Preparation for Fertilization

In vitro fertilization (IVF) is a complex series of techniques used to help with fertility or prevent genetic problems and assist with the conception. During IVF, oocytes are collected from ovaries and fertilized by spermatozoa in a laboratory. The fertilization can be done using the patient’s oocytes and the partner’s sperm or donor oocytes and partner’s/donor sperm. All gametes (spermatozoa and oocytes) should be correctly prepared and selected before the initiation of the fertilization process.

Sperm Preparation (for IUI, IVF, ICSI)

For assisted reproductive technology (ART), semen samples must first be processed in a procedure that imitates the natural conditions, where the best viable spermatozoa are separated from other elements of the ejaculate and actively migrate through the cervical mucus. To maintain fertilization capacity of sperm, viable sperm cells must be separated from other elements of the ejaculate within 30–60 minutes of ejaculation [1]. It is also recommended by the World Health Organization (WHO) and suggested to limit damage from cells (leukocytes, dead and dying sperm cells) present in the semen. All sperm preparation procedures should be conducted under aseptic conditions, i.e., in a biological class II laminar flow biosafety cabinet.

Sperm must be personally delivered to the IVF laboratory by the ovum pickup (OPU) patient’s male partner presenting a photo ID card as proof of the specimen ownership. The sperm specimen should be collected into a sterile container (tissue grade and sperm-toxicity tested) by masturbation following a 24–72-hour period of abstinence [2–3], or by 1–3 hours of abstinence according to other references [4], and delivered to the laboratory within 1 hour of ejaculation (Figure 9.1). After liquefaction (sperm liquefies typically within 5–20 minutes of ejaculation [5]), the ejaculate volume is measured, and sperm cells are counted and tested for motility, all this visually observed under a light microscope (Figure 9.2). Following complete liquefaction, sperm wash medium is added to the ejaculate to remove the seminal plasma containing prostaglandins (to prevent uterine contractions in case of intrauterine insemination [IUI]), seminal particulate debris, crystals of spermin phosphate, proteins, and microbial contamination, and to minimize reactive oxygen species (ROS) and keep spermatozoa at neutral pH. Also, the presence of large numbers of nonviable spermatozoa in the sample can inhibit the capacitation of viable spermatozoa [6]. Prolonged sperm cell exposure to seminal plasma is not recommended.

Figure 9.1 The sperm specimen should be collected into a sterile container (tissue grade and sperm-toxicity tested) following a 24–72-hour period of abstinence and delivered to the laboratory within 1 hour of ejaculation. Each sperm specimen should be treated in separate stands.

Figure 9.2 After liquefaction the ejaculate volume is measured, and sperm cells are counted. (A) Sperm volume measurement; (B) and (C) sperm preparation in counting chamber; (D) sperm cells visually observed under a light microscope for motility.

A number of methods for spermatozoa separation and isolation from the seminal plasma have been developed, including swim-up, swim-down, two-layer discontinuous gradient centrifugation, sedimentation methods, polyvinylpyrrolidone (PVP) droplet swim-out, magnetic-activated cell sorting (MACS), fluorescence cell sorting methods, glass wool filtration, and electrophoresis. The ideal sperm preparation method should be cost-effective and allow for the processing of a large ejaculate volume, maximizing the number of spermatozoa available. Examination of spermatozoa in the ejaculate cannot evaluate the spermatozoa capacitation capacities, the acquisition of sperm cell surface proteins required for zona pellucida (ZP) binding and penetration, and the ability to fertilize the oocyte.

Density gradient centrifugation.

Density gradient centrifugation separates sperm cells by their density, which differs between morphologically normal and abnormal spermatozoa (Figure 9.3). A mature morphologically normal spermatozoon has a density of 1.10 g/ml or more, whereas an immature and morphologically abnormal spermatozoon has a density of 1.06–1.09 g/ml [7]. Density gradient sperm separation fraction includes a colloidal suspension of silica particles stabilized with covalently bonded hydrophilic silane supplied in HEPES. The two gradients used are a lower phase (90%) and an upper phase (45%). Semen is gently layered on top of the upper phase. After centrifugation, the interphases between seminal plasma and 45% density gradient, and 45% and 90% gradient containing the leukocytes, cell debris, and morphologically abnormal sperm with poor motility are discarded. The highly motile, morphologically normal, viable spermatozoa form a pellet at the bottom of the tube. Centrifugal force and time are kept at the lowest possible values (~300 × g) in order to minimize the production of ROS by leukocytes and nonviable sperm cells [8]. Sperm washing medium (with 5.0 mg/ml human albumin) is used to wash and resuspend the final pellet.

Figure 9.3 Sperm density gradient centrifugation.

Oocyte Preparation

Oocyte denudation for ICSI.

The optimal timing for oocyte denudation (or stripping) and ICSI remains under debate [16–18], and the effect of manipulating these intervals concerning the human chorionic gonadotropin (hCG)/agonist–OPU interval is currently unclear. Late OPU is associated with more available embryos than early OPU and significantly higher rates of fertilization and pregnancy. Some studies showed that the length of incubation before or after denudation did not affect fertilization and pregnancy rates, regardless of OPU timing [19]. Immediate (up to 30 minutes) and early (0.5–2 hours) denudation showed no statistically significant differences in fertilization and pregnancy rates between these groups [20].

Oocytes are denuded using enzymatic (hyaluronidase) and then mechanical (stripper tips of ~300–140 µm) techniques. Commercial denudation solution typically consists of 80 IU/ml hyaluronidase in a HEPES buffered medium supplemented with human serum albumin (5 mg/ml) and gentamicin (10 μg/ml). A lower concentration of hyaluronidase (8 IU/ml) has been suggested to improve the fertilization rate and embryo quality [21].

CCs are partially removed from retrieved oocytes within 1–2 minutes of pipetting in hyaluronidase, which is followed by further mechanical stripping of oocytes in buffered washing medium by gradually reducing the diameter of the stripper tips from ~300 µm to ~140 µm to complete denudation (Figure 9.4). The oocyte size must be appropriately defined visually as incorrect use of a narrow tip for a large oocyte may damage the oocyte. After stripping, oocytes should be thoroughly washed to remove traces of hyaluronidase. Removal of the CC mass provides the unique opportunity of evaluating oocyte morphology before the fertilization process, and in particular, the nuclear maturation status. Oocyte nuclear maturity status, as assessed by light microscopy, is assumed to be at the metaphase II (MII) stage when the first polar body (PB) is visible in the perivitelline space. Generally, 85% of the oocytes retrieved following ovarian hyperstimulation display the first PB and are classified as MII, whereas 10% present show an intracytoplasmic nucleus called the germinal vesicle (GV). Approximately 5% of the oocytes have neither a visible GV nor a first PB, and these oocytes are generally classified as metaphase I (MI) oocytes [22]. Following a morphological assessment of the stripped oocyte, all oocytes are separated by maturity level and cultured until fertilization.

Figure 9.4 Denudation of oocytes.

The current literature remains contradictory with regards to the value of oocyte morphology as a predictor of IVF outcome (Figure 9.5) [23–24]. Nuclear maturity alone is insufficient for the determination of oocyte quality. Nuclear and cytoplasmic maturation should be completed in a coordinated manner to ensure optimal conditions for subsequent fertilization. In the fully matured oocyte (MII), the meiotic spindle aligns with the first PB position. The appearance of the spindle is time dependent, with maximal appearance occurring between 39 and 40.5 hours post-hCG [25]. Mature oocytes have a short fertile life span and are highly sensitive to external conditions, e.g., pH, temperature, light, accuracy of stripping, and others (Figure 9.6).

Figure 9.5 Morphology of oocytes: (A) and (B) normal and mature (MII) oocyte; (C) immature oocyte with two GVs; (D) immature (MI) oocyte with compact CC; (E) fragmented oocyte, (F) vacuolated oocyte; (G) dark ooplasm and septum in perivitelline space; (H) non-regular edges; (I) huge PB; (J) two (or fragmented) PBs; (K) formless oocyte; (L) granulated ooplasm and a few PBs; (M) absence of perivitelline space; (N) big perivitelline space; (O) oval oocyte; (P) pear-shaped oocyte; (Q) two oocytes with common ZP; (R) connected oocytes.

Figure 9.6 Damaged oocyte after stripping process. (A) Partial extrusion of the ooplasm from damaged oolemma; (B) leaked ooplasm from damaged oolema.

Spontaneous maturation of immature oocytes, generally at the MI stage, retrieved after conventional gonadotropin stimulation, can be induced by in vitro culture, but are associated with lower fertilization and implantation rates compared with in vivo-matured oocytes [26–27]. Human oocytes matured in vitro require at least 1 hour to complete nuclear maturation after first PB extrusion [28].

Incomplete denudation (oocytes with attached CCs) was shown to increase the quality of subsequent cleavage embryo and blastocyst development by improving the cytoplasmic and nuclear maturation of retrieved oocytes and preimplantation development [29]. The oocyte would be cultured also with other autologous CCs and then transferred to the uterus. This process is called cumulus-assisted embryo transfer and has been shown to increase pregnancy rates [30].

Fertilization

Human life is created with the fertilization of an oocyte. This procedure in the laboratory is highly sensitive ethically; therefore, a double check of the identity of gametes before starting fertilization is mandatory. Once the oocyte and the sperm fuse, the oocyte and sperm chromosomes are first packaged into two separate male and female membrane-enclosed nuclei. Both nuclei then slowly move toward each other to the center of the fertilized egg, called the zygote. There, they continue occupying distinct territories in the zygote throughout the first cellular division. How the autonomy of parental genomes is retained after fertilization remains unclear, but involves male and female chromosome separation machinery, which increases the probability that chromosomes are separated into multiple, unequal groups, which may compromise embryo development and give rise to spontaneous miscarriage.

Fertilization by Intrauterine Insemination (IUI)

IUI is considered the simplest and least invasive insemination procedure, with reasonable live birth rates and costs. Inseminations have been performed in domestic and farm animals since the 1900s, while artificial inseminations in humans began only in the 1940s. Human IUIs were first reported in 1962 [31].

While sperm motility seems to be the most valuable predictor of IUI success [32], total motile sperm cell count (TMC) used for insemination has been cited as the most predictive index of conception after IUI cycles [33]. A minimal TMC of 1 million for insemination was initially reported to be required to achieve pregnancy [34], but later it was found that the native sperm TMC in the range of 5–10 million is the best correlate with IUI success [35–36].

We suggest an ejaculatory abstinence period of 1–2 days before IUI, despite a lower TMC inseminated compared with more prolonged ejaculatory abstinence, which is generally associated with increased sperm count, but decreased motility and no impact on sperm morphology [37]. An extended period of abstinence (more than 5–7 days) may be related to a higher exposure of sperm cells to ROS and, consequently, to greater sperm DNA damage.

Age is the most important factor in the success rate of IUI. The chances of achieving a live birth are negatively correlated with advanced maternal age [38]. The age-related drop in female fecundity has been well documented in women undergoing IUI with donor spermatozoa [39]. In women older than 40 years, IVF treatment is more preferred as the most successful strategy [40].

IUI performed in natural cycles has roughly half the pregnancy rate of that of cycles stimulated with either clomiphene citrate or gonadotropins [41–42]. In stimulated cycles two preovulatory follicles are suitable. Three preovulatory follicles, especially in older women, are acceptable [35], while most centers cancel the procedure if more than three mature follicles are observed. The hCG–IUI interval showed no impact on clinical pregnancy (any conception that is detected by ultrasound or serum hCG levels developed to pregnancy): 24 versus 36 hours of an interval [43] or 24 versus 48 hours of an interval [44]. Either one or two inseminations are valid for this procedure, and should ideally be performed within the window of 12–38 hours after ovulation induction. Follicle rupture and uterine contractions visualized by ultrasound following IUI are favorable prognostic factors.

In Vitro Insemination (classic IVF)

In vitro insemination (or classic IVF) provides a higher probability for sperm–oocyte collision through the availability of high concentrations of spermatozoa, which include numbers of motile and morphologically normal spermatozoa adequate to achieve fertilization [45]. Desirable sperm counts should provide for a ratio of 2000–10 000 sperm cells per oocyte, depending on semen parameters [46]. Insemination is carried out by placing one oocyte with sperm cells in each ~20 µl microdroplet of carbon dioxide pre-equilibrated culture medium overlaid with light paraffin oil and incubated in a 5% CO₂, 5% O₂, 90% N₂ humidified atmosphere, at 37°C. Up to five oocytes can be cultured with 50 000 sperm cells in a volume of 300 µl. For standard IVF procedures, oocytes are incubated with spermatozoa for 16 hours, mainly for practical reasons because it corresponds to the timing for the observation of pronuclei. According to several studies, prolonged oocyte exposure to high concentrations of spermatozoa may be detrimental, especially in male factor infertility cases, because of ROS formation [47].

In humans, sperm entry into the COC occurs within 15 minutes of in vitro insemination [48]. Following 1 hour of oocyte exposure to spermatozoa, a large part of the cumulus oophorus is disassociated, verifying an immediate interaction between the gametes. However, it is interesting to note that in some male factor cases, although the CCs are still intact after 1 hour of in vitro insemination, fertilization still occurs. Consequently, this could mean that the cumulus plays an essential role in the entrapment of spermatozoa within the vicinity of the oocyte [49]. The higher fertilization rates and enhanced embryo development and viability achieved after a short (1 hour) insemination indicate that prolonged exposure of oocytes to high concentrations of spermatozoa is detrimental [50].

Intracytoplasmic Sperm Injection (ICSI)

ICSI involves the direct injection of a single immobilized sperm cell into the oolema of a mature oocyte and is superior to PZD or SUZI, methods which have been used before 1992 (Figure 9.7). ICSI is used for the cases when sperm cells may not penetrate the oocyte (male/female factors) during natural conception and classical IVF. A randomized comparison found that ICSI doubled the fertilization rate compared with SUZI and generated embryos in 83% compared with 50% of SUZI cycles [55].

Figure 9.7 Types of fertilization.

The first births after the use of ICSI technology were reported by Palermo and colleagues in 1992 [56]. The direct injection of a single sperm cell into a mature oocyte bypasses the natural processes of sperm selection; oocyte survival rate after this mechanical intervention was 94%. The use of ICSI in IVF cycles has increased from 36.4% in 1996 to 93.3% in 2012 among cycles with male factor infertility [57]. ICSI has become a well-established universal method as an effective form of treatment for couples with infertility and has become the standard of care for patients with male factor infertility, with fertilization rates of 60–70%, similar to rates of conventional insemination in men with normal semen parameters [58].

Sperm suspensions after preparation are combined with medium containing PVP or hyaluronate to increase viscosity and to facilitate spermatozoa handling, though the efficacy and safety of these substances have been questioned [59]. Micromanipulation for ICSI involves a standard holding pipette for the oocyte and a sharpened injection pipette for the sperm cell. For injection, a single, motile, and overtly morphologically normal sperm cell is selected from the PVP drop and immobilized using the injection pipette. The tail of the sperm cell is positioned at a 90° angle to the injection pipette, which is lowered and drawn across the tail, resulting in membrane permeability as in nature (initiation of capacitation) and loss of motility; both of these factors enhance fertilization [60]. The immobilized sperm cell is aspirated tail first, into the injection pipette. The oocyte is held with the holding pipette and positioned such that the PB is adjacent to 6 or 12 o’clock, a position which prevents spindle destruction by the injection pipette (3 o’clock entry point of the injection pipette). The injection pipette is gently inserted through the ZP and into the plasma membrane of the oocyte. The membrane must be ruptured by gentle manipulation of the injection pipette. The sign of broken membrane is the ooplasm backflow into the injection pipette. The sperm cell is then carefully released into the ooplasm and the pipette is withdrawn, completing the procedure.

The spindle view (PolScope) system has been developed to identify a location of the spindle, useful information to prevent oocyte damage at the time of ICSI, thereby increasing the likelihood of successful, normal fertilization [61].

The survival rate of oocytes following the ICSI procedure is 94%, and the fertilization rate with ejaculated spermatozoa and surgically obtained spermatozoa is 75% and 69.8%, respectively [62]. Complete fertilization failure following ICSI has been linked to oocyte activation failure or incomplete sperm decondensation [63–65]. DNA fragmentation in female or male gametes is believed to be the primary cause of fertilization failure [66].

The timing of the ICSI procedure may be critical. The time between OPU and ICSI ranges from 1 to 10 hours. No effect of the OPU–ICSI interval on fertilization rate or embryo quality was found on day 2 or day 3 of embryo development [67]; no statistically significant differences were found between early (1–2 hours post-denudation) and late (5 hours post-denudation) injection on ICSI outcomes, embryo implantation, clinical pregnancy or live birth rates [68]. However, the aging of oocytes after OPU has been described and late fertilization (more than 5 hours) remains a controversial topic.

In contrast to in vivo fertilization, ICSI introduces a whole sperm cell into the ovum cytoplasm (Figure 9.8). In such a way, paternal mitochondria from sperm neck is introduced into the ovum. There is a theory that the paternal mitochondria are tagged with ubiquitin shortly after fertilization, successfully eliminating them from the cytoplasm [69].

Figure 9.8 Intracytoplasmic sperm cell injection (ICSI).

Selection of Spermatozoa for Fertilization

Sperm selection for ICSI is based on the visual morphological assessment by an embryologist. Therefore, exploring the relationship between sperm morphology and fertilization capacity is of critical importance to the success of ICSI [70]. Since structurally abnormal human sperm cells do not necessarily contain an abnormal chromosome constitution [71], it is not surprising that many normal babies have been born after ICSI using a low morphology sperm cell, including round-headed sperm cell without acrosomal caps [72], stump-tail sperm cell [73], and immotile sperm cell of men with axonemal defects [74]. It has even been possible to produce pregnancy and birth from oocytes fertilized by ICSI with immature spermatozoa as round spermatids [75].

Sperm cells from some men may be immotile due to low intracellular concentrations of cAMP. “Awakening” such spermatozoa using a phosphodiesterase inhibitor, e.g., pentoxifylline or theophylline, that elevates cAMP levels and enhances motility before performing ICSI has resulted in healthy children [76]. Some immotile spermatozoa are acceptable for ICSI use, as indicated by functional sperm tail membrane integrity, measured by a pulse of diode laser at the tail; a viable spermatozoon curls its tail in response to the laser. ICSI results are significantly improved when spermatozoa are selected through diode laser, as opposed to the hypo-osmotic swelling sperm selection technique [77].

MACS adapted to separate apoptotic spermatozoa from non-apoptotic spermatozoa was found to improve sperm specimen quality. Spermatozoa isolated by density gradient centrifugation followed by MACS have a higher percentage of motile and viable sperm cells and lower expression of apoptotic markers than samples prepared by density gradient centrifugation only [78].

It was also found that impaired morphology of the sperm cell head, which can be observed by embryologists in the routine sperm selection process, correlates with poorer ICSI outcomes. Motile sperm organellar morphology examinations demonstrated a positive correlation between the morphological normalcy of the sperm cell nucleus and the potential to achieve fertilization and pregnancy after ICSI [79].

Hyaluronic acid (HA) is naturally present in the extracellular matrix of the cumulus oophorus surrounding the oocyte during the natural human fertilization process. The extracellular matrix is a barrier that can only be overcome by mature spermatozoa that have extruded their specific receptors to HA and digest the matrix by hyaluronidase. After that, they penetrate the ZP and fertilize the oocyte [80]. Spermatozoa which are unable to bind HA exhibit many aspects of immaturity; they retain cytoplasm on the sperm neck and excess histones in the nucleus, show greater aberrant sperm head morphology, and have lower genomic integrity [81–82]. To improve fertilization results the noninvasive method, based on selective binding of the mature sperm cells to HA, was suggested. This method has been associated with a higher quality of embryo scores with significant decrease in aneuploidies and higher pregnancy rates [83]. HA-based spermatozoa selection is useful to improve the results of fertilization in some IVF cycles. Nevertheless, sperm cell morphology in conjunction with its motility is considered to be the best predictor of successful fertilization during natural conception, IUI conception, conventional IVF method, and ICSI. Selection of sperm cells for ICSI by a combination of different methods is highly recommended.

During the ICSI procedure, the whole spermatozoon is injected, including its outer plasma membrane and acrosome cap. Then enzymes within the ooplasm break down the sperm cell plasma membrane before sperm-derived oocyte-activating factor can activate the oocyte [84]. This delay of oocyte activation often results in an extension of the normal sperm head remodeling progression. Consequently, an asymmetry in sperm chromatin decondensation develops due to the persistence of perinuclear theca at the base of the acrosome. This delay in chromatin decondensation leads to a delay in the subsequent nuclear remodeling process, including the recruitment of nuclear pore constituents, and also leads to a delay in DNA replication and the first cell cycle following fertilization [85].

A sperm cell may not be able to induce Ca²⁺ oscillations if it undergoes premature chromatin condensation, or fails to undergo decondensation at the appropriate time [86]. Application of a Ca²⁺ ionophore to rescue an oocyte, which failed to activate, resulted in a healthy child delivery [87]. Implantation rates after ICSI with testicular spermatozoa improved when artificial oocyte activation using a Ca²⁺ ionophore was performed but were not improved when ejaculated or epididymal spermatozoa were used [88]. Sperm-specific cytosolic phospholipase C zeta (PLCζ) induces Ca²⁺ oscillations within the oocyte. Globozoospermic sperm cells can have deficient production or release of PLCζ, which is associated with oocyte activation failure. Injection of PLCζ with such sperm cell to oocyte could greatly improve the rate of oocyte activation and fertilization [89].

It has been noted that the rates of fertilization by cryopreserved sperm cells were consistently higher than with fresh sperm cells. This phenomenon can be explained due to the plasma membrane damaging of freeze–thaw cycles. During the cryopreservation process, the sperm cell plasma membrane weakens and ruptures under the stress of osmotic pressure and the formation of ice crystals. This membrane condition may accelerate sperm chromatin release from the sperm cell upon entry into the ovum by ICSI, resulting in higher fertilization rates [90].

Semen Viscosity

To reduce semen viscosity, specimens can be diluted with a sperm wash medium. Liquefaction achieved due to this procedure is not suitable for highly viscous samples. Alternatively, the viscous semen can be forced through a needle with a narrow gauge but it is associated with sperm cell damage [5]. A commonly used treatment to reduce viscosity involves enzymatic liquefaction using trypsin. If the semen fails to liquefy after a 20-minute incubation at 37°C, trypsin is added directly to the semen specimen. The specimen is then swirled and incubated for an additional 10 minutes, resulting in complete liquefaction of the sample. However, no data regarding the effect of trypsin treatment on integrity and health status of sperm cells are available.

Sperm Preparation after Retrograde Ejaculation

Retrograde ejaculation occurs when semen is redirected into the urinary bladder during ejaculation. The acidity of the urine, toxicity of urea, and hypotonic osmotic conditions in urine quickly affect the viability of sperm cells. In cases of low sperm volume and a low number of spermatozoa in the ejaculate, the urine needs to be analyzed for sperm cells. Upon arrival at the laboratory, the patient should empty his bladder, and drink a cup or two of water. As soon as the patient feels an urge to urinate, he should masturbate and collect the ejaculated specimen. Immediately after the semen collection, the patient should empty his bladder into a large container. The total volume and pH of the urine are measured and recorded, and the urine is poured into 50 ml conical centrifuge tubes. Following centrifugation for 10 minutes at 800–1000 × g, the supernatant is discarded and pellets are resuspended in a small volume of sperm wash medium. The treated retrograde and antegrade specimens are tested for sperm cells.

For clinical application of retrograde sperm for fertilization, the patient is asked to drink one tablespoon of bicarbonate with a glass of water 2 to 3 hours before masturbation. If urine pH is still too low, increase the amount of bicarbonate to two tablespoons at the next evaluation. The concentrated retrograde specimen and the antegrade specimen are usually prepared using density gradient centrifugation [91].

Sperm Preparation after Assisted Ejaculation

Direct penile vibratory stimulation (PVS) [92] or indirect rectal electro-stimulation [93] are used to retrieve semen from men who have disturbed ejaculation or who cannot ejaculate due to health conditions, such as spinal cord injury (SCI), or young boys (before oncology treatments). Infertility is a significant complication of SCI in men, 90% of whom cannot create children via sexual intercourse. Patients with SCI often have ejaculates with a high sperm concentration, low sperm motility, and contaminations of red blood cells and white blood cells. It has been reported that semen obtained by PVS has better quality than semen obtained by rectal electro-ejaculation for men with SCI [94]. Obtained ejaculates are most effectively prepared with density gradient centrifugation [91].

Variation in sperm preparation methods is available to process sperm for ART use. A suitable sperm preparation method should be carefully examined and chosen for each infertile couple.