Human Reproduction Update Advance Access originally published online on July 14, 2007
Human Reproduction Update 2007 13(6):515-526; doi:10.1093/humupd/dmm024
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Intracytoplasmic sperm injection (ICSI) in 2006: Evidence and Evolution
Correspondence address. P.G. Crosignani, II Department of Obstetrics and Gynecology, University of Milano, Via Commenda 12, 20122 Milano, Italy. Tel: +39-025-032-0256; Fax: +39-025-032-0255; E-mail: piergiorgio.crosignani{at}unimi.it
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Abstract |
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TOP
Abstract
IntroductionThe introduction of intracytoplasmic sperm injection (ICSI) in 1992 has dramatically changed the management of severe male infertility. In severe male infertility, live birth rates with ICSI are superior to those with other non-donor treatments. In non-male infertility, however, pregnancy rates are not better with ICSI than with in vitro fertilization (IVF). With obstructive or non-obstructive azoospermia, reasonable pregnancy rates are now possible with ICSI after recovery of sperm from the testes followed by ICSI. Genetic counselling is indicated for severe male infertility, whether or not ICSI is considered. ICSI is indicated in preimplantation genetic diagnosis (PGD) to avoid contamination by extraneous DNA in the case of PCR-based testing and to increase the number of embryos available for testing. In turn, PGD may be indicated in pregnancies that are at high risk of aneuploidy because of genetic factors associated with azoospermia. As with IVF, not all couples succeed, but 2% of couples with failed ICSI cycles will conceive without treatment. ICSI outcome studies indicate that there is a significant increase in prematurity, low birthweight, and perinatal mortality associated with single and multiple births, similar to the outcomes of conventional IVF. However, as evidenced in long-term follow-up studies, the higher rates of urogenital abnormalities and increased use of healthcare may be associated with paternal characteristics.
Key words: intracytoplasmic sperm injection / male infertility / preimplantation genetic diagnosis
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Introduction |
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The main indication for intracytoplasmic sperm injection (ICSI) is severe male infertility due to a limited number of spermatozoa or to a higher proportion of dysfunctional sperm cells (Devroey and van Steirteghem, 2004
). ICSI is used in couples with many other reproductive indications, even if clinical trials indicate that ICSI is no more effective in terms of clinical pregnancy rates than in vitro fertilization (IVF) (Bhattacharya et al., 2001). In patients with azoospermia, spermatozoa for ICSI can be retrieved from the epididymis or the testes. Even in men with non-mosaic Klinefelter syndrome, pregnancies have occurred (Palermo et al., 1998); notably, all but one conceptus to date have had a normal karyotype (Friedler et al., 2001; Staessen et al., 2003; Schiff et al., 2005). Nevertheless, in couples considering ICSI, genetic counselling is indicated because of the frequency of genetic disorders in men with severe male infertility and other semen abnormalities. Preimplantation genetic diagnosis (PGD) can be useful in these ICSI cycles because of the potential increase in aneuploid embryos (Bonduelle et al., 2002a). In turn, ICSI may be preferred in PGD cycles because the higher fertilization rate with ICSI increases the number of embryos available for testing and the number of normal embryos available for transfer (Jun et al., 2006) and especially because ICSI can avoid contamination by extraneous DNA. The outcomes of ICSI for newborns and families have been studied in a systematic manner since the procedure was introduced. Although fertilization rates with ICSI may be higher than with IVF, current embryo transfer practices aim at limiting the occurrence of any higher pregnancy rates. The higher fertilization rates and the fact that ICSI female partners usually have normal ovarian function are an opportunity, however, to consider and evaluate less aggressive ovarian stimulation and embryo transfer protocols. Mild ovarian stimulation and single embryo transfer (SET) protocols are consistent with the ability to select the best oocytes, which is made possible by the ICSI technique.
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Indications and Effectiveness |
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ICSI has been proposed as a treatment for severe male infertility (Table 1), borderline male infertility, non-male factor infertility and unexplained infertility. In registry reports, ICSI is used in more than half of assisted reproduction procedures with fresh non-donor oocytes or embryos (Table 2) (Centers for Disease Control, Prevention, 2005
; Andersen et al., 2006). Only half of the ICSI cycles involve a male factor and success rates are lower in the cycles with non-male factor infertility (Centers for Disease Control, Prevention, 2005). Despite the lower live birth rates reported in registries and despite the evidence from randomized studies, many continue to advocate the use of ICSI for all cases of assisted reproduction.
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Severe male factor infertility
ICSI is the first intervention for severe male factor infertility to achieve pregnancy rates that are equivalent to IVF pregnancy rates for non-male factor infertility (van Rumste et al., 2000).
Since the 1992 publication on four pregnancies after injection of single spermatozoa into an oocyte (Palermo et al., 1992), no randomized controlled trials with pregnancy or birth as outcomes have been done to evaluate ICSI for severe male infertility. In this clinical setting, however, trials are not necessary because live birth rates with ICSI are far better than those with intrauterine insemination (IUI), IUI with ovarian stimulation, conventional IVF, partial zona dissection or subzonal sperm insertion (Devroey and van Steirteghem, 2004).
Furthermore, it has become apparent that the technique can be applied after surgical retrieval of sperm from the male reproductive tract, even when there are only a few individual sperm. In cases of non-obstructive azoospermia, however, fertilization rates are less than that with ejaculated sperm (Devroey and van Steirteghem, 2004). In general, if spermatozoa have matured to the point of being identified as such (e.g. elongated spermatids), then fertilization is likely to be successful. Fertilization and normal embryogenesis with round sperm cells has not yet been convincingly demonstrated.
Non-male infertility and non-severe male infertility
ICSI has also been used in the case of borderline or moderate male infertility. Most of the trials comparing IVF and ICSI have sibling oocytes as the unit of randomization and fertilization rate as a surrogate outcome. Thus, these trials cannot offer information on pregnancy rates with either intervention. In two systematic reviews, fertilization rates were better with ICSI than with IVF (van Rumste et al., 2000; Tournaye et al., 2002). In one sibling oocyte trial, fertilization rates with a higher concentration of spermatozoa for IVF were similar to those with ICSI: 60% with 20 000 sperm per oocyte for IVF versus 68% with ICSI (not significant), compared with 37% using 5000 sperm per oocyte for IVF versus 64% with ICSI (P < 0.007) (Tournaye et al., 2002). To date, the potential of high insemination concentration has not been evaluated in a trial with live birth as the primary outcome.
Although fertilization rates were better after ICSI than IVF in sibling oocyte trials, pregnancy rates are not better with ICSI. A systematic review excluded 14 of 15 controlled trials, either because they randomized oocytes rather than women or because of problems with the randomization itself (van Rumste et al., 2000). The single included study was a large trial with implantation rate as the primary outcome (Bhattacharya et al., 2001). There were 72 (33%) clinical pregnancies in 219 IVF cycles and 53 (26%) in 204 ICSI cycles (RR 1.27; 95% CI 0.95–1.72). The implantation rate was 1.35-fold higher in IVF cycles (95% CI 1.04–1.76). The authors concluded that ICSI should be reserved for severe male factor infertility. An accompanying leading article indicated that although ICSI should be reserved for severe male factor infertility, studies were needed in couples with poor ovarian responses or oocyte quality (Oehninger, 2001). Unfortunately, although ICSI usage for such indications continues, no such studies have been reported.
Failure of fertilization in all oocytes is unusual and probably associated with suboptimal culture conditions. Total fertilization failure occurs in no more than 1–2% of IVF cycles (Ola et al., 2001).
ICSI has been considered for primary and secondary prevention of fertilization failure. Primary prevention can be evaluated in the British trial because couples with <20% fertilization in a previous IVF cycle were excluded. Failed fertilization occurred in 11 (5%) of 206 IVF patients and in 4 (2%) of 209 ICSI patients (Bhattacharya et al., 2001). This 3% difference implies that 33 ICSI cycles would be necessary to prevent a single fertilization failure in cycles not at high risk for fertilization failure.
No recent randomized studies have considered ICSI for secondary prevention of failed fertilization in couples with a history of failed or poor fertilization. A prospective cohort study involved 24 retrievals in patients with previous failed fertilization and 14 retrievals in patients with previous fertilization rates <25%. There were 16 (67%) and 7 (50%) failed fertilizations with IVF in the 24 and 14 retrievals, respectively, and the fertilization rate was uniformly better with ICSI, even when the comparison was limited to the successful IVF cycles (van der Westerlaken et al., 2005). The differences suggest that a trial should be done with patients as the unit of randomization and live birth as the outcome.
In PGD, where contamination by sperm could affect the PCR-based diagnosis, the likelihood of contamination is minimized by the use of ICSI (Sermon et al., 2004; Thornhill et al., 2005).
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Surgical Retrieval of Sperm |
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A prerequisite to appropriate management of azoospermia is to distinguish between the obstructive form due to post-testicular lesions and the non-obstructive one, usually involving disorders of spermatogenesis. The patient's history, physical and endocrine evaluation can help to make this distinction, but only testicular histology can provide the definitive diagnosis.
Epididymal sperm were recovered surgically and used in association with IVF, even before the introduction of ICSI (Temple-Smith et al., 1985; Patrizio et al., 1988). Pregnancies were reported, since epididymal sperm can be sufficiently motile to generate normal fertilization if they are placed in proximity to the oocyte. The discovery of ICSI facilitated this success because sperm motility was no longer a limiting factor. Within 2 years of the first ICSI paper, it was reported that up to 50% of sperm recovery procedures followed by ICSI with fresh epididymal sperm were associated with pregnancies (Tournaye et al., 1994). Epididymal sperm can be retrieved either by microsurgery or by blind aspiration (Shrivastav et al., 1994; Jansen et al., 2000). The acquisition and use of testicular sperm was a logical development, since progressive motility is not a requirement for success in ICSI cycles. Testicular sperm were initially acquired by testicular open biopsy and sperm extraction (TESE) (Devroey et al., 1994; Schlegel et al., 1997). The results with fresh and frozen sperm samples are similar (Devroey et al., 1995a; Tournaye et al., 1999).
In practice, for men with obstructive azoospermia, micro-surgical epididymal sperm aspiration can retrieve sufficient sperm both for immediate insemination and for cryopreservation. Percutaneous epididymal sperm aspiration may yield only enough sperm for one ICSI procedure, but it is less invasive and, therefore, is often the first choice. Men with non-obstructive azoospermia usually do not have sperm in the epididymis and require testicular retrieval, either by fine needle aspiration (FNA) (TESA) or open TESE. The open procedure may involve either random biopsies or microdissection (Ramasamy and Schlegel, 2007). Although both open testicular procedures involve a temporary decline in testosterone and rise in FSH, there were fewer acute and chronic ultrasound changes in the microdissection group and the sperm retrieval rate was 57% using microdissection compared with 32% using random biopsy (Ramasamy et al., 2005).
When FNA was introduced, there was no need for scrotal exploration, epididymal sperm removal or TESE in most cases (Bourne et al., 1995). It is essential, however, to ensure that the obstruction is confirmed: an efficient protocol involves testicular biopsy with cryopreservation and, once the diagnosis is confirmed, FNA can be done or sperm can be extracted from the frozen tissue. If the obstructive azoospermia is due to post-infective epididymal obstruction or vasectomy, no specific additional testing is necessary. If, however, there is congenital absence of the epididymis, the couple must be tested for cystic fibrosis 
F508 mutations (Foresta et al., 2002; Huynh et al., 2002; Lissens et al., 2007).
Non-obstructive azoospermia is usually congenital but may follow chemotherapy or radiotherapy. By 1995, pregnancy after testicular biopsy and sperm extraction for ICSI in non-obstructive azoospermia had been reported (Devroey et al., 1995b; Palermo et al., 1999). Testicular biopsy is essential to define whether the diagnosis is germ cell aplasia (Sertoli-cell-only syndrome) or maturation arrest. In germ cell aplasia, FSH is elevated and in maturation arrest, FSH levels are in the normal range. Although testicular biopsy will provide the diagnosis, it cannot predict whether sperm cells will be found in the testicle because of heterogeneity in testicular tissue (Su et al., 1999). If spermatozoa are found, they can be used immediately or stored frozen for future use (Tournaye et al., 1999). On-going pregnancy rates are 
15% per transfer with either germ cell aplasia or maturation arrest. No pregnancy has been reported after insemination with cells described as round spermatids. Among men with non-obstructive azoospermia, karyotyping and testing for Y-deletions are indicated. The frequency of Klinefelter syndrome is 0.2% of male newborns and 11% of azoospermic men (Schiff et al., 2005). Klinefelter syndrome is a specific chromosomal abnormality (47XXY) and when it is non-mosaic, there are elevated FSH values and only rare focal areas of spermatogenesis. Nevertheless, sperm are found in 50% of cases on testicular exploration and in the couples who have ICSI, pregnancy rates range from 30% to 50% (Palermo et al., 1998). As in other men with non-obstructive azoospermia, none of the clinical assessments can predict whether sperm retrieval will be successful. The births of more than 60 children have been reported, of which 50 had karyotype analysis, all of which were normal; one fetus was diagnosed 47XXY after prenatal diagnosis (Friedler et al., 2001; Staessen et al., 2003; Schiff et al., 2005). The upper 95% confidence interval would be 6% for a zero incidence of aneuploidy in 50 cases. Yq deletions, mainly in the AZFc region, are found in 10% of azoospermic men and 6% of men with severe oligozoospermia (Stouffs et al., 2005). Sperm have not been recovered in men with lesions in the AZFa or AZFb region, and with the AZFc deletion, sperm may be recovered in just over half of those men (Choi et al., 2004; Katagiri et al., 2004). In 27 cycles of ICSI, there were four on-going pregnancies (Stouffs et al., 2005).
In pregnancies after surgical retrieval of sperm and ICSI in men with non-obstructive and obstructive azoospermia, the incidence of chromosomal abnormality is so far unknown because of limited testing (Vernaeve et al., 2003). If the couple would undergo prenatal testing for this level of risk, PGD also could be considered. PGD is necessary if the couple wish to prevent the birth of sons who will have a Yq deletion. Table 3 summarizes the results obtained with the different sperm used.
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Investigations and Genetic Counselling for ICSI Couples |
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The causes of azoospermia, obstructive or non-obstructive, and oligozooasthenoteratospermia (OAT) can be unknown, acquired or genetic (The ESHRE Capri Workshop Group, 2000
).
Establishing a correct diagnosis is mandatory for adequate counselling. Specifically, before starting an ICSI procedure, the male investigations should include: a personal and familial history, a physical examination, a sperm analysis, hormone measurements and a karyotype. The results of these investigations may suggest further tests for cystic fibrosis transmembrane conductance regulator (CFTR) mutations or a Yq deletion (Table 4). Genetic testing may be indicated for such rare conditions as myotonic dystrophy or the polyglutamine repeat expansion responsible for Kennedy disease (Foresta et al., 2002). When necessary, the female partner should provide a personal and familial history, undergo a physical examination and have CFTR testing, if congenital bilateral absence of the vas deferens (CBAVD) was diagnosed in the male partner. The female also may require a karyotype analysis depending on the pedigree information. However, routine karyotype analysis in the infertile woman is not very productive, as only 0.58% (95% CI 0.28–1.19) have an abnormality (Papanikolaou et al., 2005). At the end of the diagnostic procedure, specific genetic counselling is mandatory in order to allow the patients or the couples to make their own decision concerning their future treatment.
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Cause of male infertility unknown
If the diagnosis of the cause of infertility is idiopathic, the couple should be counselled that the cause can still be genetic and therefore transmitted especially to the male offspring. In a few cases, there may be a need to search for de-novo chromosomal aberrations through prenatal diagnosis. Aneuploidy screening of spermatozoa prior to ICSI treatment can identify chromosome abnormality in up to 14% of spermatozoa, but whether this is a useful prediction remains uncertain (Palermo et al., 2002; Gianaroli et al., 2005a; Petit et al., 2005).
If the diagnosis involves a chromosomal aberration, usually a Robertsonian or reciprocal translocation in the case of OAT, PGD can allow for the selection of balanced embryos in order to increase the chance of a healthy child. The counsellor should mention that the success rate of PGD per started cycle is low because often no embryos are available for transfer. Segregation studies show that reciprocal translocation carriers may produce more unbalanced sperm than normal or balanced sperm, and such studies in these men may help in counselling. If the male partner is a non-mosaic Klinefelter patient, ICSI can be successful without PGD (Palermo et al., 1998; Denschlag et al., 2004; Schiff et al., 2005), but when PGD is done, only 54% of embryos are normal (Staessen et al., 2003).
If the diagnosis involves a Yq deletion, the condition will be transmitted to the male offspring. Usually, testing is recommended if the count is <1 million sperm per millilitre and offered if the count is below 5 million, when the chance of Yq is 14%. Couples may opt for PGD with the transfer of XX-embryos only (Stouffs et al., 2005). With respect to cystic fibrosis genes, if the man has a low sperm count, it is useful to test whether the woman is a carrier and then test the man (Schlegel et al., 1995). With unilateral absence of the vas deferens, some men have cystic fibrosis gene abnormalities. If the woman is normal, the likelihood of an anomaly is minimal (Lissens et al., 2007). For couples where both partners carry at least one CFTR mutation, PGD is indicated to avoid the conception of a child with cystic fibrosis (Goossens et al., 2003). Table 5 summarizes the options for the management of the different clinical conditions of male infertility.
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ICSI for Preimplantation Genetic Diagnosis |
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PGD is used as an alternative to prenatal diagnosis to avoid transmission of serious recurrent genetic disorders. Specific gene mutations can be identified from single blastomeres after PCR or whole genome amplification (Renwick et al., 2006
; Spits et al., 2006). The sex of the embryo in X-linked disorders and abnormalities of sperm number can be identified using fluorescence in situ hybridization (FISH). Couples should be informed prior to PGD about the risk of misdiagnosis and failure of diagnosis, the need for confirmatory prenatal diagnosis and the risks to the health of resulting children as far as known today.
PGD requires the removal and testing of a representative sample of the embryo or oocyte in order to identify those at high risk of transmitting the disorder. Although this has been accomplished by testing of one or more polar bodies, polar body testing is diagnosis by inference; if an abnormal allele is not present in the polar body, then it is assumed to be present in the oocyte. This reasoning applies only in recessive or sex chromosome disorders, or in dominant disorders carried by the female. Biopsy of the embryo itself at cleavage stages allows an examination of the zygotic genome, and takes into account errors that might develop during cleavage (Braude et al., 2002; Sermon, 2002; Sermon et al., 2004).
The necessary molecular tests are performed on single cells that contain, effectively, one or two double strands of DNA, and these must be amplified by PCR. Even where two cells are removed in an attempt to improve diagnostic accuracy and decrease misdiagnosis, each cell is still tested independently. Any extraneous DNA, most often from cumulus cells or sperm DNA, also would be amplified and thus contamination presents a significant risk since free DNA and DNA present in aerosols in the laboratory and elsewhere may contaminate the DNA product (Pickering and Muggleton-Harris, 1995). The strategies developed to minimize this risk include: analysing wash-drops to detect early contamination of media or reagents, analysing DNA polymorphic markers as a ‘fingerprint’ of the source of DNA and analysing two cells and requiring concordance for a secure diagnosis. The development of preimplantation haplotyping substantially improves accuracy, making wash-drops unnecessary, and reducing the fear of misdiagnosis, provided the markers used are informative (Renwick et al., 2006; Spits et al., 2006). ICSI is still required, however, to ensure monospermy, enhance fertilization and minimize contamination during insemination.
Where FISH is used for diagnosis, contamination by sperm is unlikely because the sperm nucleus is easily identified, especially with modern fluorescence microscopes when the zonae pellucidae have been appropriately stripped of cumulus cells. Although ICSI is not essential to prevent contamination, there is the omnipresent concern that despite adequate sperm parameters, unexpected failure of fertilization may occur, or insufficient oocytes might be fertilized. Since the likelihood of achieving a pregnancy is proportional to the number of embryos available for biopsy (Vandervorst et al., 1998; Grace et al., 2006), there may be a preference for the use of ICSI.
Benefits of PGS in ICSI cycles
The increasing use of preimplantation genetic aneuploidy screening (PGS) in order to avoid the transfer of chromosomally abnormal embryos has led to its use in severe oligozoospermia and azoospermia, where aneuploidy is more frequent, (Palermo et al., 2002; Platteau et al. 2004; Gianaroli et al., 2005b) and in the case of specific sperm abnormality, such as Klinefelter syndrome (Kahraman et al., 2003). The value of PGS in ICSI is uncertain for several reasons: aneuploidy rates depend mainly on oocyte quality, mosaicism is common among blastomeres and only a limited number of chromosomes can be examined (Baart et al., 2004; Staessen et al., 2004; Twisk et al., 2006). Nevertheless, the frequency of new chromosomal abnormalities in prenatal testing after ICSI for poor sperm quality is 1.6%. This questions whether the ICSI itself increases the incidence of aneuploid embryos, and could in part explain the higher rate of abnormality found in follow-up studies (Bonduelle et al., 2002a). Studies of the benefit of PGS in ICSI cycles are needed, especially after sperm recovery for azoospermia, which involves the highest risks of aneuploidy (Donoso et al., 2006). So far, the usefulness of PGS has not been proven for advanced maternal age or any other indication. Also, to date, there is no clear evidence that the incidence of aneuploid offspring is higher following TESE. Therefore, aneuploidy screening to prevent the transfer of aneuploid embryos after ICSI with spermatozoa isolated from azoospermic men also should be done only in clinical trials until it has been proven.
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What to do When ICSI Fails? |
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ICSI failure can occur at three levels: failure to obtain sperm for injection, failed fertilization after successful ICSI and lack of pregnancy after successful fertilization and embryo transfer.
In men with azoospermia, there could be a failure to obtain sperm from the testis that could be used in the ICSI procedure. Sperm are recovered in virtually all attempts in men with obstructive azoospermia, but in only 50–70% of men with non-obstructive azoospermia (Palermo et al., 1999; Tournaye et al., 2002). If sperm cannot be recovered, donor sperm can be considered.
Failure of fertilization after ICSI
Fertilization failure occurs in 2% of ICSI cycles, whether there is male infertility or non-male infertility (Liu et al., 1995; Bhattacharya et al., 2001). The reasons for a failure of all oocytes to fertilize following micromanipulation techniques remain unclear, but may involve poor viability of the spermatozoa at the time of ICSI.
Attempts can be made to enhance chances of fertilization in selected cases by utilizing sperm activating factors, or chemical factors (Palermo et al., 1997; Morozumi et al., 2006). Proposed approaches to failure of fertilization and failure to conceive after ICSI include assisted oocyte activation, which involves injecting calcium chloride with the spermatozoon and repeated exposure to calcium ionophore after injection (Heindryckx et al., 2005). These approaches have to be considered as clinical research and not as routine practice.
The implications of fertilization failure for later ICSI attempts are not necessarily bleak. When couples with failed ICSI fertilization attempted further ICSI cycles, their success rates were similar to other couples undergoing repeat ICSI cycles (Liu et al., 1995; Moomjy et al., 1998).
Failure to conceive after ICSI fertilization
As with IVF, some couples fail to conceive, even after repeated ICSI cycles where the causes range from centrosomal dysfunction to DNA abnormalities (Palermo et al., 1994, 1997; Evenson and Wixon, 2006). Two hundred couples who had 433 cycles of ICSI treatment without success subsequently had 23 (12%) live births with no further treatment within 4 years of the last ICSI cycle (Osmanagaoglu et al., 2002). The only predictor of live birth after ICSI failure was shorter duration of infertility, suggesting that unknown factors contributing to longer duration of infertility were barriers to success not only in the ICSI cycles but also afterward.
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Outcome of ICSI |
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Even though great concern was voiced at the introduction of IVF, no formal and systematic evaluation of the outcome of this high-tech procedure was carried out. IVF was accepted as a safe technique, mostly on the basis of the information in registries with retrospectively collected data. Before long, it became clear that IVF children are more often born prematurely and with a low birthweight. Although these problems stemmed primarily from the higher incidence of multiple pregnancies, they were also more frequent in singleton IVF pregnancies (Jackson et al., 2004
). When ICSI was introduced, new concerns were raised related to the invasiveness of the procedure and to the type of sperm used.
Twins as well as singletons born after IVF and ICSI have a 2-fold increased risk for perinatal mortality, preterm delivery and low birth weight and a 3-fold risk for very low birthweight (Helmerhorst et al., 2004; Jackson et al., 2004). Vanishing twins after multiple embryo replacement could be one of the causes of adverse neonatal outcome (Pinborg, 2005) and infertility by itself can play a causative role (Saunders et al., 1988; Doyle et al., 1992; Pandian et al., 2001; Schieve et al., 2002). IVF and ICSI outcomes are similar (Bonduelle et al., 2002b; Neri et al., 2006).
A higher rate of inherited chromosomal anomalies was found, mainly due to paternal structural chromosomal anomalies, as well as a higher rate of de-novo chromosomal anomalies, related to paternal sperm characteristics (Table 6) (Bonduelle et al., 2002a). Malformation rates were comparable between ICSI and IVF in most studies, but were higher compared with the general population (Bonduelle et al., 2002b; Hansen et al., 2005; Lie et al., 2005). An increase in malformations of specific organ systems was reported in a few studies and not confirmed by others. Controlled studies also indicate a possible higher malformation rate in ICSI compared with the general population, mainly related to paternal variables and genetic background (Katalinic et al., 2004). By the age of 8 years, 15 major congenital malformations were diagnosed in 150 ICSI children, compared with 5 in 147 matched controls (Belva et al., 2007). Inguinal hernias accounted for four of the excess ICSI cases and a large naevus was diagnosed in two ICSI cases and no controls.
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Long-term follow-up
Recent long-term follow-up studies indicate that although singleton ICSI and IVF 5-year-olds seem largely comparable with their naturally conceived peers, they are more likely to need health service resources in the first 5 years, though the impact of over-attentive parenting cannot be ruled out. The ICSI children had more congenital anomalies, due partially to an excess of male urogenital abnormalities that were not detected in the newborn period (Bonduelle et al., 2005). Health parameters such as growth and cognitive and emotional development were reassuring, as was the psychological well being of the families involved (Barnes et al., 2004; Ponjaert-Kristoffersen et al., 2005). Questions on whether sperm quality or source have an influence on the outcome remain to be solved and particular interest should be given to the use of sperm from a testicular biopsy with non-obstructive pathology (Wennerholm et al., 2006). Prospective monitoring of the health of children born after assisted conception needs to be continued as they grow up, with special attention to puberty and future fertility. In 150 8-year-old ICSI children, pubertal staging, neurological status, need for more remedial therapy, surgery or hospitalization were comparable with 147 matched spontaneously conceived controls (Belva et al., 2007).
Reports of imprinting-related diseases, such as Angelman and Beckwith–Wiedemann syndromes, in children born after IVF as well as ICSI suggest a possible risk of in vitro culture procedures and should be further investigated (Maher et al., 2003). A systematic survey aimed at those syndromes and defined phenotypes linked to imprinted genes may clarify whether epigenetic anomalies play a role in ART (or subfertility) more often than in the general population (Ludwig et al., 2005). Moreover, basic research in this field will clarify possible effects of hormone stimulation and in vitro culture.
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Mild Ovarian Stimulation Protocols for ICSI |
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For many years, IVF has involved aggressive ovarian stimulation to maximize pregnancy rates per cycle. The process is complex, time-consuming and uncomfortable for patients and involves unacceptable risks associated with iatrogenic twinning and the frequency of ovarian stimulation syndrome is also worrying (Fauser et al., 2005
; Macklon et al., 2006). ICSI cycles, however, could be a natural venue for the use of mild ovarian stimulation protocols because the female partner usually has normal ovarian function. The rationale for mild ovarian stimulation
Mild ovarian stimulation is less complex, less time-consuming, less costly and, by reducing side effects, more acceptable to patients (Edwards et al., 1996; Fauser et al., 1999). Lower cost and reduced dropout rates could allow women to undergo more cycles and have additional chances to achieve a pregnancy. The pregnancy rate per IVF cycle may be slightly reduced, but if the patient can undergo slightly more cycles without additional cost, the overall outcome could be similar to but safer than aggressive stimulation (Heijnen et al., 2004).
The physiological rationale for mild ovarian stimulation is that FSH must remain above the threshold which is essential for single dominant follicle development during the normal menstrual cycle (Fauser and van Heusden, 1997). With the use of GnRH antagonists, which are given only during the period at risk for a premature LH rise, some of the follicles which would undergo atresia in a normal cycle will be rescued by low-dose FSH (Fauser and Devroey, 2005).
FSH in a dose of 100–150 IU/d starting on cycle day 5, with GnRH antagonist co-treatment in case any follicle exceeds 14 mm diameter, can induce growth of multiple dominant follicles, allowing for retrieval of 1–15 oocytes in most patients. Duration of stimulation is significantly reduced. Most of the pregnancies occur in women, where only 1–6 oocytes are obtained (Hohmann et al., 2003). Even with this short exposure to GnRH analogue, however, luteal phase supplementation is required (Beckers et al., 2003).
The mild stimulation concept was tested in a multi-centre RCT involving 400 couples and almost 800 IVF cycles, which compared four cycles of mild ovarian stimulation (and SET) with three cycles of conventional GnRH agonist long-protocol ovarian stimulation (and two embryo transfer). The cumulative pregnancy rates leading to term live birth were similar 45% over a 1 year period. The expected reduced outcome per cycle in the mild protocol was balanced by the greater number of IVF cycles that could be undertaken within a year. Multiple pregnancies virtually disappeared in the mild protocol and the overall treatment costs were reduced (Heijnen et al., 2006).
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Single Embryo Transfer for ICSI |
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SET is the most effective means of improving safety in IVF cycles through the minimization of multiple pregnancy. For the same reason that less aggressive ovarian stimulation protocols may be optimal in ICSI cycles, a less aggressive embryo transfer policy also may accord with the aims of ICSI cycles.
Several SET RCTs have been reported in selected good prognosis patients (Gerris et al., 1999; Martikainen et al., 2001; Gardner et al., 2004; Thurin et al., 2004; Lukassen et al., 2005). With different SET interventions but similar double embryos transfer (DET) interventions the results confirmed the expected: when similar good prognosis patients got either SET or DET, pregnancy rates were lower with SET, although the difference was not significant in all studies. (Table 7).
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In a trial of elective SET in all consenting patients irrespective of age and embryo quality, the ongoing pregnancy rate was significantly lower than after DET (21% versus 40%), but the twin rates were 0% versus 21% (Van Montfoort et al., 2006
). Thus, SET is not for all patients, but when applied in good prognosis patients any possible reduction in the pregnancy rate is balanced by a substantially lower twin pregnancy rate as well as by a higher number of good quality embryos to freeze. This obviously will offer additional pregnancy chances to those patients failing to conceive in their fresh cycle. SET effectiveness in ICSI cycles should be the basis of continuing study.
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Recommendations |
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1. Indications. The evidence shows that the main indications for ICSI anticipate a high or increased possibility of fertilization failure primarily because of severe seminal deficiency. In the case of non-male factor or unexplained infertility, however, ICSI offers no apparent advantage over routine IVF with respect to clinical outcomes. Although better fertilization rates are promising, sibling oocyte trials are an insufficient basis for clinical practice. Only one patient-based trial has been reported.
- Effectiveness trials are needed with patients as the unit of randomization and live birth as the primary outcome.
2. Genetics. The quality of genetic counselling for men with severe male infertility depends on the accuracy of the clinical diagnosis. The cause of severe male infertility is unexplained in many men and in each of these cases there may be a genetic cause. More aggressive investigation may help the individual patient and lead the way to a better understanding of gene-related severe male infertility. Segregation studies show that reciprocal translocation carriers produce excess unbalanced sperm.
- Studies are needed to determine the role of sperm aneuploidy testing to aid genetic counsellors in predicting chromosomal abnormalities in embryos.
3. Impact of testicular sperm retrieval on children. In pregnancies among men with non-obstructive azoospermia, there are more aneuploid embryos and there may be covert minor and major congenital abnormalities. Reports of systematic follow-up on the outcome of children born after ICSI with testicular sperm have not been published.
- There is a need for follow-up among children born after testicular sperm retrieval procedures.
4. PGD. When performing PGD for recurrent serious genetic disorders using DNA analysis, contamination by extraneous DNA from sperm or aerosols is an ominipresent risk. Besides the routine use of ICSI in these cases, laboratories should have in place appropriate procedures to detect non-embryonic sources of DNA in the samples being tested.
- Follow-up studies of the children born following embryo biopsy and preimplantation diagnosis and screening are needed and should be encouraged.
5. Follow-up studies of ICSI children. Long-term follow-up is difficult because of the dropout rate, especially when parents have high interurban motility. The time commitment is not inconsiderable for meaningful study of congenital abnormalities, general health and social and educational progress.
- More follow-up studies are needed and current studies should be extended.
- Studies are needed to clarify whether newborn and long-term health of ICSI offspring differ from typical births in the population, and if so, whether those differences are common to both IVF and ICSI.
- Studies are needed in which the comparison group involves children of infertile couples who have used various non-ART treatments or conceived without treatment.
6. Optimal ICSI protocols. Multiple follicular recruitment may be less important in ICSI cycles because the female partners generally have normal ovulatory function and the metaphase II oocytes can be identified. In ICSI cycles, mild ovarian stimulation may be a realistic and sensible alternative. Protocols which might have a slightly lower pregnancy rate per cycle but enable couples to have more cycles can only be implemented where there is a holistic approach to eligibility for funding.
- Registry end-points such as pregnancy rates per cycle should be replaced by the likelihood of a healthy child per treatment over a period of time which may involve more than one cycle.
- Further trials are needed to convincingly establish the medical, health, economic and psychological benefits of mild ovarian stimulation protocols in different clinical settings.
7. SET. Multiple pregnancy is the most common serious adverse effect of IVF and ICSI treatment. The risk is due to the perception that transferring more than one embryo increases pregnancy rates per cycle. Indeed, overall pregnancy rates are slightly higher with transfer of two embryos, but singleton pregnancy rates (i.e. proportion of pregnancies that are singletons rather than twins) are higher with transfer of one embryo. With higher fertilization rates, ICSI cycles generate more embryos from which to choose those which should be transferred or cryopreserved.
- Studies are needed to define which embryos should be selected for SET.
- There is a need for qualitative studies of how couples make decisions about the number of embryos to transfer and studies aimed at developing decision aids for application in ART clinics.
- Studies are needed to evaluate SET in more clinical practice settings.
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Acknowledgements |
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The secretarial assistance of Mrs Simonetta Vassallo is gratefully acknowledged.
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Footnotes |
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* A meeting was organized by ESHRE (Capri, 2–3 September 2006) with an unrestricted educational grant from Institut Biochimique SA to discuss the above subjects. The speakers included: M. Bonduelle (Centre for Medical Genetics, Universitair Ziekenhuis Vrije Universiteit Brussel, Belgium), P. Braude (Department of Women's Health, King's College London, School of Medicine at Guy's King's and St Thomas' Hospitals, London, UK), J. Collins (McMaster University, Hamilton, Canada), P. Devroey (Centre for Reproductive Medicine, Universitair Ziekenhuis Vrije Universiteit Brussel, Belgium), J.L.H. Evers (Department of Obstetrics and Gynecology, Academic Hospital Maastricht, The Netherlands), B.C.J.M. Fauser (Department of Reproductive Medicine and Gynecology, University Medical Center, Utrecht, The Netherlands), I. Liebaers (Centre for Medical Genetics, Universitair Ziekenhuis Vrije Universiteit Brussel, Belgium), G.D. Palermo (Andrology and Assisted Fertilization, Cornell Institute for Reproductive Medicine, New York, USA), A. Templeton (Department of Obstetrics and Gynecology, University of Aberdeen, Mat. Hospital, UK). The discussants included: D.T. Baird (Centre for Reproductive Biology, University of Edinburgh, UK), J. Cohen (8 rue de Marignan, Paris, France), P.G. Crosignani (II Department of Obstetrics and Gynecology, University of Milano, Italy), E. Diczfalusy (Karolinska Institutet, Stockholm, Sweden), K. Diedrich (Klinik für Frauenheilkunde und Geburtshilfe, Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Germany), L. Fraser (Reproduction and Rhythms Group, School of Biomedical and Health Sciences, Kings College London, UK), L. Gianaroli (S.I.S.Me.R., Bologna, Italy), A. Glasier (Family Planning and WW Services, Edinburgh, UK), G. Ragni (U.O.C. Sterilità di Coppia ed Andrologia, Fondazione Policlinico, Mangiagalli e Regina Elena, Milano, Italy), A. Sunde (Department of Obstetrics and Gynecology, University of Trondheim, Norway) B. Tarlatzis (Infertility and IVF Center, Thessaloniki, Greece), A. Van Steirteghem (Centre for Reproductive Medicine, Universitair Ziekenhuis Vrije Universiteit Brussel, Belgium). The report was prepared by J. Collins (Hamilton) and P.G. Crosignani (Milano).
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