In Vitro Fertilization (IVF)
The Embryology Laboratory at the Strong Fertility Center is a state-of-the-art facility accredited by the NYS Department of Health. We offer a wide variety of laboratory techniques to help patients achieve success with assisted reproduction. These include in vitro fertilization 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 long GnRH-Agonist (Lupron) 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 done by HCG administration. Egg retrieval is scheduled 34-36 hours after HCG injection.
Eggs surrounded by
cumulus complex at
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 utilized 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.
|A normally fertilized egg is called a zygote. Two pronuclei are seen in the center.||Intracytoplasmic Sperm Injection (ICSI) procedure where a single spermatozoa, as shown by an arrow, is in the process of being injected into a mature egg's cytoplasm.|
|Embryos at the cleavage stage: A 4-cell embryo (left) and a 6-cell embryo (right)|
|An expanded blastocyst (left) and a hatching blastocyst (right)|
Step 4: Embryo Quality
There are several criteria used to asses 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 as well as 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.
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 four represents the best quality embryos:
|Grade 4: A good quality cleavage embryo||Grade 1: A poor quality cleavage embryo|
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 just how expanded these embryos are. The more expanded is the better the quality of the embryo. These embryos are also classified by a number scale, 1 through 6. Grade six represents the best quality blastocyst:
|Grade 6: A nicely expanded blastocyst||Grade 1: A poorly expanded 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.
Additional IVF Procedures
An assisted hatching procedure where a part of the
zona is thinned by expelling acidified medium by
An embryo must hatch out of its outer membrane (zona pellucida) before implanting in the uterine wall (endometrium). Sometimes the zona is abnormally thick. Assisted hatching allows a small part of the membrane to be thinned chemically using a micromanipulation technique. It is a technique that is performed on day 3 of embryo development. 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.
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. Under certain circumstances, we can also freeze embryos at the cleavage stage. Embryos frozen at any stage can be stored for several years under subzero temperatures using liquid nitrogen. These embryos can be thawed and transferred to patients in a frozen embryo transfer cycle.
Microsurgical Epididymal Sperm Aspiration (MESA) or Testicular Sperm Extraction (TESE)
Some patient’s 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.
Preimplantation Genetic Diagnosis (PGD)
Preimplantation Genetic Diagnosis (PGD) is a procedure that is performed in conjunction with in vitro fertilization (IVF). It is designed to help detect genetic abnormalities in embryos before implantation, thereby avoiding the transfer of affected embryos.
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, then 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 Klinefelters syndrome (XXY).
Indications for PGD
PGD is indicated for the patients who have a history of recurrent miscarriages, advanced maternal age (≥38 yrs), 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 50 types of single gene mutations that can be diagnosed.
An embryo biopsy procedure where one
cell is removed from an 8-cell embryo
In order to perform genetic testing on an embryo, a single cell is removed from the embryo on the third day of development. Embryos must have 5 or more cells. This procedure is called embryo biopsy. It has been shown that removal of one or two cells from an 8-cell embryo does not impede its development. This biopsied cell is lysed, fixed on a glass slide to expose the nuclear material and sent to a reference lab by overnight courier for Fluorescence In Situ Hybridization (FISH) analysis to detect chromosomal anomalies. If these cells have to be analyzed for single gene mutations, they are transferred to tubes in a lysis buffer and sent to a reference lab for polymerase chain reaction (PCR) analysis. We get results on the fifth day after egg retrieval and the embryos that are normal based on the genetic analysis results are transferred.
|PGD of X-linked Disorders Using FISH
Two nuclei that have been hybridized with probes that are complementary to sequences on chromosomes X (green), Y (red), and 18 (blue).
A nucleus from the blastomere of a normal female embryo has two green and two blue signals (top).
Whereas, a nucleus from a normal male has one red, one green, and two blue signals (bottom).
|Normal FISH result (left): Two signals for 13, 16, 18, 21 & 22. Red=13, Aqua=16, Blue=18, Green=21, Gold= 22||Trisomy 21 (right): Three signals for green (21)|
Benefits of PGD
Patients who are carriers of single gene mutations can avoid transmitting those disorders to their offspring by testing the embryos at the preimplantation stage and choosing not to transfer those which are affected. Patients who have had several miscarriages in the past can benefit from PGD for aneuploidy screening (PGD-AS) 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 yrs). PGD 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 PGD-AS. Another group of patients who can benefit from PGD are those who carry translocations, which are detected by karyotyping.
There are certain risks associated with any micromanipulation procedure but these are minimal and are outweighed by the benefits. Occasionally nuclear material from the blastomeres is lost during processing. Due to poor hybridization of chromosome probes, sometimes results for all chromosomes are not able to be determined. This often happens when embryos are of poor quality on day 3 of development and exhibit extensive fragmentation. In single gene mutation analysis, if DNA material from the biopsied cells is degraded, amplification will be poor and sometimes no results can be obtained. A 10% misdiagnosis rate is not uncommon in aneuploidy screening due to mosaicism, a condition where one cell of a multicellular embryo is genetically different than another cell.