A brief history of technical developments in intracytoplasmic sperm injection (ICSI). Dedicated to the memory of J.M. Cummins
J. G. Thompson
A B C * , H. J. McLennan A , S. L. Heinrich A , M. P. Inge A , D. K. Gardner D E and A. J. Harvey D E
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B
C
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Abstract
Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technology for treatment of severe male infertility introduced into clinical practice in 1992. This review provides a brief history of the development of ICSI by acknowledging major developments in the field. The review addresses key developments in pre-clinical and early studies, how ICSI compares with in vitro fertilisation, long-term consequences, how the mechanistic approach to ICSI has changed in both manual and semi-automated approaches, and how sperm selection procedures are integrated into ICSI. From the beginnings using animal models in the 1960–1970s, the development of ICSI is a remarkable and transformative success story. Indeed, its broad use (70% of cycles globally) exceeds the need required for treating infertile males, and this remains a controversial issue. There remain questions around the long-term health impacts of ICSI. Furthermore, advances in automation of the ICSI procedure are occurring. An estimated 6 million children have been born from the ICSI procedure. With further automation of sperm selection technologies, coupled with automation of the injection procedure, it is likely that the proportion of children born from ICSI will further increase.
Keywords: assisted reproduction, automation, fertilisation, ICSI, IVF, male infertility, piezo, spermatozoa, sperm injection, sperm selection.
Introduction
Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technology (ART) that provides the most effective treatment solution for severe male infertility circumventing the use of conventional in vitro fertilisation (IVF) in an ART cycle. ICSI as an infertility treatment has been transformational (Palermo et al. 1992; Schlegel and Girardi 1997; Esteves et al. 2018) in resolving even the most intractable male infertility prognosis, including azoospermia. Since its first use, improvements have led to an accepted oocyte survival rate after ICSI of 90–95%, and an accepted fertilisation rate of greater than 65%, with an achievable benchmark of 80% (ESHRE Special Interest Group of Embryology and Alpha Scientists in Reproductive Medicine 2017). Although initially intended for use in male factor infertility (contributing to ~50% of all ART cycles), global application has exceeded the proportion of sub-fertile males and accounts for almost 70% of all treatment cycles, although this figure varies widely between individual countries (Dyer et al. 2016; Banker et al. 2021). The purpose of this review is to bring a (brief) historical perspective to the clinical development and subsequent widespread adoption of ICSI, as it now can claim to be the most impactful ‘add-on’ laboratory IVF treatment. A recent statement by the Chair of The International Committee Monitoring Assisted Reproduction (ICMART – https://www.icmartivf.org/) proposed that, as of 2023, an estimated 12 million children had been born through IVF/ICSI, thereby suggesting ICSI was involved in an estimated 6 million births. But throughout its relatively short clinical history, questions have emerged and remain over its use, trending to overuse, and what are the consequences of being conceived by ICSI compared with conventional IVF. This was a subject of concern to James (Jim) M. Cummins (1943–2023), to whom we dedicate this review. Jim was a significant figure in Australian male reproduction research. An obituary can be found in the Society for Reproductive Biology’s April 2023 Newsletter (https://www.srb.org.au/newsletters). Jim was a frequent visitor to Ryuzo Yanagimachi’s laboratory in Hawaii, who also passed away in 2023, and we also acknowledge his significant contribution in the development of ICSI (Wakayama and Ogura 2024).
Methodology
A literature search was conducted using the following search terms: ICSI/intracytoplasmic sperm injection AND PolScope OR Oosight OR spindle/polar body (±orientation) OR polarised light OR Piezo OR laser-assisted OR sperm selection OR PICSI OR SpermSlow OR cytoplasmic aspiration OR oolemma damage/oocyte degradation OR blastocyst (AND human). This review discusses the literature returned from these search criteria to present a brief history of the developments made in the use of ICSI. Additional references were added where significant gaps remained as adjudged by the authors.
Pre-clinical and early observations
It is often cited that ICSI was developed serendipitously by the team at Vrije Universiteit Brussel (Palermo et al. 1992). There can be no question of the significance of their publication in Lancet, as it was the first to demonstrate that children could be conceived by this technique. But descriptions of techniques for sperm microinjection into oocytes were first reported for sea urchin and amphibia as early as the 1960s (Hiramoto 1962; Graham 1966; Brun 1974). The first demonstration of mammalian sperm injection into a mammalian oocyte was a hamster sperm nucleus, validated by injecting a human sperm into hamster oocytes (Uehara and Yanagimachi 1976), utilising micro-injection equipment and tools that are not dissimilar to those currently used. In both cases, clear evidence of male pronuclei (PN) formation were observed. Furthermore, frozen–thawed and freeze-dried human sperm were also able to form PN (Uehara and Yanagimachi 1976). It was also the first account of the use of polyvinylpyrrolidone (PVP) to prevent ‘sticking of sperm’ in the injection pipette.
The hamster oocyte was found to be an ideal ICSI model to examine the microinjection of sperm from other species, such as the mouse, rat, rabbit, bull, and human, which were all shown to form PN (reviewed by Iritani 1991). Furthermore, live births were achieved from ICSI of rabbit and cattle oocytes with homologous sperm (Iritani 1991). However, the first evaluation of human oocyte survival from microinjection of a human sperm was performed by Lanzendorf et al. (1988). They showed PN formation was able to occur (6/20 injected MII oocytes) but remarked on the necessity of breaking the oolemma to ensure the sperm was injected in the cytoplasm. At the same time, there emerged two alternative insemination methods for sub-fertile men: sub-zonal insemination (SUZI) (Ng et al. 1988) and partial zona dissection (PZD) (Cohen et al. 1988). However, although pregnancies were achieved through both, with reports over the next 5–6 years, neither were as effective as ICSI for fertilisation (reviewed by Tarín 1995), in particular, the foundational reference of Palermo et al. (1992) that compared SUZI to ICSI in four couples with significant male infertility indicated. Of 70 MII oocytes, three were fertilised by applying SUZI compared to 31 of 47 oocytes fertilised by ICSI, despite 19% (9/47) oocytes ruptured following injection. Moreover, 18 oocytes produced good quality embryos, of which four pregnancies were established. This was followed by two further studies of 300 consecutive cycles (Van Steirteghem et al. 1993a) and 150 cycles (Van Steirteghem et al. 1993b), respectively, between SUZI and ICSI where previous cycles of conventional IVF insemination had failed fertilisation. It clearly showed the utility of ICSI to deliver pregnancies over SUZI.
Efficacy of IVF versus ICSI
The success of ICSI to resolve severe male infertility was subsequently validated in several studies, including the use of epididymal and testicular sperm to support fertilisation and live births (e.g. Silber et al. 1994; Levran et al. 1995). A common theme in these studies is that the initial loss of oocytes from injection-mediated damage is offset by the improvement in subsequent fertilisation rates and embryo production. Indeed, the success of ICSI led some to suggest that all IVF cycles should be ICSI cycles (e.g. Fishel et al. 2000). This was given some impetus from studies demonstrating equivalence to IVF in live birth rates and post-natal markers (Bonduelle et al. 1995). However, questions began being raised about the efficacy and safety outcomes of ICSI relative to IVF, particularly when there was no evidence of male sub-fertility (Staessen et al. 1999; Dumoulin et al. 2000). As an example, Bhattacharya et al. (2001) compared 415 randomly assigned IVF and ICSI cycles that had similar patient characteristics and non-male factor infertility which demonstrated conventional IVF remained superior in these patients. This conclusion was based on data indicating that fertilisation rate per oocyte retrieved was significantly higher after IVF (58%) compared with ICSI (47%), despite the fertilisation rate per oocyte inseminated/injected being significantly lower after IVF (58%) compared with ICSI (65%). Notably, the implantation rate was significantly higher following IVF (30%) compared with ICSI (22%). However, they also recorded the laboratory time taken for each procedure, which was significantly less for IVF (22 min) compared with ICSI (74 min), considerably impacting workflow within the IVF laboratory. They concluded that the data did not support the expanded use of ICSI in ART. This was further supported by two Cochrane Reviews (van Rumste et al. 2000; Lepine et al. 2019) and recommended by the Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology (2020).
Between IVF and ICSI inseminated oocytes, ICSI oocytes completed morphological development markers earlier, demonstrating a difference in the kinetics of development (Dumoulin et al. 2000; Van Landuyt et al. 2005; Lemmen et al. 2008). Compared to conventional IVF, time-lapse recordings showed that the 2-cell stage occurred approximately 2.5 h earlier following ICSI (Lemmen et al. 2008). Most studies attribute this to the difference in timing of fertilisation (e.g. Cruz et al. 2013), and there are suggestions that later cell cycles of IVF embryos are more rapid despite normalisation to PN fading (Bodri et al. 2015; De Munck et al. 2022).
A variant in the timing of ICSI is ‘Rescue ICSI’, applied when IVF fertilisation has failed completely or in very low numbers relative to the mature oocyte numbers collected. Failure of fertilisation is most likely caused by sperm defects but may also be a malfunction within oocytes (reviewed by Beck-Fruchter et al. 2014). Successful pregnancies are markedly lower following Rescue ICSI, especially when performed the day after IVF insemination, but is more successful the earlier ICSI is performed (Beck-Fruchter et al. 2014; Ohgi et al. 2016).
Long-term health consequences of ICSI versus IVF
Questions over the long-term health of children born from ART have been raised, from both the association with infertility and conception within an in vitro environment. Such concerns were raised from animal studies, where many examples of perturbed growth and physiology have been published (reviewed by Beilby et al. 2023). Of significance is a recent publication investigating generational impacts of mouse ICSI, which revealed behavioural and congenital abnormalities in F2 offspring, as well as supporting results on behavioural differences in ICSI-derived F1 males (Kanatsu-Shinohara et al. 2023). Even as early as 1995, Cummins and Jequier (1995) voiced concern over the long-term impacts of ICSI, especially with testicular-sourced cells. Linking birth registry data with patient data from two IVF units, Davies et al. (2012) found that registered birth defects were significantly higher in both IVF and ICSI children compared to natural births, but for IVF births this relationship was lost when parental factors were accounted for. However, a higher incidence of birth defects was maintained in ICSI-conceived children.
Two reviews of long-term studies of ICSI-conceived children (Catford et al. 2017, 2018) found little evidence of differences in neurological assessment, growth, vision, and hearing compared with naturally conceived or IVF-conceived children, but remained cautious in claiming there were no broader health impacts. A further review (Esteves et al. 2018) concluded it remained unclear whether ICSI has a negative impact on long-term health. There are however indications of altered reproductive health, including a significant reduction in sperm count and quality in young male adults conceived through ICSI to treat infertility in their fathers (Belva et al. 2016). A more recent study by Sonntag et al. (2020) revealed a higher incidence of hypergonadotropic hypogonadism (1.4%) in female ICSI adolescents compared with the normal population, and male ICSI adolescents had significantly higher estradiol levels and a significantly lower testosterone-to-estradiol ratio. These data add to a similar report by Belva et al. (2017), indicating comparable levels of follicle-stimulating hormone (FSH), luteinising hormone (LH), testosterone, and inhibin B in ICSI-conceived males, despite trending towards lower inhibin B and higher FSH levels than their spontaneously conceived counterparts. These data may indicate altered testicular function that reflects a heritable health issue; therefore, the long-term health of ICSI children remains an open question justifying further ongoing monitoring.
Methodological aspects – manual injection process
Mechanics of injection
Following on from the initial success of Palermo et al. (1992), efforts were made to understand the mechanistic determinants for successful ICSI. Prominent is the study of Nagy et al. (1995), who analysed the impact of the injection process. Firstly, they examined the position of the first polar body and its impact on subsequent fertilisation that supported the 12 and 6 o’clock alignment, and then they assessed the depth and positioning of the injection pipette during the injection. As recognised by Lanzendorf et al. (1988), puncturing the oolemma was essential for success, as was the need for aspirating a small volume of cytoplasm into the injection pipette prior to injection. The elasticity of the oolemma varies between oocytes and therefore experience of the operator is necessary. Palermo et al. (1996a) provided observations on the degree of oolemma elasticity and related it to injection damage. They observed that 74% of oocyte oolemma membranes broke as expected, 12% suddenly broke, and 14% were difficult to break. Sudden breakage was associated with a higher rate of oocyte lysis and reduced pronuclear formation. Similar results were obtained by Danfour and Elmahaishi (2010). Likewise, McLachlan et al. (1995) related fertilisation success to increasing levels of cytoplasmic withdrawal. Further, in a highly insightful study, Tesarik and Sousa (1995) demonstrated that much higher fertilisation rates were achieved through ‘vigorous aspiration’, mediated by increasing the intracellular Ca2+ load at the time of injection, thereby increasing the impact of subsequent intracellular Ca2+ pulses associated with oocyte activation. Nevertheless, less vigorous aspiration appears to support higher rates of blastocyst development (Dumoulin et al. 2001; Hiraoka et al. 2012), plausibly though reduced cytoplasmic damage.
Visualisation of the spindle
The development and application of polarised light microscopy (‘PolScope’) to oocytes for imaging the meiotic spindle was first described using hamster oocytes (Silva et al. 1999). The adoption of polarised light microscopy was aimed at preventing damage to the spindle during the injection procedure. The revelation from this study was to dismiss the long-held view that the meiotic spindle was adjacently aligned to the first polar body. This was verified for human ICSI by Wang et al. (2001a), where only 20% of polar bodies aligned with the spindle. Furthermore, not all oocytes had clear spindles and these oocytes were less capable of forming embryos (Wang et al. 2001b; Cohen et al. 2004). A follow-up meta-analysis determined that fertilisation rate was improved when a spindle was visible (Petersen et al. 2009), but other studies concluded otherwise (Moon et al. 2003; Konc et al. 2004). More recently, Tilia et al. (2020) found that a normal spindle morphology is twice as likely to result in a euploid blastocyst.
Immobilisation of sperm prior to injection
A feature of ICSI methodology is the immobilisation of sperm by striking or touching the sperm tail with the injection pipette. This promotes PN formation, whereas injection of motile sperm results in significantly lower 2PN rates (Fishel et al. 1995). The immobilisation induces permeabilisation of the sperm membrane and enhances subsequent nuclear decondensation (Dozortsev et al. 1995). Aggressive immobilisation significantly increased 2PN and clinical pregnancy rates for both fresh and frozen–thawed ejaculated, epididymal, and testicular sperm (Palermo et al. 1996b). Several studies showed the importance of breaking the sperm membrane as a part of the immobilisation prior to injection (e.g. Svalander et al. 1995; Vanderzwalmen et al. 1996), with the latter demonstrating improved fertilisation rates from reducing the diameter of the injection pipette, an observation also made by Yavas et al. (2001). Piezo-mediated immobilisation also improved fertilisation rates (Yanagida et al. 2001). An explanation of Piezo motion is provided in the following section.
Semi-automated ICSI
Intracytoplasmic sperm injection remains a highly skilled laboratory task (Tiegs and Scott 2020), and even partial automation of the ICSI procedure would have several advantages over conventional ICSI, such as decreasing laboratory workload and training inputs, while assisting in standardising the procedure between different embryologists. Indeed, technician skill is documented to significantly impact blastocyst rates (Dumoulin et al. 2001). Such an automated ICSI system was first described for human sperm injection into hamster oocytes by Lu et al. (2011). However, their approach in this often-cited publication of combining image guidance sperm manipulation in conjunction with a fabricated chip, appears not to have been furthered for clinical assessment. Since then, other approaches have been developed, as described below.
Piezo-ICSI
Piezo actuators for ICSI deliver high-frequency micro-oscillations with high force to a modified injection pipette. The piezo motion acts as a ‘drill’ to remove a core of the zona pellucida, thereby the pipette easily enters the perivitelline space and reduces oolemma invagination during injection (Yanagida et al. 1999). Originally applied to overcome the limitations with mouse ICSI due to the large size of murine sperm heads (Kimura and Yanagimachi 1995), human oocyte Piezo-ICSI was first reported by Huang et al. (1996), with a comparison study between Piezo-ICSI and conventional ICSI reported 2 years later (Yanagida et al. 1999). This latter study demonstrated that compared to conventional ICSI, Piezo-ICSI enabled significantly more oocytes surviving injection (66% vs 88%), more fertilised oocytes, more good quality day 3 embryos, and significantly higher pregnancy rates (23% vs 15%); partly by the use of thin-walled glass pipettes (Hiraoka and Kitamura 2015). Piezo injection also improved results from ICSI patients with previous poor outcomes (Caddy et al. 2023). Nevertheless, initial clinical adoption was limited due to the risk of contamination of the toxic hydraulic fluids (mercury or ‘Fluoronert’) required for Piezo actuation (Yanagida et al. 1999). Despite this encumbrance, several studies have followed, all reporting benefits to fertilisation and/or subsequent developmental outcomes (Takeuchi et al. 2001; Furuhashi et al. 2019; Fujii et al. 2020; Zander-Fox et al. 2021). Recently, the use of an alternative actuator fluid, perfluoro-n-octane, has alleviated the potential toxicity risk (Zander-Fox et al. 2021). Nevertheless, Piezo-ICSI takes longer to perform and requires additional costly equipment expenditure and training (Zander-Fox et al. 2021). A recent study combined Piezo-ICSI with image-guided automated control of the injection pipette and injection sequence. After testing in animal models, two clinical pregnancies from 14 injected oocytes were achieved (Costa-Borges et al. 2023), demonstrating that automation of the injection process is feasible.
Laser-assisted ICSI
An alternative to Piezo-ICSI, laser-assisted ICSI (LA-ICSI) also creates a suitably sized hole in the zona pellucida but with a high-energy laser beam, providing an unimpeded path for the injection pipette, thereby reducing invagination. Rienzi et al. (2001) and Nagy et al. (2001) simultaneously reported this approach in case studies to overcome poor oocyte survival following four and two conventional ICSI cycle attempts respectively. Both produced embryos and reported successful pregnancies. In a small study of 32 patients with one or more previous ICSI-induced degeneration of oocytes, where oocytes were split between C-ICSI (conventional ICSI) and LA-ICSI, Abdelmassih et al. (2002) reported a significant increase in day 3 embryo development with LA-ICSI (77% vs 57%). A further, yet inadequately powered, comparison between C-ICSI and LA-ICSI concluded that there was no advantage (Richter et al. 2006). This differed from the results of Choi et al. (2011) in a larger study (106 patients in each arm), which found that in patients less than 38 years old, LA-ICSI reduced the oocyte degeneration rate and increased fertilisation and embryo developmental rates. However, LA-ISCI did not affect these outcomes in older patients (≥38 years of age). Furthermore, LA-ICSI did not improve outcomes for patients with more than three previous IVF cycle attempts. Finally, a large and appropriately powered study (966 couples) of first and second ICSI cycle attempts demonstrated that LA-ICSI improved oocyte survival and fertilisation rate, but not subsequent development, with even a suggestion that the blastocyst rate was lower (Fawzy et al. 2020). This is possibly from a residual influence of the laser treatment, a concern that has likely prevented significant uptake of this technology to date, but, like Piezo-ICSI, no long-term studies of LA-ICSI-conceived children have been conducted.
Modified holding pipettes
While much attention has been focussed on automation of the injection sequence, devices designed to provide improved support to the oocyte during injection have been limited. Alternative designs to the conventional ‘heat-polished end’ hand-made or micro-engineered holding pipette have been published (Lyu et al. 2010; Ma et al. 2019; Fernández et al. 2020). These glass-holding pipette designs all feature varying degrees of a flanged end that enclosed mouse oocytes, in some cases significantly distorting the shape of the oocyte. Nevertheless, in a mouse oocyte ICSI model, Fernández et al. (2020) observed that cytoplasmic withdrawal into the injection pipette was not required, seemingly causing less invagination through better support for the oocyte. A different approach involved a V-shaped support for the oocytes, created through micron-sized 3D-printing fabrication (Yagoub et al. 2022). This device allowed for up to three mouse oocytes to be loaded in an array, which provided greater traceability following microinjection and required the use of only one micromanipulator to complete the microinjection. Further development of this fabricated concept has recently been reported, enabling up to 20 oocytes to be injected within a 20 micro-well array, delivering an improved blastocyst rate in a porcine model (McLennan et al. 2024).
The contribution of sperm and sperm selection technologies
There is now a consensus view that poor DNA integrity of human sperm, due to, and not limited to these alone, one or more factors associated with increasing paternal age, poor diet, and lifestyle choices, are associated with poor early development outcomes (reviewed by Aitken and Bakos 2021). Companioning with this understanding are rapid advancements in the development of devices and methods to select the most viable and least DNA damaged sperm for use in ICSI. There is a World Health Organization guide for human sperm selection, which is currently in its sixth edition (Boitrelle et al. 2021). Two specific techniques have been investigated in recent years to select sperm specifically for ICSI: physiological intracytoplasmic sperm injection (PICSI) and zeta sperm selection.
The PICSI method evolved from observations that mature sperm bind to a hyaluronic acid (HA) matrix (Jakab et al. 2005). HA is a naturally occurring compound present in cervical mucus, cumulus cells, and follicular fluid (Jakab et al. 2005). The PICSI dish uses a conventional polystyrene culture dish enhanced with three microdots of hyaluronan where the sperm suspension is added. Sperm binding to the hyaluronan substrate may then be selected for injection whereby bound (mature) sperm have more desirable traits such as improved viability and motility and lower frequency of chromosomal aneuploidies (Jakab et al. 2005; Nasr-Esfahani et al. 2008). In addition, immature sperm do not bind to HA as they lack the maturation-related remodelling, cytoplasmic extrusion, and nuclear replacement of histamines with protamines required for optimal zona binding (Huszar et al. 2003; Jakab et al. 2005). In the study by Huszar et al. (2003), HA increased sperm velocity and facilitated the retention of motility and viability in freshly ejaculated and cryopreserved–thawed sperm.
Studies using PICSI in a clinical setting are scarce and some results are contradictory (Jakab et al. 2005; Hasanen et al. 2020; West et al. 2022). PICSI has delivered significant improvements in pregnancy rate for patients with male factor infertility, particularly teratozoospermia (Erberelli et al. 2017). Likewise for patients with low sperm binding scores, PICSI selection positively impacted clinical success rate (Mokánszki et al. 2014). However, the Cochrane Review on the efficacy of PICSI assessed eight randomised control trials including a total of 4147 women and found no conclusive evidence that live birth rates were improved, although the miscarriage rate may be decreased (Lepine et al. 2019). This matched the findings of a previous systematic review of seven studies that also found no consistent benefit of PICSI (Beck-Fruchter et al. 2016). In contrast, a randomised controlled trial by Miller et al. (2019), with further analysis of the data in a follow-up study by West et al. (2022), showed that the PICSI cohort had a lower fertilisation rate when compared to the ICSI cohort with no significant difference between biochemical or clinical pregnancy and premature birth for PICSI and ICSI. However, and of clinical significance, there was a reduced miscarriage rate in the PCSI group (Miller et al. 2019). This is plausibly due to PICSI reducing chromosomal abnormalities in ICSI offspring, as the frequency of aneuploidies and diploidies excluding Y disomy declined in HA-bound sperm, putting HA-selected sperm within the range for normospermic men (Jakab et al. 2005). It seems apparent though that the use of PICSI is beneficial for patients in older age groups, as only after the age of approximately 35 years did the use of PICSI begin to show higher predictive live birth rate, which may reflect elevated DNA damage in the gametes of older patients (West et al. 2022). Novoselsky Persky et al. (2021) also suggests that PICSI is more suited for subsets of the infertile population, namely those with male factor and unexplained infertility (Mokánszki et al. 2014; Erberelli et al. 2017). Future studies should investigate how PICSI would benefit these subsets of infertility patients in a larger study with more cycles.
Another selection technique specific to ICSI is zeta sperm selection. Chan et al. (2006) pioneered this technique to separate sperm by their electrokinetic potential, first referred to as the zeta method, which attracts morphologically normal and highly motile sperm to the charged surface of the tube. With samples from eight men, zeta-processed sperm had double the amount of normal motility and hyperactive motility, with a 1.5-fold increase in progressive motility. However, the sperm concentration of recovered sperm was 8.8% of the original concentration (Chan et al. 2006). However, this is less of an issue for ICSI, which only requires a single sperm to achieve successful fertilisation. When zeta selection was used in combination with ICSI on sibling oocytes, fertilisation rate improved while cleavage rate, embryo score, and pregnancy rates were equivalent and the technique was suggested to have greater efficiency on mature sperm with minimal DNA damage (Kheirollahi-Kouhestani et al. 2009). A clinical trial documents embryo quality and pregnancy rate improvements, with lower miscarriage rate but no difference in implantation rate. However, in the same study, significantly more girls were born from the zeta-sorted cohort (Nasr Esfahani et al. 2016).
Comparisons of PICSI and zeta sperm selection for couples with male factor infertility are contradictory. Both selection techniques were shown to improve the proportion of normal sperm, and reduce the percentage of sperm with protamine deficiency, but only zeta selection was found to reduce DNA fragmentation index (DFI; Razavi et al. 2010). However, in a recent study by Vahidi et al. (2022), both methods were shown to reduce sperm DFI and PICSI was superior to zeta selection. While many techniques to sort sperm exist, each with their own advantages and disadvantages, a systematic review has shown that no single technique has emerged as the best at delivering higher pregnancy rates (Baldini et al. 2021).
An emerging area is fabricated microfluidic devices specifically designed for separating motile sperm with low DNA fragmentation (reviewed by Nosrati et al. 2017 and Goss et al. 2023). Examples of such devices utilised for ICSI sperm selection to successfully improve embryo ploidy and pregnancy rates include the use of porous membranes to select for progressive motility and higher chromatin integrity (Parrella et al. 2019), as well as commercially available devices such as The ZyMōt Multi (850 μL) Sperm Separation Device (Kocur et al. 2023).
In cases where no ejaculated sperm is available in patients with non-obstructive azoospermia, the option of obtaining epididymal and testicular sperm cells provides a further option as a source of cells for ICSI (Verheyen et al. 2017). Much work has been conducted in optimising the preparation and selection for ICSI of these sources of cells, with results providing similar results to mature sperm for fertilisation rate and embryo production (for reviews see Verheyen et al. 2017; Aydos and Aydos 2021). The injection of round spermatids (ROSI) has also been explored (reviewed in Hanson et al. 2021), but results using these cells show limited clinical success.
Conclusions
From basic research beginnings undertaken in invertebrate and animal models to understand the mechanisms of fertilisation, ICSI has revolutionised the treatment of malefactor infertility, accounting for an estimated 6 million births since its application to humans in 1992. Over the last three decades, technological advances to ICSI have largely focused on meeting challenges of balancing the need to minimise oocyte degeneration with the need to ensure fertilisation signalling occurs. This involves balancing factors that alleviate the stress applied to the oolemma and zona pellucida during injection while ensuring sperm are deposited within the cytoplasm of the oocyte and capable of initiating oocyte activation. Recent advances include mechanical assistance modifications like Piezo-ICSI (and LA-ISCI) that especially appear useful for treating patient oocytes with hard zonas or ‘abnormal oolemmal breakage’, also referred to as ‘fragile oocytes’. However, despite mounting evidence of a beneficial effect on fertilisation and embryo development, slow adoption of Piezo-ICSI likely stems from past concerns over toxicity within the device, along with technical skill requirements and increased time per oocyte required for injection, which likely is a concern for all current advances discussed in this review. Likewise, while studies exploring the use of polarised light to reduce spindle damage indicate no apparent negative effects from the light exposure itself, adoption has been limited as the oocyte is rarely readjusted upon identifying spindle location due to a lack of consensus for which spindle position is optimal. Despite limited adoption of many of these technological advances, fertilisation rates and developmental outcomes following ICSI have nonetheless progressively improved through the incorporation of methodological modifications, including cytoplasmic aspiration and sperm immobilisation. Advances have also been made in methods to select the ‘best’ sperm for ICSI, though no single technique has emerged as having consistently improved ICSI outcomes. Such modifications have contributed to ICSI’s success worldwide, even extending its use to non-male factor infertility cases in an attempt to minimise fertilisation failure. However, most data support restricted use of ICSI for male factor infertility or prior fertilisation failure cases only (Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology 2020). Given broad concerns around the long-term effects of ICSI, future studies must focus on long-term outcomes for both standard ICSI and the advances discussed in this review. Such studies will help inform on not only the safety of the procedure, but also the utility across infertile cohorts, particularly for non-male factor cases and with patient age. Similarly, improvements to the handling of eggs during ICSI will be critical to improving outcomes, particularly embryo utilisation.
Conflicts of interest
Fertilis Pty Ltd is developing a product for use during intracytoplasmic sperm injection. J.G.T. is a co-founder and shareholder of Fertilis Pty Ltd; H.J.M., S.L.H., and M.P.I. are employees of Fertilis Pty Ltd; and D.K.G. is a shareholder of Fertilis Pty Ltd. A.J.H. declares no conflict of interest.
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