Each individual treatment cycle involves a number of different stages and manipulations in IVF laboratory, and each case must be assessed and prepared for in advance. The afternoon prior to the procedure (the day after hCG administration) is a convenient time to make the preparations. IVF laboratory staff must ensure that all relevant consent forms are signed by both partners, including consent to special procedures and storage of cryopreserved embryos.
Details of any previous assisted conception treatment should be studied, including response to stimulation, number and quality of oocytes, timing of insemination, fertilization rate, embryo quality and embryo transfer procedure, and judgments regarding whether any parameters at any stage could be altered or improved in the present cycle can be assessed.
The risk of introducing any infection into the IVF laboratory via gametes and samples must be absolutely minimized: screening tests such as human immunodeficiency virus (HIV 1 and 2: Anti-HIV 1, 2) and hepatitis B (HbsAg/Anti-HBc) and C (Anti-HCV-Ab) should be confirmed, as well as any other tests indicated by the patients’ history. If donor gametes are to be used, additional tests for the donor are required: chlamydia, cytomegalovirus and a validated testing algorithm to exclude the presence of active infection with Treponema pallidum for syphilis testing.
The IVF laboratory case notes, media, culture vessels and tubes for sperm preparation, with clear and adequate labeling throughout, are prepared in advance of each case. All labeling should have a minimum of the patient’s full name and a unique identifier, for example, patient number. When donor sperm is used, the donor code must uniquely identify that specific donor. Tissue culture dishes or plates can be equilibrated in the culture incubator overnight. The choice of culture system used is a matter of individual preference and previous experience.
Oocyte Retrieval (OCR) and Identification
A Class II biological safety cabinet is recommended for handling of follicular aspirates to avoid risk of infection.
If follicular aspirates cannot be examined immediately, they should be collected into test tubes that are completely filled with fluid, tightly sealed and rigorously maintained at 37 °C until they reach the IVF laboratory. Aliquot the contents of each test tube into two or three Petri dishes, forming a thin layer of fluid that can be quickly, carefully and easily scanned for the presence of an oocyte, using a stereo dissecting microscope with transmitted illumination base and heated stage. Low-power magnification (x6-12) can be used for scanning the fluid, and oocyte identification verified using higher magnification (x25-50).
The IVF laboratory staff always works quickly and carefully, with rigid attention to sterile technique, maintaining the correct temperature and pH at all times.
The oocyte usually appears within varying quantities of cumulus cells and, if very mature, maybe pale and difficult to see. Granulosa cells are clearer and more “fluffy,” present in amorphous, often iridescent clumps. Blood clots, especially from a collecting needle, should be carefully excised with 23-gauge needles to check for the presence of cumulus cells.
The presence of blood clots within the cumulus-oocyte complex (COC) may be a reflection of poor follicular development, with an effect on the competence of the corresponding oocyte. When a COC is identified, assess its stage of maturity by noting the volume, density and condition of the surrounding cumulus cells and the expansion of coronal cells. It is unlikely that the oocyte itself can be seen, since it will most commonly be surrounded by cumulus cells. However, when an oocyte can be observed with minimal cumulus cells, the presence of a single polar body indicates that it has reached the stage of metaphase II. The appearance of the COC can be used to classify the oocyte according to the following scheme:
Germinal vesicle: The oocyte is very immature. There is no expansion of the surrounding cells, which are tightly packed around the oocyte. A large nucleus (the germinal vesicle) is still present and may occasionally be seen with the help of an inverted microscope. Maturation may occasionally take place in vitro from this stage, and the COC can be assessed later in the day prior to insemination.
Metaphase I: The oocyte is surrounded by a tightly opposed layer of corona cells. Tightly packed cumulus with little extracellular matrix may surround this with a maximum size of approximately five oocyte diameters. If the oocyte can be seen, it no longer shows a germinal vesicle. The absence of a polar body indicates that the oocyte is in metaphase I, and these immature oocytes maybe pre-incubated for 6–24 hours before insemination.
Pre-ovulatory (harvested from Graafian follicles): This is the optimal level of maturity, appropriate for successful fertilization. Coronal cells are still opposed to the oocyte but are fully radiating; one polar body has been extruded. The cumulus has expanded into a fluffy viscous mass that can be easily stretched, with abundant extracellular matrix.
Mature: The oocyte can often be seen clearly as a pale orb; little coronal material is present and is dissociated from the oocyte. The cumulus is very profuse but is still cellular. The latest events of this stage involve a condensation of cumulus into small black (refractile) drops, as if a tight corona is reforming around the oocyte. The perivitelline space often shows granularity.
Luteinized: The oocyte is very pale and often difficult to find. Cumulus clouds decay and turn into a gel-like mass around the egg. These oocytes have a low probability of fertilization and usually inseminate with a slight delay.
Atretic: The oocyte is very dark and can be difficult to identify. Granulosa cells are fragmented and have a lace-like appearance.
Gross morphological assessment of oocyte maturity is highly subjective and open to inaccuracies. In preparation for intracytoplasmic sperm injection (ICSI), the oocytes are completely denuded of surrounding cells using hyaluronidase, allowing accurate assessment of nuclear maturity and cytoplasm; this process has made it apparent that gross COC morphology does not necessarily correlate with nuclear maturity, and there is considerable conflict in the data available regarding the association between oocyte morphology and treatment outcome. A number of dysmorphic features can be identified in denuded oocytes, including areas of necrosis, organelle clustering, vacuolation or accumulating aggregates of smooth endoplasmic reticulum (sER). Anomalies of the zona pellucida and nonspherical oocytes can also be seen. In practice, a wide variety of unusual and surprising dysmorphism are often observed.
Some features of dysmorphism may be associated with the endocrine environment during ovarian stimulation, in particular the structure of the zona pellucida and/or oolemma. Although aberrations in the morphology of oocytes are not necessarily of any consequence to fertilization or early cleavage after ICSI, it is possible that embryos generated from dysmorphic oocytes have a reduced potential for implantation and further development. Repeated appearance of some dysmorphic features such as sER aggregation, central granulation or vacuoles in an individual patient’s oocyte cohort may indicate an underlying intrinsic problem in the process of oocyte development within the ovary.
Oocytes are routinely inseminated with a concentration of 100,000 progressively motile sperm per milliliter. If the prepared sperm shows suboptimal parameters of motility or morphology, the insemination concentration may be accordingly increased. Some reports have suggested that the use of a high insemination concentration of up to 300,000 progressively motile sperm per milliliter may be a useful prelude before deciding upon ICSI treatment for male factor patients. Traditionally, inseminated oocytes were incubated overnight in the presence of the prepared sperm sample; however, sperm binding to the zona pellucida normally takes place within 1–3 hours of insemination, and fertilization occurs very rapidly thereafter. A few hours of sperm contact with the eggs lead to the same course of events that occurs after incubation overnight, and oocytes can be washed without excess sperm after a three-hour incubation.
Scoring of Fertilization on Day 1
Inseminated oocytes are dissected 17–20 hours following insemination in order to assess fertilization. Oocytes at this time are normally covered with a layer of dispersed coronal and cumulus cells, which must be carefully removed so that the cell cytoplasm can be examined for the presence of two pronuclei and two polar bodies, indicating normal fertilization. The choice of dissection procedure is a matter of individual preference, and sometimes a combination of methods may be necessary for particular cases. Whatever the method used, it must be carried out carefully, delicately and speedily, taking care not to expose the fertilized oocytes to changes in temperature and pH. Scoring for pronuclei should be carried out within the appropriate time span, before pronuclei merge during syngamy: cleaved embryos with abnormal fertilization are indistinguishable from those with two pronuclei.