Should ICSI be the treatment of choice for all cases of in-vitro conception?

No, not in light of the scientific data

Sergio Oehninger1 and Roger G. Gosden

The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA, USA


    Abstract
 Top
 Abstract
 Introduction
 Our experience and...
 Fertilization disorders: lessons...
 What gamete defects are...
 Conclusions
 References
 
There is an ongoing debate among reproductive endocrinologists and embryologists about the indications for ICSI in the management of the infertile couple. Analysis of published results indicates that there are no data to suggest that ICSI should be performed in all cases of in-vitro conception. If the results of the basic semen analysis and sperm function tests demonstrate an impairment of sperm fertilizing capacity, couples should be directed to ICSI. In cases of previous fertilization failure, ICSI usually results in an improved outcome. This suggests the presence of ‘occult’ male or female gamete defects that can be bypassed by ICSI. We stress that efforts should be geared toward the identification of the aetiology and pathophysiology of sperm and oocyte lesions/dysfunctions responsible for fertilization impairment and their potential contributions to defective embryogenesis. A better definition of the indications for ICSI is needed, together with the development of directed, simpler, less expensive and safer alternatives.

Key words: fertilization/ICSI/oocyte defects/pregnancy/sperm function tests


    Introduction
 Top
 Abstract
 Introduction
 Our experience and...
 Fertilization disorders: lessons...
 What gamete defects are...
 Conclusions
 References
 
There is ongoing debate among reproductive endocrinologists and embryologists about the indications for ICSI in the management of the infertile couple. Although ICSI is relatively new (Palermo et al., 1992Go), no other technique since IVF itself has had such a positive and almost explosive impact in helping infertile patients to achieve conception. This is demonstrated by the increased trend in the ratio of ICSI to IVF cycles, according to Human Fertilisation and Embryology Association (HFEA) statistics (Patient Guide, 1999Go), and by the report from the Centers for Disease Control and Prevention showing use of ICSI in ~40% of 53 154 assisted reproduction technology cycles performed in 1998 in the USA (Centers for Disease Control and Prevention, 2000Go).

There is general agreement that ICSI should be used when male factor infertility is diagnosed from an abnormal semen analysis. Typically, such cases are expressed clinically as varying degrees of oligoasthenoteratozoospermia, presence of anti-sperm antibodies, or azoospermia (obstructive or non-obstructive, where ICSI is combined with testicular or epididymal sperm retrieval). ICSI is also being performed successfully in cases with fertilization failure in a previous IVF cycle. In many cases, such failures may be an expression of an ‘occult male factor’. Occasionally, however, they may be due to undiagnosed oocyte defects or, alternatively, may be of truly unknown origin. On the other hand, and on a purely pragmatic basis, some assisted reproduction technology centres have extended the indications for microsurgically assisted fertilization with ICSI to (i) cases of unexplained infertility (in the presence of normal semen parameters), and (ii) even generalized to all cases of in-vitro conception. This appears to be a significant departure from principles of evidence-based medicine.

Few well-designed studies have addressed this important issue in a prospective fashion and some have investigated fertilization, but not implantation or pregnancy outcome. Staessen et al. reported that in couples with tubal infertility but with normozoospermic semen, there were no differences in fertilization rate when comparing IVF and ICSI (Staessen et al., 1999Go). Fishel et al. concluded that ICSI resulted in a superior fertilization rate and should be used in all cases with previous fertilization failure of idiopathic origin and in cases with unsuitable semen parameters (male factors) when compared with either standard IVF or IVF with a high insemination concentration (HIC) (Fishel et al., 2000Go). These authors also suggested that ICSI offers the advantage of bypassing barriers for fertilization, which may be of oocyte or sperm origin; especially if of sperm origin, such risk could be minimized by ICSI. Nonetheless, these authors did not conclude that ICSI is the answer to all cases of infertility requiring in-vitro conception.

Although some studies showed poorer embryo development in ICSI as compared with IVF (either because of the use of low quality sperm or due to the technique itself) (Ola et al., 2001Go; review), others have shown similar implantation rates in randomized controlled studies in non-male factor cases (Aboulghar et al., 1996Go). In our programme, we have observed similar embryo quality and pregnancy rates in ICSI and IVF (Hsu et al., 1999Go), and no impact of severe oligoasthenoteratozoospermia on implantation outcome when comparing ICSI with IVF in the oocyte donation model (optimal model for control of oocyte quality) (Oehninger et al., 1998Go).

Barratt’s group performed an elegant analysis of the ICSI literature with particular emphasis on fertilization (success and failure) and embryo development, cost-effectiveness and safety (Ola et al., 2001Go). Two main conclusions were drawn: (i) more studies are required on non-male factor patients to address the question of whether ICSI results in significantly higher fertilization rates (and embryo development) than IVF (standard or HIC) in men with apparently normal semen; and (ii) in light of the available scientific data, cost-effectiveness and safety considerations, the authors did not support the routine use of ICSI in all IVF treatments. Bhattacharya et al. compared ICSI and IVF in a multicentre randomized controlled trial in couples with non-male factor infertility (Bhattacharya et al., 2001Go). They reported that implantation rates were similar for both techniques and concluded that ICSI offers no advantage in the non-male factor population or in couples with unexplained infertility.

It is a well-established fact that some cases of severe male infertility are associated with chromosomal aberrations and/or anomalies at the gene level, such as microdeletions of the Y chromosome, mutations associated with congenital absence of the vas deferens, and others (Kent-First et al., 1996Go; Martin, 1998Go; St John, 1999Go; Rovio et al., 2001Go). In addition to aneuploidy and microdeletions, sperm from infertile men have been shown to contain other nuclear alterations such as an abnormal chromatic structure and DNA strand breaks. DNA damage in sperm could occur at the time or as a result of anomalies during spermiogenesis, and could be the consequence of direct oxidative damage, or be caused by apoptosis (Aitken et al., 1989Go, 1998Go; Barroso et al., 2000Go). A few assays have been validated for the assessment of chromatin/DNA integrity (Evenson et al., 2002Go; review).

Because of such findings and the fact that ICSI is usually performed in the absence of a defined aetiological or pathophysiological diagnosis, the use of this technique has raised the concern of risk of transmission of chromosomal or genetic disease (Cummins and Jequier, 1994Go, 1995Go). Evidence also exists that reproductive failure can be the result of male-mediated teratogenicity (Brinkworth, 2000Go) and, therefore, uncertainty also exists about the consequences of damaged DNA in sperm and potential negative contributions during embryogenesis (Sakkas et al., 1998Go). Furthermore, non-human primate data suggest that the technique of ICSI itself may be responsible for alterations in the process of fertilization that could potentially have an impact on later embryogenesis stages (Hewittson et al., 2000).

Notwithstanding those concerns, the health of children conceived after ICSI has been, overall, reassuring (Bonduelle et al., 1999Go). Additionally, studies of neuro-developmental capacities of children born after ICSI revealed no differences compared with controls in well designed case–control studies (Sutcliffe et al., 2001Go). Nevertheless, more studies are needed, particularly controlling for maternal age. Vigilance is required to monitor the technique and to identify any potential negative impact on the long-term health of children and trans-genertional effects (Faddy et al., 2001Go; Oehninger, 2001Go).


    Our experience and recommendations
 Top
 Abstract
 Introduction
 Our experience and...
 Fertilization disorders: lessons...
 What gamete defects are...
 Conclusions
 References
 
Based on published reports and experience in the management of infertility, our programme selects patients for ICSI according to the following indications (Table IGo): (i) poor sperm parameters, which are predictive of fertilization failure [including (a) <1.5x106 total motile sperm with adequate progressive motility after separation, (b) teratozoospermia with <4% normal forms by strict criteria when associated with <5x106 total motile sperm after separation, and/or (c) a poor sperm–zona pellucida (ZP) binding capacity in the hemizona assay (HZA) with a hemizona index (HZI) <30]; (ii) failure of intrauterine insemination (IUI) therapy for 4–6 cycles with <5x106 total motile sperm recovered for IUI after wash, with or without the presence of anti-sperm antibodies examined by direct tests (World Health Organization, 1999Go); (iii) presence of obstructive or non-obstructive azoospermia where ICSI is combined with surgical sperm extraction from the testes or the epididymis; and (iv) previous failed fertilization in the presence of normal sperm parameters and no identifiable oocyte factors (unexplained fertilization failure) (Oehninger et al., 1997aGo,bGo; Oehninger, 2000Go, 2001Go).


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Table I. Indications for ICSI
 
On the other hand, patients with teratozoospermia but with a high recovered-motile-sperm fraction are offered IVF with HIC (Oehninger et al., 1988aGo, 1996aGo). Couples with fertilization failure or very low levels of normal fertilization, morphological oocyte abnormalities (either cytoplasmic, nuclear or at the level of the ZP) and normal semen, represent a clinical dilemma. We recognize the absence of data to prove that ICSI improves fertilization and embryo implantation potential in such cases. A large prospective study randomizing sibling oocytes to either ICSI or IVF needs to be performed to answer this issue.


    Fertilization disorders: lessons from IVF-ICSI
 Top
 Abstract
 Introduction
 Our experience and...
 Fertilization disorders: lessons...
 What gamete defects are...
 Conclusions
 References
 
In the IVF-ICSI setting, failure of fertilization can be attributed to sperm anomalies (in quality and/or quantity), oocyte abnormalities (intrinsic or ovarian stimulation-derived) and/or technical factors (Oehninger et al., 1988bGo).

Sperm defects
Because fertilization entails a complex series of events, we hypothesized previously that a more exhaustive ‘functional’ testing of sperm features crucial for gamete interaction would aid in the management of male infertility. Consequently, in order to identify sperm dysfunctions, we have been supporters of a sequential, multistep strategy that includes the ‘basic’ semen analysis followed by bioassays that directly examine aspects of sperm–oocyte interaction (Oehninger et al., 1991Go, 1992Go).

The diagnostic power of a variety of sperm functional/biochemical assays has been emphasized by working groups (ESHRE Andrology Special Interest Group, 1996Go, 1998Go). Importantly, a meta-analysis demonstrated that sperm–ZP binding assays and agonist-induced acrosome reaction tests had the highest predictive power for IVF outcome (Oehninger et al., 2000Go). The validated sperm–ZP binding assays include the HZA (Burkman et al., 1988Go) and a sperm zona binding test (Liu et al., 1988Go). Calcium-ionophore use remains the favoured test for acrosome reaction assessment (Cummins et al., 1991Go; ESHRE Andrology Special Interest Group, 1996Go). Liu and Baker and Liu et al. have described a relatively high frequency of disordered ZP-induced acrosome reactions in a newly described bioassay using human oocytes (Liu and Baker, 2000Go; Liu et al., 2001Go). At the same time, Franken et al. developed a microassay using solubilized human ZP for the assessment of the acrosome reaction (Franken et al., 2000Go), and obtained an excellent predictive value for IVF (Esterhuizen et al., 2001Go).

As proven in the clinical setting, the interaction between the spermatozoon and the ZP is a critical event leading to fertilization. This step reflects multiple sperm functions (Liu and Baker, 1992Go; Oehninger et al., 1992Go, 1997aGo,bGo). The limitation to the use of such assays is that they require a constant supply of human oocytes. Recombinant technology has allowed for the cloning and expression of human ZP proteins and several laboratories have published on bioactive human recombinant ZP3 obtained from different sources (van Duin et al., 1994Go; Chapman and Barratt, 1996Go; Harris et al., 1999Go; Dong et al., 2001Go). Not withstanding some limitations in the present availability of large amounts of fully characterized recombinant products, new biotechnology approaches should allow for the optimization of zona binding and acrosome reaction tests.

Clinical evidence points to other potentially altered steps. Capacitation anomalies may render the sperm incapable of becoming hyperactivated or interacting successfully with the zona (Florman et al., 1998Go; Visconti et al., 1998; Ho and Suarez, 2001Go). Post-ZP defects include alterations of sperm–oolemma fusion, failure of oocyte activation by the fertilizing spermatozoon perhaps due to absence or dysfunctions of the putative oocyte-activating factor, failure of sperm head decondensation, and/or abnormal pronuclear formation (Fissore et al., 1999Go; Rawe et al., 2001Go).

In addition to the effects secondary to membrane fusion and the release of oocyte activating factor(s) by the spermatozoon, the oocyte uses molecules that induce sperm head decondensation (male pronucleus growth factor) and the substitution of protamines by histones (Yanagimachi, 1994Go). Advances in fluorescent imaging by laser scanning confocal microscopy and other novel techniques permit a sophisticated high-resolution examination of gametes and embryos. Recent evidence from the ICSI setting demonstrates that post-gamete fusion abnormalities are sometimes associated with failed fertilization cases. Such problems include, but are probably not limited to, the sperm centrosome (Moomjy et al., 1999Go) and the paternal mitochondria (Cummins, 2000Go; St John et al., 2000Go), the oocyte’s microtubule organizing centre and mitochondrial distribution, and the initiation of embryo cleavage (Schatten, 1994Go; Hewitson et al., 2000Go; Van Blerkom et al., 2000Go, 2001Go).

Oocyte defects
The number of recruited growing follicles depends upon an inherent ovarian reserve, related to genetic factors, the woman’s chronological age and basal serum FSH levels (principally controlled by feedback inhibition by inhibin B and estradiol). Oocyte quality, on the other hand, is a direct consequence of adequate genetic control of the mechanisms leading to growth differentiation and to inhibition of programmed cell death, chromosomal spindle apparatus intactness and an optimized metabolism. Controversial data exist related to an increased incidence of apoptosis in poor quality oocytes, such as is observed in advancing age (Perez et al., 1997; Van Blerkom and Davis, 1998Go).

Each embryo has a unique developmental potential and only a relatively small proportion of cleavage stage embryos is competent to implant following IVF and develop through gestation (Acosta et al., 1988Go). There are developmental defects that occur in the female gamete prior to IVF insemination. Mature oocytes frequently have numerical chromosomal abnormalities such as aneuploidism that may or may not be lethal. ZP and cytoplasmic structural defects (vacuolization, fragmentation, presence of refractile bodies, excessive granularity, etc.), and suboptimal accumulation of adequate reserves of mRNAs, proteins and others, may also predispose the oocyte to fail fertilization or result in early embryonic developmental arrest (Veeck, 1991Go; Van Blerkom, 1997Go; Dean, 2002Go).

These abnormalities may be due to altered intrafollicular conditions during preovulatory oocyte maturation, occurring either naturally (during the previous stages of follicular development in vivo) or during controlled ovarian stimulation. The extent to which aneuploidies detectable in mature human oocytes are a consequence of chromosomal defects that occur prior to the arrest of meiosis at the prophase I stage is unknown (Van Blerkom et al., 1997Go). Clearly, the presence of pre-existing follicular defects and/or poor culture conditions, including use of suboptimal culture media, may be additive resulting in defective fertilization or embryogenesis.

Follicular fluid biochemistry (ATP content, pO2, pH, vascular growth factors), granulosa cell/oocyte biology (i.e. presence and polarization of regulatory proteins) and perifollicular blood flow (using colour pulsed Doppler ultrasonography) have been analysed and correlated with various embryo developmental capacities in the IVF setting (Van Blerkom, 1997Go, 1998Go; Barroso et al., 1999Go; Huey et al., 1999Go). The demonstration of an association between follicular homeostasis, changes in perifollicular blood flow and embryo quality could provide a non-invasive and indirect indication of follicle health and possibly of the developmental competence of the corresponding oocyte. Unfortunately, no single follicular factor secreted into the circulation or present in the follicular fluid has been demonstrated to provide a definitive prediction of the developmental competence of the oocyte or early embryo.

The structure of the human ZP has been examined using antisera generated against synthetic ZP3 peptides (Hinsch, et al., 1994aGo). Human metaphase II oocytes studied under a variety of conditions normally reveal a strong immunoreactivity to the specific anti-ZP3 antisera (Hinsch, et al., 1994bGo). Interestingly, in clinical cases of failed fertilization with either a poor ovarian response or morphologically defective oocytes (and in the presence of normal sperm parameters), we observed a marked alteration in ZP immunoreactivity to the antisera (Oehninger, et al., 1996bGo). This finding demonstrates the presence of structural defects of the protein backbone of the ZP in the oocytes of women with a poor ovarian response and suggests that this defect may be a possible cause of fertilization failure.


    What gamete defects are we then treating with ICSI?
 Top
 Abstract
 Introduction
 Our experience and...
 Fertilization disorders: lessons...
 What gamete defects are...
 Conclusions
 References
 
ICSI bypasses sperm abnormalities associated with an inadequate sperm concentration (oligozoospermia) and/or motility defects (asthenozoospermia due to structural or functional deficiencies) as it bypasses dysfunctions related to the male gamete, including migration and zona penetration. ICSI results in successful fertilization in men with poor morphology (teratozoospermia); those cases are typically associated with dysfunctions of the sperm membrane and/or acrosomal compartments, resulting in an inadequate interaction with the ZP and/or the oolemma. ICSI is helpful when anti-sperm antibodies are present, perhaps by impeding their negative effects on motility and membrane–oocyte interactions. We do not know if ICSI bypasses capacitation defects when using ejaculated sperm. Although epididymal and even testicular sperm (morphologically mature) fertilize successfully with microsurgically assisted fertilization, are such sperm adequately capacitated? Is ICSI successful when certain degrees of ‘immaturity’ of the membrane–cytoplasmic–nuclear compartments are present?

It is tempting to speculate that ICSI bypasses most of the sperm lesions/dysfunctions (and their respective cellular/molecular causes) that result in fertilization failure due to an inappropriate interaction with the ZP, binding and fusion with the oolemma, and even an inadequate release of the putative oocyte activating factor. Conversely, it is unlikely that ICSI helps in cases where fertilization failure is associated with sperm nuclear (chromatin or DNA) alterations, when the spermatozoon fails to undergo head decondensation or is unable to form the male pronucleus, or when centrosome anomalies are present.

At the oocyte level, we can speculate that ICSI might allow fertilization to ensue when isolated ZP deficiencies are present. These may include structural (disarrangement or poor assembly of the zona microfilaments or an abnormal protein backbone) or functional (deficient ligand function) abnormalities of the various ZP glycoproteins. In addition, ICSI might bypass isolated oolemma deficiencies. However, it is known that abnormalities of the ZP, particularly of genetic origin, are associated with more severe follicular/oocyte developmental abnormalities (Greenhouse et al., 1998Go) and in such cases ICSI would probably be unsuccessful. It is doubtful that ICSI could be of use in cases of oocyte aneuploidy or DNA damage, or when severe cytoplasmic aberrations are present (either structural or functional) resulting in failure of the processes of oocyte activation, formation of the female pronucleus, and initial cleavage divisions. Because the expression of the male genome occurs at the 4- to 8-cell stage (Braude et al., 1988Go) and because oocyte dysfunctions may also be expressed after fertilization, the most adequate outcome measure to determine ICSI success should be embryo development including implantation competence.


    Conclusions
 Top
 Abstract
 Introduction
 Our experience and...
 Fertilization disorders: lessons...
 What gamete defects are...
 Conclusions
 References
 
There are no data to suggest that ICSI should be performed in all cases of in-vitro conception. If the results of the semen analysis and sperm function tests indicate an impairment of sperm fertilizing capacity, couples should be directed to ICSI. Cases with previous fertilization failure in IVF therapy usually benefit from ICSI, suggesting the presence of ‘occult’ male and/or female factors that can be bypassed by the technique. Therefore, and because of concerns about potential negative effects of the technique itself and the use of gametes of poor quality, efforts should be geared toward identification of the aetiology and pathophysiology of sperm and oocyte lesions/dysfunctions responsible for fertilization impairment and their potential contributions to defective embryogenesis. We remain enthusiastic that the results of ongoing studies and expected biotechnology advances will unravel the cellular/molecular mechanisms involved in gamete dysfunction, and that such achievements will have a significant impact on the elucidation of the causes of human reproductive failure.


    Notes
 
1 To whom correspondence should be addressed. E-mail: oehninsc{at}evms.edu Back


    References
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 Abstract
 Introduction
 Our experience and...
 Fertilization disorders: lessons...
 What gamete defects are...
 Conclusions
 References
 
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