Embryology Lab Services
In Vitro Fertilization
In-Vitro Fertilization (IVF) is the treatment of choice for infertile couples. From the embryology laboratory perspective, IVF is a set of procedures that includes isolation of oocytes from follicular aspirates at the time of oocyte retrieval, maturation of oocytes, insemination of oocytes with or without ICSI, confirmation of fertilization, culture of fertilized oocytes (zygotes), morphological assessment of embryo quality, and finally intrauterine transfer of embryos on day 3 or day 5. If it is indicated, assisted hatching of embryos is done before their transfer. If supernumerary embryos are available, they are cryopreserved for future cycles. There are three factors responsible for infertility: female, male and unexplained factors. Under female factor, tubal disorders, ovulatory dysfunction, and endometriosis are the main causes of infertility. The age of the female patient has direct influence on the success rate of an IVF cycle. With the advancement of age especially after 40 years, the pregnancy rate starts declining. However, after the introduction of donor oocytes program, the pregnancy rates in older patients are comparable with the pregnancy rate in younger patients. In the last three decades since the birth of the first child from IVF, substantial progress has been made. Especially, after the development of ICSI, assisted hatching, and lately blastocyst transfer, significantly high pregnancy rate has been achieved.
Gamete Intrafallopian Transfer/Zygote Intrafallopian Transfer
The Gamete Intrafallopian Transfer (GIFT) involves the placement of oocytes (retrieved from a stimulated cycle) and freshly prepared sperm into the fallopian tube(s) via laparoscopy. This procedure benefits those patients with cervical abnormalities that hinder transvaginal transfer and allows for the proper placement of oocytes and spermatozoa for fertilization in the fallopian tube. GIFT is also an ethical or moral alternative for the patient who is not able to undergo IVF on such grounds. This procedure requires at least one patent fallopian tube.
Zygote Intrafallopian Transfer (ZIFT) requires the fertilization of the eggs in the IVF laboratory, and then a certain number of zygotes are placed in one or both fallopian tube(s). Like GIFT, ZIFT requires at least one patent tube. GIFT and ZIFT are rarely, if ever, being done currently. With the high pregnancy rates with conventional IVF, these procedures have now become obsolete.
Assisted Fertilization by Intracytoplasmic Sperm Injection (ICSI)
ICSI involves the insertion of single selected spermatozoa directly into the oocyte, thereby crossing not only the zona pellucida (shell) but also the cell membrane (oolemma). This method bypasses all of the preliminary steps of fertilization. Fertilization rates after ICSI had been reported to be significantly better than a conventional IVF for male factor infertility. Moreover, ICSI resulted in the production of more embryos with higher implantation rates. As a result, ICSI has been used worldwide and successfully to treat infertility due to impaired testicular function or obstruction of the excretory ducts resulting in abnormal spermatozoa or azoospermia. ICSI is currently a routine procedure in the context of IVF.
Retrograde Ejaculate Testing
Retrograde ejaculation involves reflux of spermatozoa in the backward direction into the bladder instead of the normal forward movement along the urethra to the exterior. This is often caused by an incompetent bladder neck. Some of common causes include: diabetes, iatrogenic surgical damage to the bladder neck innervation, and as a side effect of hypertensive therapy using alpha-adrenergic blockers. Clinically, men with retrograde ejaculation will present with either aspermia or no apparent ejaculate. In these cases diagnostic confirmation involves examination of the post-ejaculatory urine samples for the presence of spermatozoa.
Preparation of Fresh and/or Frozen-Thawed Sperm for Intra-Uterine Insemination (IUI)
The rationale for the use of IUI is to reduce the effect of factors that may impede the progress of spermatozoa such as vaginal acidity and cervical mucus hostility, and to benefit from the deposition of a bolus of concentrated highly motile, morphologically normal sperm as close as possible to the oocytes. The use of washed prepared sperm for IUI has resulted in a significant reduction in the side effects associated with the use of undiluted semen for IUI, such as painful uterine cramps, and infection. For therapeutic IUI, a variety of sperm preparation methods are available. The most important consideration is to obtain a debris-free sperm suspension of adequate concentration and motility. The normal and motile sperm for IUI are separated by the Discontinuous Density Gradient and followed by repeated washing of the final suspension. Finally, the sperm pellet is mixed with a minute quantity of culture media, and then inseminated into the uterus using a special sterile catheter.
Cryopreservation of Semen
Cryopreservation of semen may be advantageous to couples in instances where the male partner is to undergo irradiation or chemotherapy, when sperm are retrieved surgically, or by electro-ejaculation, when the male partner is unavailable at the time of follicular aspiration due to previous commitment or situation, when the male partner is planning to undergo vasectomy, and/or is unable to produce on the day of follicular aspiration during IVF. The semen analysis is performed in the fresh ejaculate, after diluting with freezing media, and before and after the freezing process. The semen is then loaded in cryo-vials, and frozen in liquid nitrogen vapors. The frozen semen vials are then transferred very carefully into the liquid nitrogen tank (temperature -196’b0C) until used for IUI or IVF.
The blastocyst is the embryonic stage at which human implantation occurs. Blastocysts first form about 5 days after ovulation and fertilization. In the laboratory, the embryos are usually cultured for 3 days. However, to develop embryos up to the blastocyst stage, embryo culture is extended up to 5 or 6 days. As the embryo grow for 5 days, it divides from a two-cell stage to a 75-120 cells blastocyst. A two-step formulation of culture medium recently developed is used to culture blastocyst. Embryos that survive to the blastocyst stage are likely to be of better quality and survive the uterine environment much better than day 3 embryos. Blastocyst transfer has achieved significantly higher implantation rate as compared to day 3 transfer. The main advantage of this procedure is that rate of multiple pregnancies could be reduced by transferring less embryos. On the other hand, there is a great risk to lose some of the embryos because only 20-50% embryos make it to the blastocyst stage. At VCRM we only do blastocyst transfers and stopped doing day 2 or day 3 transfers in early 2013.
Cryopreservation of Embryos and Intrauterine Transfer of Frozen-thawed Embryos
Embryos may be cryopreserved in an effort to maximize the chances of pregnancy from any one IVF treatment attempt and prevent wastage of embryos. It is possible to achieve implantation and pregnancy rates with frozen-thawed embryos as high as those achieved with fresh embryos. The condition for this is to transfer good quality 8-cell frozen-thawed embryos. However, most of the time the pregnancy rate of frozen-thawed embryos is relatively lower compared to those of fresh cycles.
Embryos may be frozen at stages ranging from zygotes through the blastocyst stage, with preference for day 3 freezes. Embryos after freezing are stored under liquid nitrogen at -196°C.
Cryopreservation of Unfertilized Oocytes (Egg freezing):
Freezing of human oocytes is now possible. However, techniques to freeze and thaw oocytes still have room for improvement. Although, the survival rate of frozen-thawed oocytes is lower than that of frozen embryos, a handful of pregnancies have been reported after freezing and thawing mature oocytes. Several possible explanations have been offered for low survival rate of oocyte. It has been shown that the cytoskeleton of the oocyte is damaged by the Cryopreservation process, which could lead to significant changes in the organization and trafficking of molecules and organelles. Of greater concern is the possibility of aneuploidy caused by depolymerization of the spindle apparatus (genetic material, DNA) during cellular cooling. Despite the limitations, freezing of unfertilized oocytes would be extremely useful to some individuals. A young woman about to undergo radiation treatment or facing the loss of her ovaries could benefit greatly. Donor oocytes banks could be set up much in the same way as sperm banks are now to assist an ever-growing population of women requesting donor eggs.
Preimplantation Genetic Diagnosis and Screening
Preimplantation genetic diagnosis (PGD) and screening (PGS) can be used to test embryos for genetic disorders prior to their transfer to the uterus. PGD can help to the couples having serious inherited disorders to decrease the risk of having a child who is affected by the same problem. The disorders such as cystic fibrosis, sickle cell anemia, Down’s syndrome, Turner syndrome, Tay-Sachs, and other commonly occurring genetic disorders may be detected by PGD. Moreover, the sex of a preembryo can now reliably be determined by this technique. PGS tests for genetic errors or aneuploidies and ensures that the transferred embryos are genetically normal. The first step of PGD/PGS is the collection of informative genetic material from each embryo. For this, microscopic guided biopsy of embryo is done at the balstocyst (or day 5) stage. By using advanced techniques such as PCR, the genetic material is then analyzed for the presence of genetic disorders. A diagnosis is obtained within day or so of the test and only the unaffected embryos are replaced to the female’s uterus.
Surgical Recovery of Sperm by Testicular Sperm Aspiration, Testicular Sperm Extraction, Microsurgical Epididymal Sperm Aspiration and Percutaneous Epididymal Aspiration for ICSI
Surgical sperm recovery for ICSI is indicated for irreversible obstructive azoospermia, primary testicular failure, necrozoospermia, and some cases of ejaculatory failure. In men with sperm obstructions, sperm recovery is possible by Testicular Sperm Aspiration (TESA), Microsurgical Epididymal Sperm Aspiration (MESA) or Percutaneous Epididymal Aspiration (PESA). A 19 or 21 G butterfly needle attached with a 10 ml syringe is used to aspirate sperm from the testicle or epididymis. In non-obstructive azoospermia, Testicular Sperm Extraction (TESE; Testicular Biopsy) is usually necessary for sperm recovery. In testicular biopsy, about 5 mm tissue is dissected to facilitate sperm isolation. In men with no previous sperm-positive testicular histology, bilateral or multiple biopsies may be required. A Urologist, with the help of an embryologist, conducts these procedures in the operating room under local or general anesthesia. Recovered spermatozoa are processed and cultured in the embryology laboratory and may be used fresh or can be cryopreserved for future ICSI cycles.