The Role of the Embryologist
Printer-friendly versionJim Meriano, BSc, RT, MSc
Lab Director, LifeQuest Centre for Reproductive Medicine
The science of assisted reproduction goes back as far as 1790, with the first reported human birth from artificial insemination recorded in Britain. The first attempts at in vitro fertilization of mammalian eggs go back to the late 1870s! The spring of 1968, however, saw Robert Edwards and Barry Bavister successfully fertilize a human egg using in vitro fertilization (Trounson and Gardener 2000). Ten years later, in 1978, after many scientific and ethical hurdles, Patrick Steptoe and Robert Edwards announced the birth of the world’s first human baby born from in vitro fertilization: Louise Brown.
Today, the science of human embryology has evolved significantly. Louise Brown was conceived from a natural cycle of IVF and was transferred to her mother’s uterus as a Day 2 embryo. Advances in the last twenty five years have drastically changed the methods of embryo culture, sperm recovery, egg retrieval and embryo transfer, and have significantly increased the rates at which an infertility patient goes home with a baby. In this article, I will discuss some of these advances and how they have changed this wonderful field of ours. I will briefly give an overview of the IVF cycle, ICSI and embryo selection.
After the delicate and elegant process of ovarian stimulation, the embryology staff goes to work with the gametes we receive and we do our best under controlled conditions not to add any negative variables to the culture of these gametes and the resulting embryos. The stimulation is an important aspect of the cycle and one that can potentially affect the infertility patient’s results. The physician carefully stimulates the patient’s ovaries so that the ovarian follicles receive proper oxygenation and appropriate growth relative to hormone levels resulting from this stimulation (VanBlerkom et al 1997). The quality of the oocyte and eventually the quality of the embryo will depend on the environment of the follicle and proper triggering of egg maturation with the administration of the hormone hCG. The hCG injection mimics the natural LH surge by sending a chemical message to the oocyte’s nurse cells, which then cue the oocyte to continue its maturation cycle to a certain stage in its cell cycle, making the egg fertilizable by the sperm, whether by ICSI or conventional IVF. The physician removes the mature eggs from the follicle by needle aspiration. The follicular fluid is searched by the embryologist, and all eggs found are carefully washed in fresh culture media and placed directly into our incubators. The incubators provide the embryologist the environment in which to culture the eggs and embryos. The incubator is set to body temperature and is 100 percent humid inside. The mixture of gas (CO2 and O2) allows the nutritive media used to maintain physiological pH similar to conditions inside the body, so that the embryos grow and metabolize in an environment of minimal stress.
The method of insemination of the oocyte depends on the type of infertility that has been diagnosed. Blocked fallopian tubes, for example, are among the most common indications for conventional IVF. The semen fraction is washed off the sperm using centrifugation and 20,000 to 30,000 progressively motile sperm are placed into a culture dish containing 3 to 5 eggs and their nurse cells (cumulus cells). The cumulus cells that surround the egg are needed for the IVF to work well. Scientists have been pondering the specific function of these cells in fertilization for years and some minor functions have been identified. But it is possible that they are simply a medium for the sperm to swim through, in preparation for entrance and fertilization when it reaches the egg. As the sperm battles its way through these cells, many changes in its membranes and receptors take place, making it ready for fertilization once it reaches the outer vestments of the oocyte.
Conventional IVF assumes that the sperm count, motility and normal form concentration are all close to or within the normal ranges. Male factor patients with subnormal sperm were also treated using IVF in the mid-1980s. It was found that if sufficient sperm was recovered, some fertilization was possible, however, these fertilization rates were much lower depending on just how poor the sperm quality and/or quantity was.
Sperm that was recovered from the epididymis in patients who had absent or blocked vas deferens (the male version of “blocked tubes”) was also used in conventional IVF, but was found to yield very low or even zero fertilization rates. Until the early 1990s, the only hope for severe male factor infertility was donor sperm, if all else failed.
One of the most significant developments in assisted reproduction was introduced in IVF clinics in 1992 (Van Steirtegham et al 1993). Intracytoplasmic sperm injection (ICSI) is a micromanipulation technique designed for use in those patients who have a severely depressed sperm count, motility or concentration of normal forms. The sperm from the patient is immobilized and injected into the egg, using a fine microscopic needle. The procedure is controlled from a special microscope workstation. Introduction of ICSI to the field really changed the way we looked at eggs, sperm and embryos (Figure 1). The simple act of removing the outer cumulus cells from the egg before ICSI allowed us to look at the visible characteristics of the egg and correlate certain visible characteristics with quality (VanBlerkom et al 1992, Meriano et al 2001). Fertilization using ICSI has been shown to be very successful, with pregnancy rates as good as and in some cases better than conventional IVF. Patients who would formerly have needed donor sperm may now have a good chance of having a biological child.
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After insemination is completed, developmental checks of the zygotes and embryos are carried out by the embryologist on a daily basis. Most laboratories culture eggs and embryos independently so that they know exactly how a certain embryo is developing. The embryologist has a list of visible characteristics to observe at different stages of embryo development in order to select the most likely normal embryos from the cohort for the fresh embryo transfer. We group these observations into relative importance as the embryo is developing. We select out the abnormal embryos and select those embryos with the characteristics that most often denote a normally developing embryo.
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For example, when fertilization is checked at 18 hours after insemination, the presence of two “pronuclei” is observed in normally fertilized zygotes (Figure 2). Three pronuclei are abnormal, as are no pronuclei. Recent studies have shown that the orientation of the small bodies inside the pronuclei is predictive of a higher possibility of a normal embryo (Scott et al 2000). This is an observational advance that has met with some controversy. The orientation of these bodies is not static, therefore if the oocytes are fertilized by IVF (we do not know exactly when the sperm has entered to the oocytes) one has to either make two relative observations or make one observation and use that piece of information loosely ( Tesarik et al 1999 ), however this observation is slightly more meaningful with ICSI embryos since we know when those oocytes were fertilized. We observe development of the normally fertilized oocytes over the next few days to Day 5.
Cell number (how many cells per embryo) and nucleation (how many nuclei per cell) are two important observations. An embryo that has undergone ICSI should be at a certain stage at 48 hours, 72 hours, etc. Some embryo cohorts are more diverse than others in their development and normality. The certainty of visible cues is not an absolute. We do know that there are cues for abnormal embryos such tripronucleate zygotes (three nuclei) and multinucleated embryos (embryos whose cells have more than one nucleus; this is abnormal); these can be selected out immediately. Completely fragmented embryos and arrested embryos (embryos that do not develop past a certain point) are also selected out at the time of observation (Figure 3).
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Cytoplasmic fragmentation (not to be confused with DNA fragmentation) is a phenomenon that is very commonly encountered when assessing the quality of embryo. The fragmentation refers to the cytoplasmic blebbing (small nodules of cytoplasm) that occurs occasionally when embryos (other cells too) divide. The embryologist looks at the various sizes of the fragmentation and how it is positioned within the embryos. Most fragmentation (small and localized to a spot in the zona) is not important, as long as the fragments do not contain any DNA. Highly fragmented embryos have been shown to have a higher arresting rate and also a lower rate of development to blastocyst, possibly because the fragments physically hinder growth of the blastocyst structure. It has also been postulated that highly fragmented embryos have programmed themselves for cell death, a natural procedure designed to remove abnormal cells from the mix ( Jurisicova et al 1996).
The developmental stage at which an embryo is transferred has been an issue over the last decade. In late 1980s and early 1990s, we transferred embryos at the 42- to 48-hour stage of development. In the mid-1990s, as culture systems became better and more consistent, we moved away from Day 2 transfers and progressed toward a 72-hour transfer. This increased results significantly. The Day 3 human embryo is “one day more developed” and at a very special stage. Until now, the embryo has used energy that it has stored when it was in the follicle as an oocyte. As the embryo compacts and becomes more developed, its own genome is turned on. At this point, some very special things happen. The embryo divides very quickly, growing to 60–120 cells in the next two days. It becomes differentiated into two cell lines: the precursor to the placenta and the precursor to the baby. And it becomes a hatching blastocyst staged embryo, which is the stage at which it should be in the uterus physiologically (Figure 4).
Another characteristic of the blastocyst is its need for different nutrients after the embryonic genome has turned on. This need has sparked another advance, called sequential culture (Gardener et al): at Day 3 we add more nutritive media to the embryos so that they are not stressed as they enter their next stage. Sequential culture allows us to grow more advanced embryos without adding negative variables (which is the reason we haven’t done this earlier). Growing embryos to blastocyst allows us to observe their development for two more days, and also allows the embryos to accumulate more cells and become hardier.
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Fifty to sixty percent of all fertilized zygotes (2PN) should achieve the blastocyst stage on Day 5. Hatching blastocysts have very high pregnancy rates. It is common practice to replace only two blastocysts to the uterus on Day 5 or 6. Statistically, if the pregnancy rate were to stay the same, at this point, the implantation rate per embryo would be increased given the lower amount of embryos transferred. However, recent literature does show rather consistently the benefit of a proper blastocyst program.
From the patient’s point of view, the field of embryology has certainly changed for the better. Pregnancy rates are higher with fewer embryos transferred; therefore triplets are almost nonexistent in those patients that receive two blastocysts. Monozygotic twinning can still occur approximately 3.8% of the time (this causes a triplet pregnancy when only two embryos are replaced. One of the embryos “splits” into identical twins and the other is fraternal). We are able to check embryos for their chromosomal number in those patients who are prone to recurrent loss. We can also screen embryos for genetic disorders and replace genetically normal embryos (for a specific gene). We are able to simply remove a cell from the embryo so that we can test it genetically, without harming the embryo (Figure 5). This, in itself, is a remarkable feat.
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Today, more than ever before, embryologists can call upon a wealth of scientific resources to observe, collect and correlate information that lets us select the most implantable embryos, and provide our patients with the best and safest chance of getting pregnant and giving birth to a healthy child. Of course many challenges still lie ahead. But given our high standards of achievement, our patients’ high hopes and the immense satisfaction we share in a successful conception, I truly can say we have the best job in the world.
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References:
Gardener,D., Schoolcraft W., Waglet, L., et al., A prospective randomized trial of blastocyst culture and transfer in in vitro fertilization.
Hum Reprod., 1998, 13,3434-3440.
Jurisicova A., Varmuza S., and Casper R.F.,
Programmed cell death and human embryo fragmentation.
Mol. Hum. Reprod. 1996, 6, 801-809.
Meriano J., Alexis J., Visram-Zaver, S., Cruz, M., Casper R.F.,
Tracking of oocyte dysmorphisms for ICSI patients may prove relevant to the outcome in subsequent patient cycles.
Hum. Reprod., 2001, 10, 2118-2123.
Scott L., Alvero R., Leondires M., Miller B.T.,
The morphology of human pronuclear embryos is positively related to blastocyst development ad implantation.
Hum Reprod. 2000, 15:2394-2403.
Tesarik J., Greco E.,
The probability of abnormal preimplantation development can be predicted by single static observation on pronuclear stage morphology.
Human Reprod. 1999, 14: 1318-1323.
Trouson A.O., Gardner D.K., ( edited by) Handbook of In Vitro Fertilization
Copy right 2000, CRC Press LLC, 200 N.W. Corporate Blvd., Boca Raton, Fla
Van Blerkom J, Antczak M, Schrader R.
The developmental potential of the human oocyte is related to the dissolved
oxygen content of follicular fluid: association with vascular endothelial growth
factor levels and perifollicular blood flow characteristics.
Hum Reprod. 1997 May; 12(5):1047-55.
Van Blerkom J., Henry G.,
Oocyte dysmorphisms and aneuploidy in mieotically mature human oocytes after ovarian stimulation.
Hum Reprod. 1992, 7,379-390.
Van Steirteghem, A.C., Nagy Z.P., Joris, H., et al High Fertilzation ad implantation rates after intracytoplasmic sperm injection, Hum Reprod., 8,1061,1993.
About the author: Jim Meriano is the lab Director at The LifeQuest Centre for Reproductive Medicine.
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Figure 1. A mature oocyte just before ICSI. Polar body is at 12 o’clock position.
Figure 2. A normally fertilized oocyte. The two nuclei in the centre are called pronuclei. One is from the sperm, and the other is from the oocyte.
Figure 3. A highly fragmented embryo.
Figure 4. Hatching blastocyst.
Figure 5. Blastomere biopsy; visible nucleus.