low numbers of embryos and poor embryo quality reduce the chances for good blastocyst development. A day 3 embryo transfer is recommended for cycles with low numbers and/or poor quality.

Step 4: Embryo Quality

There are several criteria used to assess the quality of the embryo. This is especially important when trying to decide which embryos to choose for embryo transfer. Early in the morning on the day of your transfer, the embryos are evaluated and photographed by the embryologist. The embryologist and your physician will decide, based on the rate of development and appearance of the embryos, which and how many embryos are recommended to be transferred.

Typically, embryos are transferred at the cleavage stage (day 3 after oocyte retrieval) or at the blastocyst stage (day 5). In the lab, a grading system is used to asses the quality of the embryos.

Cleavage stage

Cleavage Stage

Day 3 Transfers

Day three embryos are called cleavage stage embryos and have approximately 4 - 8 cells. When analyzing these embryos, we not only look at the number of cells but also how symmetrical they are and whether there is any fragmentation. Fragmentation occurs when the cells divide unevenly, resulting in cell-like structures which crowd the embryo. No fragmentation is preferable but some is acceptable. In our lab, we classify embryos into grades 1 through 4. Grade 1 represents the best quality embryos.

Day 5 Blast

Day 5 Blast

Day 5 Transfers

Day 5 embryos are called blastocyst embryos. At this stage, the embryos have increased in size and are even more developed. They resemble a ball of cells with fluid inside. One of the things we look for at this stage is how expanded these embryos are. The more expanded, the better the quality of the embryo. These embryos are also classified by a number scale, 1 through 6. Grade 6 represents the best quality blastocyst.

Step 5: Embryo Transfer

Embryos are transferred on day 3 when they are at the cleavage stage (6 - 8 cells) or on day 5 when they have reached the blastocyst stage. Embryo transfer is a simple procedure that does not require any anesthesia. Embryos are loaded in a soft catheter and are placed in the uterine cavity through the cervix under ultrasound guidance.

Additional IVF Procedures

Hatching Blast

Hatching Blast

Laser Assisted Hatching

An embryo must hatch out of its outer membrane (zona pellucida) before implanting in the uterine wall (endometrium). Sometimes, the zona is abnormally thick. Laser assisted hatching is a technique that allows a small gap in the zona pellucida to be made. This will aid the embryo in breaking out of this membrane and facilitates implantation. It is a technique that is performed before embryo transfer and when doing trophectoderm biopsies. Assisted hatching is specifically recommended for patients who are over 37 years of age, have diminished ovarian reserve as determined by a day 3 FSH level, or have lower antral follicle counts. Patients who are poor responders to gonadotropin stimulation, or have had previous failed implantation may also benefit from this procedure. Studies have shown that assisted hatching improves IVF success rates in both fresh embryo transfers and frozen embryo transfers.


Embryo cryopreservation (freezing) may be available to patients that have an excess number of normally fertilized embryos or high quality blastocysts that remain following embryo transfer. Embryos may be frozen at the zygote stage one day after egg retrieval, or on day 5 or 6 at the blastocyst stage. 

Microsurgical Epididymal Sperm Aspiration (MESA) or Testicular Sperm Extraction (TESE)

Some patients' semen samples contain no spermatozoa due to a congenital obstruction of the sperm ducts, vasectomy, failed vasectomy reversal, or primary testicular failure. In these conditions, a urologist can obtain sperm surgically from the epididymis (MESA) or from the testis (TESE). This sperm can be frozen and used for fertilization by ICSI.

Embryo Biopsy

Preimplantation Genetic Testing (PGT)

PGT is a procedure that is performed in conjunction with IVF. It is designed to help detect genetic abnormalities/inherited genetic diseases in embryos before implantation, thereby minimizing the transfer of affected embryos.

Preimplantation Genetic Testing - Aneuploidy (PGT-A)

Normal human cells contain 46 chromosomes located in the nucleus of the cell. Chromosomes carry the genetic information in the form of DNA. Every human being receives 23 chromosomes from each parent. If an error occurs leading to the egg or sperm having a missing or extra chromosome, the embryo created will also have a missing or extra chromosome. This condition is called aneuploidy. Most of the aneuploidies will not result in implantation of the embryo, but certain aneuploidies, such as trisomy 21, can implant and lead to Down’s syndrome. Some other common aneuploidies include trisomy 13, trisomy 18, and Klinefelter's syndrome (XXY).

Indications for PGT

PGT is indicated for  patients who have a history of recurrent miscarriages, advanced maternal age (≥38 years), repeated IVF failures in spite of high grade embryos, unexplained infertility, severe male factor infertility, or inherited genetic disorders (e.g., cystic fibrosis, Tay Sachs disease, Myotonic dystrophy, etc.). Currently, there are more than 1000 types of single gene mutations that can be diagnosed.


In order to perform genetic testing on an embryo, several cells from the trophectoderm layer are extracted on fifth, sixth or seventh day of development. This procedure is called an embryo biopsy. The extracted cells are sent out for analysis to Cooper Genomics and/or Ingenomix. Subsequently, the biopsied embryo is vitrified. After the results are received, patients will have the normal embryo transferred back.

Benefits of PGT

Patients who are carriers of single gene mutations can avoid transmitting those disorders to their offspring by testing the embryos and choosing not to transfer those which are affected or carriers.

Patients who have had several miscarriages in the past can benefit from PGT-A for aneuploidy screening by avoiding the transfer of embryos that are aneuploid and will eventually fail to implant. The likelihood of having a trisomic pregnancy increases with advanced maternal age (>38 years). PGT-A allows selection of normal embryos at the pre-implantation stage and reduces the chance of detecting abnormal fetal development during an aminocentesis in the second trimester. Patients who failed several IVF attempts and are known to generate good cohorts of embryos might have a high rate of aneuploidy and can benefit by PGT-A. Another group of patients who can benefit from PGT are those who carry translocations, which are detected by karyotyping.


There are certain risks associated with any micromanipulation procedure, but these are minimal. Trophectoderm biopsy does require embryos to develop to the blastocyst stage before the biopsy is performed. Not all couples will have embryos that develop to this stage. Occasionally, results are not able to be determined.