Summary
Since the establishment of in vitro fertilization, it became quickly apparent that approximately half of the couples treated presented with a dysfunctional male gamete. To alleviate this issue, intracytoplasmic sperm injection (ICSI) was introduced to treat men with compromised semen parameters or azoospermia, and more recently high sperm chromatin fragmentation or sperm-linked oocyte activation deficiency. Because of its success, ICSI has been extended for cases with low egg yield, oocyte cryopreservation, and often for preimplantation genetic testing. Due to its versatility and reliability, ICSI has become the most popular ART and will be invaluable for emerging technologies such as in vitro gametogenesis and heritable genome editing. In this chapter, we discuss the development of ICSI, its current applications, and ongoing research that will contribute to the future of reproductive medicine.
Background
In 1934, Gregory Pincus and E. V. Enzmann demonstrated normal development of mammalian oocytes cultured in vitro [Reference Pincus and Enzmann1]. Ten years later, a study reported on the initial stages of in vitro fertilization (IVF) [Reference Menkin and Rock2]. These early achievements made the delivery of the world’s first IVF baby possible [Reference Steptoe and Edwards3]. Through efforts to enhance the efficiency of IVF, and particularly to expand this indication to the most severe form of male factor infertility, intracytoplasmic sperm injection (ICSI) was clinically applied in 1991 [Reference Gilchrist, Lane and Thompson4]. This evolution of assisted reproductive technology (ART), starting from IVF in the 1970s to micromanipulation in the 1990s, delineates the rapid progress of the reproductive medicine field. ART has become a panacea for couples struggling with infertility. ART procedures have evolved over time and have contributed to the increasing number of births by IVF throughout the decades [5]. Nevertheless, scientists continue to strive to develop new forms of treatment for infertility.
Early Micromanipulation
The proof that IVF was a viable option to infertile couples arrived in 1978, when the first live birth was achieved in a woman with bilateral tubal occlusion [Reference Steptoe and Edwards3, Reference Steptoe and Edwards6]. From there onward, conventional IVF has been applied to treat infertile couples, particularly women with tubal indications. However, other forms of infertility did not benefit from standard in vitro insemination. Indeed, suboptimal spermatozoa with compromised kinetic or morphologic characteristics were unable to penetrate the glycoprotein layer surrounding the oocyte, and therefore failed to fertilize by standard IVF [Reference Forman, Hong, Ferry, Tao, Taylor, Levy, Treff and Scott7, Reference Cohen, Malter, Wright, Kort, Massey and Mitchell8].
Even with the adoption of microdroplet insemination, an IVF technique [Reference Svalander, Wikland, Jakobsson and Forsberg9] in which a higher sperm concentration is incubated with oocytes in microdroplets under oil, the number of oocytes that were fertilized was still low. The need to treat men with impaired spermatogenesis led reproductive scientists to explore more radical alternatives to accomplish better fertilization results.
It was initially believed that the main barrier hindering the penetration of the spermatozoa into the oocyte was the zona pellucida (ZP). Therefore, an early proposal was removal of the ZP to facilitate fusion of the spermatozoa to the oolemma. However, as the ZP serves as a scaffold to support embryo growth, this led to polyspermia and decreased embryo developmental competence. A subsequent approach was to soften the ZP with trypsin or pronase to yield some fertilization; however, the embryos were unable to cleave [Reference Kiessling, Loutradis, McShane and Jackson10]. In a separate study, zona drilling (ZD), a procedure in which the oocyte is exposed to acid (Tyrode’s medium) to create a hole in the ZP, was used to facilitate the entrance of the sperm to the oocyte [Reference Palmerola, Vitez, Amrane, Fischer and Forman11]. Although a fertilization rate of 32% was achieved, chemical damage from the 2.3 pH of the medium and the collateral issue of polyspermia limited the use of this application. As a result, partial zona dissection (PZD), requiring use of a mechanical measure to produce a virtual opening in the ZP, was developed. Although the fertilization rate improved to 45%, the rate of polyspermia was about 48%, and severely asthenozoospermic and teratozoospermic patients still reported total fertilization failure [Reference Tucker, Bishop, Cohen, Wiker and Wright12].
To facilitate penetration through the ZP while reining in polyspermia, a technique was developed to overcome male factor issues and improve clinical results. Subzonal insertion (SUZI) was developed to facilitate sperm–egg interactions bypassing the ZP and placing the sperm directly into the perivitelline space [Reference Ng, Bongso, Ratnam, Sathananthan, Chan, Wong, Hagglund, Anandakumar, Wong and Goh13]. The fertilization rate increased and even couples with complete fertilization failure (CFF) obtained embryos for transfer. In a study including 43 couples with a history of CFF with IVF, the fertilization rate increased to 30.9% with an 80% cleavage rate, although the pregnancy rate remained low, ranging between 2.9% and 16.3% [Reference Palermo, Joris, Devroey and Van Steirteghem14]. These early gamete manipulation techniques were capable of addressing only mild forms of male infertility, mostly as a result of semen parameters, but were virtually powerless in cases of male gamete dysfunction [Reference Palermo and Rosenwaks15].
In the late 1980s, more radical attempts were carried out by injecting the spermatozoon directly into the oocyte. This approach was more effective, especially in cases with severe oligo- or teratozoospermia. The earliest experiments were performed in the sea urchin [Reference Hiramoto16, Reference Hiramoto17] and the Chinese hamster [Reference Hayashi, Ohta, Kurimoto, Aramaki and Saitou18, Reference Neuhaus and Schlatt19], although most of the mammalian oocytes did not survive. In a later study, however, the first pregnancy was achieved in the rabbit, followed by an unexpected, isolated birth in a bovine species [Reference Goto, Kinoshita, Takuma and Ogawa20, Reference Iritani, Utsumi, Miyake, Hosoi and Saeki21]. In 1987, human oocytes were used for this technique, but no embryos were replaced [Reference Lanzendorf, Maloney, Veeck, Slusser, Hodgen and Rosenwaks22]. Although this technique was promising to alleviate male factor infertility, its routine application was very difficult because refined tools were needed and the key steps of the injection were poorly understood. It was only in 1992 that the correct procedure and proper tools were established (Figure 2.1), giving rise to the revolutionary technique now known as intracytoplasmic sperm injection, or ICSI [Reference Palermo, Joris, Devroey and Van Steirteghem23].
The Dawn of ICSI
Since the initial attempts at gamete micromanipulation in the 1940s and early breakthroughs in the murine model in the 1960s [Reference Lin24], micromanipulation strategies in human ART remained challenging because of technological limitations resulting in oocyte damage and poor embryo implantation [Reference Lanzendorf, Maloney, Veeck, Slusser, Hodgen and Rosenwaks22]. Inspired by early gamete micromanipulation procedures such as PZD and SUZI, a more direct and efficient approach was advanced to overcome spermatozoa abnormalities. To limit the amount of damage to the oocyte during micromanipulation, a more delicate and precise injector was required. Refinements of the micromanipulator included modification of the microinjector tubing to deliver consistent output to a 5-micron diameter needle, as well as modified holding and microinjection pipettes [Reference Pereira, Cozzubbo, Cheung and Palermo25].
The first ICSI occurred during a SUZI procedure in which the oolemma was unintentionally penetrated by the microinjection pipette, and a single spermatozoon slipped inside the ooplasm. This first oocyte was not damaged by the injection, and two pronuclei observed the next morning confirmed successful fertilization [Reference Pereira, Cozzubbo, Cheung and Palermo25]. The first live birth following ICSI reported in 1992 sparked the field’s interest, and ICSI has since revolutionized the practice of reproductive medicine [Reference Palermo, Joris, Devroey and Van Steirteghem23]. This effective method ensured the precise introduction of a single spermatozoon into the oocyte, preventing polyspermy, and also demonstrated the possibility of fertilizing any mature oocyte despite abnormal sperm morphology [Reference Palermo and Rosenwaks15, Reference Palermo, Joris, Derde, Camus, Devroey and Van Steirteghem26, Reference Palermo, Neri, Schlegel and Rosenwaks27]. In terms of effectiveness in fertilization, ICSI was immediately able to yield a fertilization rate of 44% compared to its preceding technology, SUZI, which was able to achieve a fertilization rate of 18% [Reference Palermo, Joris, Derde, Camus, Devroey and Van Steirteghem26]. The emergence of ICSI allowed a better understanding of the timing between sperm penetration and pronuclei appearance [Reference Palermo, Neri and Rosenwaks28, Reference Palermo, Neri, Takeuchi and Rosenwaks29], including one and three pronuclei fertilization. These findings demonstrated that the first zygotic mitosis is controlled by the sperm centrosome (Figure 2.2) [Reference Palermo and Rosenwaks15, Reference Palermo, Munne and Cohen30].
Figure 2.2 Pericentrin staining on spermatozoa assessing paternal centrosome.
The early development of ICSI involved a great deal of fine-tuning and several protocol improvements. One important refinement was the aggressive immobilization of spermatozoa by creasing the flagellum firmly prior to injection. A study published in 1996 demonstrated that aggressive sperm immobilization improved fertilization and pregnancy rates compared to standard sperm immobilization [Reference Palermo, Colombero, Schattman, Davis and Rosenwaks31]. After several upgrades and technical revisions, ICSI became a reliable method to treat severe male factor infertility cases such as hypospermatogenesis or extreme teratozoospermia [Reference Palermo and Rosenwaks15], and soon thereafter, azoospermic men [Reference Palermo, Neri, Schlegel and Rosenwaks27]. Indications for ICSI were not limited to male factors, but also benefited non-male factor couples, including those with oocyte dysmorphism, low numbers of mature oocytes, and HIV/hepatitis C-discordant couples [32].
ICSI Applications for Male Factor Infertility
ICSI Utilizing Ejaculated Spermatozoa
The original indication for ICSI was to treat mainly male factor infertility such as suboptimal sperm concentration, severely impaired sperm kinetics, and poor sperm morphology (Figure 2.3). During its development, ICSI became a versatile treatment for most types of infertility. Currently, 90% of all ICSI cycles performed at our center utilize ejaculated specimens. ICSI was developed to enable the male gamete to bypass natural barriers surrounding the oocyte, such as cumulus cells, the ZP, as well as the oolemma, and it has been proven to be the most effective method to treat couples presenting with oligo-, astheno-, and/or teratozoospermia [Reference Palermo, Cohen, Alikani, Adler and Rosenwaks33]. In addition, ICSI is the only viable method to treat cryptozoospermia, a condition in which spermatozoa are extremely scarce. In these cases, an extensive sperm search is required to identify all spermatozoa needed to inseminate the oocytes retrieved [Reference Palermo, Neri, Schlegel and Rosenwaks27].
ICSI with Surgically Retrieved Spermatozoa
Because ICSI requires only one spermatozoon to fertilize an oocyte regardless of the morphological, kinetical, and maturational status of the male gamete, it represents the sole insemination approach for azoospermic men for whom spermatozoa can only be retrieved by surgical intervention. Epididymal aspiration has become an effective way to retrieve relatively mature spermatozoa for ICSI in men with obstructive azoospermia (OA), whether it is acquired (i.e., vasectomy) or results from genetic defects such as congenital bilateral absence of the vas deferens [Reference Ma, Marti-Gutierrez, Park, Wu, Lee, Suzuki, Koski, Ji, Hayama and Ahmed34, Reference Schlegel, Palermo, Alikani, Adler, Reing, Cohen and Rosenwaks35]. Non-obstructive azoospermia (NOA), whether caused by hypogametogenesis, maturational arrest, or germ cell aplasia, requires surgical retrieval directly from the testicular seminiferous tubule [Reference Schlegel, Palermo, Alikani, Adler, Reing, Cohen and Rosenwaks35, Reference Chan, Palermo, Veeck, Rosenwaks and Schlegel36]). At our center, ICSI has allowed testicular spermatozoa to achieve a 48.4% fertilization rate and a 35.9% clinical pregnancy rate. Even in cases with Klinefelter’s syndrome, ICSI has helped men with a non-mosaic XXY genotype to father offspring with a normal karyotype [Reference Palermo, Schlegel, Sills, Veeck, Zaninovic, Menendez and Rosenwaks37].
High DNA Fragmentation of the Male Gamete
While some male infertility etiologies are straightforward, unexplained infertility because of a subtle male factor remains challenging. To understand the etiology of these peculiar cases, supplementary tests may be needed prior to ICSI treatment [Reference O’Neill, Parrella, Keating, Cheung, Rosenwaks and Palermo38]. Even though the sperm genome is highly compacted, oxidative damage during the transit through the male genital tract can result in DNA breakage (Figure 2.4). Moreover, sperm chromatin fragmentation (SCF) has been shown to be negatively correlated with sperm motility [Reference Palermo, Neri, Schlegel and Rosenwaks27]. High SCF, resulting in recurrent pregnancy loss, can therefore be prevented by ICSI, as only the most motile spermatozoa are selected for insemination. In conjunction with ICSI, surgical retrieval of spermatozoa provided another approach to ameliorate the effect of high SCF [Reference Xie, Keating, Parrella, Cheung, Rosenwaks, Goldstein and Palermo39].
Figure 2.4 TUNEL assay depicting spermatozoa with fragmented DNA (green fluorescent signal).
Assisted Oocyte Activation
Although ICSI has been able to greatly alleviate male factor infertility, rare cases of fertilization failure after ICSI can still occur. While this can result from asynchronous oocyte maturation, it may also be attributed to a lack of a specific oocyte-activating factor in the spermatozoa. Assisted oocyte activation (AOA) can therefore be used to achieve successful fertilization and even clinical pregnancy. AOA can be carried out by exposing the oocytes, post-ICSI, to either a chemical agent or an electrical pulse [Reference Yanagida, Katayose, Yazawa, Kimura, Sato, Yanagimachi and Yanagimachi40]. Moreover, sperm-derived activating extracts or calcium-releasing compounds can be used to further enhance fertilization in these problematic cases. Oocyte activation agents include electroactivation, calcium ionophore, and strontium chloride [Reference Yanagida, Katayose, Yazawa, Kimura, Sato, Yanagimachi and Yanagimachi40–Reference Yanagida, Morozumi, Katayose, Hayashi and Sato42]. During fertilization, a series of distinct Ca2+ oscillation spikes occurs in the oocyte. These cytosolic Ca2+ spikes release waves from the endoplasmic reticulum, initiated by a sperm-bound labile protein that has been shown to settle onto a sperm-specific phospholipase C zeta (PLCζ) [Reference Wolny, Fissore, Wu, Reis, Colombero, Ergun, Rosenwaks and Palermo44] (Figure 2.5). Treatment with an oocyte-activating agent enhances this process by increasing Ca2+ permeability at the cell membrane, allowing an influx of extracellular Ca2+ into the ooplasm and thereby inducing Ca2+ release from intracellular calcium stores (Figure 2.6).
Figure 2.5 PLCζ, the putative soluble cytosolic factor, required for fertilization examined by immunofluorescence microscopy.
Figure 2.6 Mechanism of assisted oocyte activation.
One pioneering study on AOA reported 17 couples with previously failed fertilization following ICSI. For these patients’ subsequent ICSI cycles, AOA was carried out by injecting the spermatozoon with medium containing a high concentration of CaCl2, followed by exposing the oocyte to calcium ionophore. As a result, couples with this peculiar defect were able to achieve an overall fertilization rate of over 70% [Reference Heindryckx, Van der Elst, De Sutter and Dhont45]. While this treatment can effectively trigger oocyte activation, allowing concurrent sperm nuclear decondensation and thereby promoting zygote development, it is important to document a clear sperm-dependent oocyte activation deficiency (OAD), as is typical for globozoospermic cases (Figure 2.7). The demonstration of a specific sperm disturbance serves as a proper indication for AOA, identified in couples with a history of fertilization failure with ICSI and confirmed with the mouse oocyte activation test (MOAT) to measure the oocyte activation capacity of the spermatozoon. Couples with a history of failed fertilization caused by OAD were categorized as suspected, somewhat related, or clearly related to the spermatozoon. ICSI fertilization and pregnancy outcomes after AOA were then compared to the same patients’ previous cycles serving as controls. AOA consistently obtained higher fertilization and clinical pregnancy rates than those achieved from cycles of the same couples serving as controls [Reference Bonte, Ferrer-Buitrago, Dhaenens, Popovic, Thys, De Croo, De Gheselle, Steyaert, Boel and Vanden Meerschaut46].
Figure 2.7 Transmission electron micrograph of globozoospermic specimen.
Despite its demonstrated safety and effectiveness, it is imperative to remember that prior to using AOA treatment, the culpable gamete must first be identified. Therefore, AOA should be reserved only for those cases where a clear sperm-linked OAD has been diagnosed, and it should not simply be regarded as a remedy whenever unexplained fertilization failure occurs.
ICSI Applications for Non-Male Factor Infertility
Oocyte Dysmorphism
ICSI is a reliable technique that can also benefit non-male factor infertility. Using oocyte morphology to predict clinical outcome is questionable, but several studies have shown that oocyte quality reflects embryo developmental potential [Reference Gilchrist, Lane and Thompson47]. In conjunction with ICSI, it is possible to evaluate detailed oocyte morphology immediately after decoronization using high-magnification microscopy. This would not be feasible with standard IVF, in which the evaluation is limited to observation of the cumulus–oocyte complex with limited information on the oocyte itself. Indeed, with standard in vitro insemination, the oocyte can be properly evaluated once the cumulus corona cells have been dispersed by the inseminating spermatozoa.
The normal metaphase II (MII) oocyte morphology is defined as an oocyte with a spherical structure surrounded by a homogenous ZP, with a cytoplasm free of inclusions and with a uniform polar body (PB). However, not all MII oocytes display this perfect morphology (Figure 2.8).
Figure 2.8 Different types of intraooplasmic dysmorphism: (A) inclusions, (B) refractile bodies, (C) smooth endoplasmic reticulum, (D) central granulation/dark center, (E) vacuoles, (F) granular ooplasm. Different types of extraooplasmic abnormalities: (G) fragmented PB, (H) large PB, (I) expanse of perivitelline space (PVS), (J) perivitelline debris. Different types of zona pellucida (ZP) deformities: (K) dark ZP, (L) thin ZP, (M) abnormal ZP, (N) bilayered ZP. Other types of oocyte dysmorphism: (O) irregular shape, (P) oval oocyte.
Oocyte dysmorphism can be characterized by an abnormal cytoplasm (dark or granular), cytoplasmic inclusions (vacuoles, refractile bodies, or smooth endoplasmic reticulum), non-spherical shapes, abnormal ZP, abnormal perivitelline space, and/or abnormal PB (fragment, giant, smooth, duplicated). Several studies have attempted to investigate the clinical outcomes of dysmorphic oocytes. Indeed, one such study showed that when these abnormal oocytes were inseminated via standard IVF, no fertilization was achieved, whereas the use of ICSI in these particular cases not only improved their fertilizing ability, but also provided a more satisfactory clinical outcome [Reference Alikani, Palermo, Adler, Bertoil, Blake and Cohen48]. This success proved that sperm decondensation and pronuclear formation/migration are not influenced by cytoplasmic features when ICSI is applied. Another study showed that oocytes with cytoplasmic defects did not influence fertilization and cleavage rates, although implantation and clinical pregnancy rates were compromised [Reference Serhal, Ranieri, Kinis, Marchant, Davies and Khadum49]. Furthermore, a similar study reported that some defects such as refractile bodies or granular cytoplasm could decrease fertilization and embryo developmental competence [Reference Xia50]. These findings were supported by the results of ICSI on oocytes with centered granularity, which achieved a 4.2% implantation rate [Reference Kahraman, Yakin, Donmez, Samli, Bahce, Cengiz, Sertyel, Samli and Imirzalioglu51, Reference Meriano, Alexis, Visram-Zaver, Cruz and Casper52]].
Lastly, a few studies also demonstrated that although fertilization is not compromised, increased pregnancy loss may result from embryo aneuploidy that is possibly caused by dysmorphic oocytes [Reference Alikani, Palermo, Adler, Bertoil, Blake and Cohen48, Reference Yakin, Balaban, Isiklar and Urman53]. For example, an assessment concluded that fertilization and embryo developmental competence could not be attributed to oocyte characteristics [Reference Swain and Pool54].
In conclusion, while many oocyte anomalies have been described that may influence embryo selection, particularly with the advent of time-lapse observation, it is still not clear what impact these morphological traits have on clinical outcome.
Low Oocyte Maturity
To achieve normal fertilization and adequate embryo developmental competence, an oocyte undergoes certain modifications and completes essential processes involving the nucleus and cytoplasm [Reference Eichenlaub-Ritter, Schmiady, Kentenich and Soewarto55]. During follicular antral formation, granulosa cells differentiate into two specialized subpopulations of cumulus complex (CC), in which the innermost layer consists of the corona radiata and the parietal granulosa cells. Communication between the oocyte and the corona cells is required for oocyte maturation. At the time of ovulation, resumption of meiosis along with an LH surge stimulates active secretion of hyaluronic acid, inducing the expansion of the cumulus corona mass, and the formation of the corona radiata, which is essential for proper spermatozoa capacitation and subsequent fertilization.
While germinal vesicles retrieved from smaller follicles are typically characterized by an unexpanded cumulus with multiple layers of granulosa cells, metaphase-I oocytes have a more subtle appearance with a well-dispersed surrounding cumulous cell. Oocyte nuclear maturity can only be assessed properly after removal of cumulus cells, and is confirmed by the extrusion of the first polar body in the perivitelline space, indicating successful completion of meiosis I and arrest at the MII stage of development. In standard IVF, the CC is retained prior to insemination; hence, oocyte morphology and, most importantly, maturity cannot be assessed. In contrast, removal of the CC is essential for ICSI and simultaneously allows for proper evaluation of oocyte maturity. MII oocytes are the optimal and sole maturational stage for ICSI. The number of MII oocytes retrieved at each cycle can be affected by follicle size, certain ovarian stimulation protocols, or the surgeon’s ability to retrieve the smallest antral follicles [Reference Parrella, Irani, Keating, Chow, Rosenwaks and Palermo56]. ICSI offers an advantage in terms of “reading” oocytes following cumulus removal and enhances fertilization chances by direct spermatozoon injection [Reference Palermo, Neri and Rosenwaks28].
Cryopreserved Oocytes
Oocyte cryopreservation provides several advantages in ART, including the ability to preserve fertility for cancer patients or for women who choose to postpone motherhood. The survival rate for cancer patients has dramatically improved in the last two decades, increasing the need for fertility preservation. Cryopreserving “young” oocytes also maintains fertility potential and improves outcomes for older women who have delayed childbearing.
Oocyte morphology prior to cryopreservation impacts the cell integrity during thawing. If the oocyte quality is suboptimal, the post-thaw survivability will be compromised [Reference Xia50]. It is important that the thawed oocytes maintain developmental competence without any major alterations, particularly of the ZP.
The cortical reaction is triggered when a single spermatozoon binds to glycoproteins on the oolemma, leading to hardening of the ZP, thus preventing polyspermy and abnormal fertilization. It has been shown that cryostress may cause an increase in intracellular calcium concentration, inducing a cortical reaction and a premature release of cortical granules, and consequently modifications of the ZP, hindering physiologic sperm–oocyte fusion by IVF. The ability to bypass natural obstacles represented by a modified ZP, and the receptivity of the oolemma during cryopreservation, make ICSI the optimal methodology to inseminate oocytes that have been frozen and thawed [Reference Kazem, Thompson, Srikantharajah, Laing, Hamilton and Templeton57].
In Vitro Maturation (IVM)
In vitro maturation (IVM) is conducted on immature oocytes retrieved from small follicles of about 10 mm diameter, and it is usually considered an optional treatment mainly for patients with polycystic ovaries (PCOs) or a history of severe ovarian hyperstimulation syndrome (OHSS). It can also be extended to many other indications mainly related to defective oocyte maturation following a standard superovulation protocol [Reference Walls, Ryan, Keelan and Hart58]. Nonetheless, ultrastructural changes and altered properties of ZP have been observed in IVM oocytes [Reference Hatirnaz, Ata, Hatirnaz, Dahan, Tannus, Tan and Tan59]. In addition, the cumulus cells surrounding IVM oocytes have shown to carry a high apoptotic potential, requiring premature removal [Reference Shu, Zeng, Ren, Zhuang, Liang, Shen, Yao, Ke and Wang60]. All these requirements may affect the ability of IVM oocytes to be fertilized by standard in vitro insemination. Thus, ICSI is the preferred insemination method for these cases, as it allows the hardened ZP of a denuded IVM oocyte to be bypassed.
Preimplantation Genetic Testing
Preimplantation genetic testing (PGT) is the primary method for detecting multiple types of genetic embryo anomalies. For instance, numerical chromosomal anomalies within the embryo can be detected by PGT for aneuploidy screening (PGT-A), common in women of advanced reproductive age. While the presence of structural anomalies such as translocation or deletions can be identified by PGT for structural rearrangement (PGT-SR), PGT for monogenic disorders (PGT-M) can screen embryos for genetic mutations, providing valuable information on conceptuses regarding a particular heritable genetic disorder (PGT-M) or aneuploidy (PGT-A).
Initially, PGT was performed on individual blastomeres of day-3 embryos at the six- to eight-cell stage. However, at this phase of development, the embryo has not completed its self-selection in terms of its ability to implant. Moreover, the removal of one or two blastomeres with a high level of pluripotency may affect the embryo’s developmental competence [Reference Kirkegaard, Hindkjaer and Ingerslev61]. Therefore, the need to assess a larger number of cells by trophectoderm biopsies has become the standard, as this does not appear to have any negative effect on the implantation rate.
Aside from the embryo stage, the genetic assessment has evolved as well. While FISH was the earliest technique used for genetic testing, followed by comparative genome hybridization (CGH) and comprehensive chromosome screening (CCS), PGT is now primarily performed by next-generation sequencing [Reference Fragouli62].
While both PGT-A and PGT-M have become increasingly common [Reference Coates, Kung, Mounts, Hesla, Bankowski, Barbieri, Ata, Cohen and Munne63, Reference Forman, Hong, Ferry, Tao, Taylor, Levy, Treff and Scott64], PGT-M is utilized by couples, often fertile, to identify the risk of a conceptus carrying a heritable defect. Conversely, PGT-A has been performed for approximately 31% of all ART cycles in 2016 alone [Reference Palmerola, Vitez, Amrane, Fischer and Forman65]. Although PGT can be used with standard in vitro insemination, it has been strongly recommended that PGT be used in conjunction with ICSI for several reasons [32]. As ICSI requires the removal of cumulus cells from the oocyte, there would be no maternal genetic material remaining in the culture media or the trophectoderm biopsy sample that could alter test results. As ICSI requires only a single spermatozoon to inseminate the oocyte, there is no risk of paternal DNA contamination, as is the case with in vitro insemination where multiple spermatozoa attach to the ZP and may contaminate the molecular genetic technique. This was demonstrated by a recent study that reported a higher incidence of mosaicism in embryos inseminated by standard IVF cycles in comparison to ICSI [Reference Palmerola, Vitez, Amrane, Fischer and Forman65]. In addition, the higher fertilization rate achieved by ICSI would provide more embryos available for genetic assessment [Reference Jun, O’Leary, Jackson and Racowsky66].
While the universal adoption of PGT remains a matter of debate – concerns remain about putative embryo injury and genetic technical error contributing to embryo “wastage” for the above-mentioned reasons – ICSI is the preferable insemination method to use with PGT to avoid DNA contaminants that may contribute to the misinterpretation of results.
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