In vitro maturation (IVM) of oocytes is a procedure in which eggs are collected from antral follicles at a stage prior to selection and dominance. These immature oocytes are cultured under conditions that facilitate the cytoplasmic and nuclear maturation of eggs to metaphase II. This procedure is especially important for cancer patients, where the time and hormonal milieu associated with a traditional IVF cycle may adversely affect the patient’s treatment and medical outcome. Likewise, patients with contraindications for ovarian stimulatory drugs such as those with polycystic ovary syndrome (PCOS) and are at higher risk for hyperstimulation with ovulation induction agents may be candidates for IVM [5].

Although the precise mechanisms that regulate the control of oocyte maturation remain obscure, it has been recognized for over 70 years that immature oocytes removed from antral follicles may undergo spontaneous maturation in culture, termed “in vitro maturation” (IVM), without the need for hormonal stimulation. With IVM, immature oocytes are typically obtained in the mid- to late-follicular phase of the menstrual cycle. To date, IVM has been most successful in young women with multiple antral follicles that typically have a high chance of pregnancy with conventional IVF. Despite this selection bias, IVM pregnancy rates remain lower than in stimulated IVF cycles [6]. As culture conditions for IVM are optimized and pregnancy rates improve, this technology may offer a safer, less expensive, more convenient alternative to stimulated IVF.

The most common method of obtaining a semen sample is through masturbation and ejaculation into a sterile cup. In cases where ejaculation cannot be achieved via masturbation, whether due to religious or psychological reasons, non-toxic condoms can be used to collect the ejaculation following sexual intercourse. In cases where there is no presence of an ejaculate following orgasm, patients are asked to immediately urinate in a sterile cup and the sample is analyzed for presence of sperm. The presence of semen in the urine is clear indicator of a retrograde ejaculation. Men with this condition may be prescribed stomach-acid buffering medications in order to neutralize the pH of the urine and thus provide a more hospitable environment for the sperm during collection and processing.

In cases where men cannot achieve an erection, or ejaculation due to neurologic or psychogenic reasons, semen can still be collected via prostate massage, electrical stimulation of the prostate or applied vibration to the penis. Samples collected from men with spinal cord injuries typically have high concentrations of sperm, poor motility and contain red blood cell contamination in the ejaculate and require rigorous washing steps to isolate highly motile sperm for ICSI.

In cases of non-obstructive and obstructive azoospermia where sperm are not present in the ejaculate, spermatozoa can be collected by way of testicular dissection or percutaneous needle biopsy. This method of collection is highly invasive and is generally the last resort in obtaining sperm for ICSI. Samples obtained testicular dissection contain large amounts of red blood cells and testicular tissues; thus requiring additional steps to isolate a clean sample of spermatozoa.

One of the oldest and most commonly used methods of sperm isolation is the swim-up procedure. This sperm separation technique is mostly used on normozoospermic males. The swim-up method is based on the active movement of motile sperm from a pre-washed pellet of sperm into an above layer of fresh medium. The first step in swim-up involves repeat dilution and centrifugation (2–3 times) of the semen sample to separate spermatozoa from seminal plasma. Following centrifugation, the pelleted spermatozoa can be both suspended and overlaid with media or the pellet can be uninterrupted and overlaid with media. If one chooses to disrupt the pellet, extreme care must be taken when overlaying with media to prevent mixing and contamination with immotile sperm, debris, and other cell types.

The swim-up method using either the intact or disrupted sperm pellet is incubated at 37 °C for 30–60 min in a buffered media to allow spermatozoa to swim from the pellet to the culture medium. Following incubation, the upper layer of culture media is carefully aspirated without disrupting the pellet and is transferred to a clean test tube for further analysis. One advantage of this technique is that it isolates a population of sperm with greater than 90% motility and without cellular debris. The disadvantages of this technique are in the low overall recovery of motile spermatozoa due to the limited surface area of the pellet and culture media . Another disadvantage of this technique is that repeat centrifugation force viable spermatozoa to be in close contact with immotile spermatozoa, cellular debris, and leukocytes, which are known to produce very high levels of ROS and affect subsequent fertilization ability.

Density gradient centrifugation is the second most common method for isolating motile sperm for ART purposes. The majority of all density gradients used to isolate spermatozoa is discontinuous and consists of two to three layers. The most commonly used materials for density gradients are colloidal silica with covalently bound silane molecules, which have a low viscosity, are non-toxic and are approved for human use.

There are many advantages in using a density gradient to process spermatozoa for IVF and ICSI. The entire ejaculate is used during the centrifugation process, resulting in a significantly higher yield of motile spermatozoa than can be obtained using other separation techniques. This makes this technique ideal for patients with suboptimal semen parameters (e.g., oligozoospermia and asthenozoospermia). Another advantage of this technique is that it produces a relatively clean sample of spermatozoa, free of cellular debris and leukocyte contamination. This property significantly reduces the ROS and problems associated with its contamination.

Fertilization assessments are performed 15–18 h after insemination for both IVF and ICSI procedures. It is necessary to examine the oocytes/zygotes within this time period to visualize the presence of pronuclei and polar bodies. Normal fertilization is characterized by the presence of two pronuclei, one male and one female, in the ooplasm and two polar bodies in the perivitelline space (Fig. 18.4). If oocytes have undergone conventional IVF, the cumulus cells must be removed to clearly see the oocyte.

Abnormal fertilization may also be represented by oligopronuclear zygotes. The term applies to zygotes that have single pronuclei. Only one pronuclei and the presence of two polar bodies may be observed in cases when the oocyte undergoes parthenogenic activation or failure of the spermatozoa head to decondense. It is possible that a second pronuclei will be developed later than the first one, therefore a repeat observation about 4 h after the first check is recommended. Failed fertilization is represented by the absence of pronuclei and presence of one or two polar bodies that may be in the process of degeneration.

Embryos can be assessed and graded daily while they are in culture. Standard morphologic methods of grading can be applied according to observations made on embryo development until their transfer to the uterus on day 3 (72 h post fertilization) (◘ Tables 18.1 and 18.2) or on day 5 or 6 at the blastocyst stage (◘ Table 18.3) [8, 9]. There are numerous scoring systems proposed for embryo development. Criteria for grading include the rate of division as judged by numbers of blastomeres, size, shape, symmetry, appearance of the cytoplasm, and presence of cytoplasmic fragments.