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

Considerations of fertilization and embryo development, cost ffectiveness and safety

Bolarinde Ola, Masoud Afnan, Khaldoun Sharif, Spyros Papaioannou, Nahed Hammadieh and Christopher L.R.Barratt,1

Assisted Conception Unit, Birmingham Women's Hospital, Edgbaston, Birmingham B15 T2G, UK


    Abstract
 Top
 Abstract
 Introduction
 Conclusion
 Acknowledgements
 References
 
There is now considerable discussion whether intracytoplasmic sperm injection (ICSI) should be used in all cases of IVF. A critical and balanced view of the current literature is presented. The difficult question is how to identify men with apparently normal semen who are likely to fail to achieve a pregnancy using IVF. In conclusion, from both the safety and scientific viewpoint, ICSI should only be used in cases where success at IVF is regarded as unlikely.

Key words: budget impact analysis/cost needed to treat/fertilization failure/intracytoplasmic sperm injection (ICSI)/number needed to treat


    Introduction
 Top
 Abstract
 Introduction
 Conclusion
 Acknowledgements
 References
 
The indication to perform intracytoplasmic sperm injection (ICSI) in the earlier days was male factor infertility. Today, this have been expanded to include fertilization failure after conventional IVF (Kastrop et al., 1999Go; Fishel et al., 2000Go), ejaculatory dysfunction, immunological infertility and treatment for cancer patients who have had chemo/radiotherapy (Naysmith et al., 1998Go; Horne et al., 2001). Conventional IVF on the other hand is performed in cases of tubal disease, anovulation, unexplained infertility, and previous failed treatment by other methods. The insemination concentration is usually about 50 000 motile spermatozoa per oocyte. However, there are reports of the use of much higher insemination concentrations (HIC) in selected groups of patients to avoid the use of ICSI (Kastrop et al., 1999Go; Fishel et al., 2000Go). In contrast to ICSI, IVF preserves the natural selection of spermatozoa, which occurs at the sperm–oocyte interface during penetration of the oocyte vestments.

Recent data from Fishel and colleagues have examined the concept that ICSI should be offered to all patients needing IVF because of the significantly higher fertilization rate (Fishel et al., 2000Go). ICSI has become more developed as a technique and popularized to a stage of routine laboratory service. For example, in the UK the Human Fertilisation and Embryology Authority (HFEA) reported a 14% rise in the use of ICSI in 1998/1999 compared with the previous year. Almost half of fresh embryo transfers (median 47% range 16–74%) in this period were a result of ICSI treatment (Human Fertilisation and Embryology Authority, 2000Go). This is consistent with data from the European register where 43% of the transfers were from ICSI (EIM/ESHRE, 2001Go). Clearly, the use of ICSI is rising throughout the world and in some clinics it is the exclusive treatment of choice. Therefore, the issue of whether to use ICSI for all in-vitro inseminations needs to be critically discussed.

In this debate, we examine the arguments for and against the use of ICSI in cases where IVF would normally be used (non male-factor infertility). The issues discussed are fertilization rate, total failure of fertilization, embryo damage/blastocyst formation, cost effectiveness and safety.

Fertilization rate as a measure of effectiveness
One group has performed a randomized, prospective, multi-centred trial using sibling metaphase II oocytes in 221 patients to try to address the question of whether ICSI should be advocated for all couples (Fishel et al., 2000Go). The patients were divided into five groups. These included: Group 1 (37 patients): idiopathic previous failed IVF, where HIC was compared with ICSI using the partnersapos; spermatozoa; Group 2 (18 patients): idiopathic previous failed IVF with HIC, where conventional IVF was compared with ICSI using donor spermatozoa; Group 3 (36 patients): patients unsuitable for conventional IVF (male infertility), where IVF using donor spermatozoa was compared with ICSI using partnerapos;s spermatozoa; Groups 4 and 5 had metaphase II oocytes that had failed to fertilize by IVF and were re-inseminated by either HIC or ICSI. The clinical bottom line for groups 2 and 3 was that conventional IVF had a fertilization rate of 65.4% and ICSI 75.6%, with an absolute treatment effect of 0.102 [95% confidence interval (CI) 0.025–0.179], generating a number needed to treat (NNT) of 10 (95% CI 6–40). The NNT is the number of sibling MII oocytes that need to be inseminated by ICSI to derive one additional zygote, compared with IVF. Although this figure is statistically significant, in clinical terms it means that, in this group of patients where normal spermatozoa were used in IVF, for every 10 sibling MII oocytes inseminated by ICSI, only one extra zygote is produced compared with insemination by conventional IVF.

In other studies, a lack of significant difference has been demonstrated in the fertilization rates obtained with ICSI and IVF in patients with non-male factor infertility (61 versus 67%) (Yang et al., 1996Go) and unexplained infertility (60.4 versus 54%) (Ruiz et al., 1997Go). Nevertheless, we need to be cautious in the interpretation of the results presented by some of these studies, as for example, at the design stage, power and sample size statistics were often not sufficiently emphasized, thereby exposing the results to possible random errors (Fishel et al., 2000Go). In addition to this, some studies (Ruiz et al., 1997Go) are not randomized controlled trials. Closer scrutiny often shows that the only control possible due to ethical considerations was the use of sibling metaphase II (MII) oocytes. Often, in these studies, no explicit descriptions were made of what happened to oocytes allocated to ICSI, but found not to be MII after denudation (i.e. was intention-to-treat analysis performed?), or how investigators who randomized and performed in-vitro inseminations were blinded to embryo grading. These potential sources of error may serve to reduce the strength of evidence presented by the authors (NHS Centre for Reviews and Dissemination, 1999Go). The implication is that often, what is presented as level Ib evidence in favour of ICSI (or in fact against) may, on critical appraisal, be found to be no better than level II or III. Therefore, larger carefully conducted studies are required on non male-factor patients to confidently address the question whether ICSI does result in significantly higher fertilization rates (and embryo development) in men with apparently normal semen.

Fertilization rate: an interim outcome measure
The use of fertilization rate instead of total failure of fertilization, or indeed clinical pregnancy rate as an outcome event has drawbacks. Fertilization rate is an interim outcome measure in an IVF programme, which may have little effect on the final outcome of a fresh cycle or that of a subsequent frozen embryo transfer. It is therefore difficult to judge whether or not to advocate ICSI over IVF based on fertilization rate alone. To illustrate the point, imagine a scenario with a mean recovery of 10 MII oocytes, fertilization rate of 65% from IVF, and 75% from ICSI. In the UK, a maximum of three embryos can be replaced in a treatment cycle. Frozen embryo–thaw success rates of 81–90% for IVF and 88–91% for ICSI have been described in prospective randomized studies (Damario et al., 1999Go; Hu et al., 1999Go). Consequently, this would allow approximately the same number of frozen embryo transfer cycles for IVF or ICSI. It would therefore seem apparent that, if decision analysis was performed based on the above scenario, an improved fertilization rate alone might not be enough to advocate ICSI over IVF per cycle of treatment.

Total failure of fertilization
From the clinician and patientsapos; points of view, the rate of total failure of fertilization is a more useful outcome measure than fertilization rate. ICSI has an advantage, which in the UK is in the form of an HFEA regulation, requiring that only MII oocytes, assessed after cleaning the oocyte–cumulus–complex, be injected. There is a prescribed oocyte quality and therefore a time limit to when insemination has to be accomplished during ICSI. For conventional IVF however, metaphase I (MI), MII, or luteinized post-maturity oocytes can be used.

Several studies have attempted to demonstrate a superiority of ICSI over IVF based on failed fertilization rates. For example, in a controlled study of 70 couples with either unexplained infertility or endometriosis who had failed to respond to intrauterine insemination, Ruiz and his colleagues, (Ruiz et al., 1997Go), found a clear benefit of ICSI over IVF (failed fertilization rates of 0 versus 11%) despite the lack of significant difference in the fertilization rates between the two methods (60.4 versus 54%). In this study, whereas metaphase II oocytes were used for ICSI, this was not the case for the IVF group, thus exposing the results to bias. In another example, an `auto-controlledapos; study of 662 sibling MII oocytes from patients with tubal disease and normozoospermic partners, found rates of total failure of fertilization of 3.6% (95% CI = 0.4 to 12.3) for ICSI and 12.5% (95% CI = 5.2–24.1) for IVF (Staessen et al., 1999Go). This would appear to present a real difference, although the small sample size may have introduced type II error. These potential sources of error may have served to reduce the strength of evidence presented by the authors, and when considered may mean that the superiority described in favour of ICSI over conventional IVF may be a chance occurrence.

However, whilst scientific rigour indicates that the above studies have potential errors it does look as though ICSI may be of benefit in cases of fertilization failure with conventional IVF that can be predicted before treatment. Data from Liu and Baker illustrate this point (Liu and Baker, 2000Go). They have reported on 160 patients who have apparently normal semen but either fail to bind to the zona pellucida (ZP) or do not acrosome react (AR) in response to the ZP (disordered ZP-induced AR) and thus fail to have successful IVF conceptions. They estimate that, in their patient population, up to a third of normozoospermic men have disordered ZP-induced AR. Interestingly, ICSI was found to overcome these defects resulting in live births (Liu and Baker, 2000Go).

In contrast, in our clinic, which is a tertiary referral centre, we do not see a high incidence of fertilization failure with IVF. The total failed fertilization rate for treatments between January 1999 and July 2000 was 1.5% (95% CI 0.04–3.8) for ICSI and 2.1% (95% CI 1.0–3.8) for IVF (Table IGo). Our data suggest that improved but flexible clinical and laboratory protocols can reduce the incidence of total failure of fertilization, although it is possible that in our centre we do not have a high incidence of normozoospermic men with dysfunctional spermatozoa (see Liu and Baker above) which may account for our low incidence of failed fertilization.


View this table:
[in this window]
[in a new window]
 
Table I. Comparisons between ICSI and IVF: January 1999–July 2000 at the Assisted Conception Clinic, Birmingham Womenapos;s Hospital, UK
 
Embryo damage and blastocyst formation
ICSI is associated with reduced blastocyst formation (Shoukir et al., 1998Go; Dumoulin et al., 2000Go; Griffiths et al., 2000Go) and a higher miscarriage rate (Aytoz et al., 1999Go). These negative influences on development have primarily been attributed to the poor quality of injected spermatozoa. There is no doubt that the spermatozoa used for ICSI have higher levels of defects which are likely to have an adverse effect on embryo development e.g. higher levels of DNA damage (Sakkas et al., 1999Go) and increased levels of aneuploidy (Bernardini et al., 1997Go). However, the technique itself may have a negative effect on development. This was illustrated by Griffiths and colleagues who showed a significantly lower (P < 0.01) development to the blastocyst stage in ICSI compared with IVF when semen from the same semen samples was used for each technique (Griffiths et al., 2000Go). Perhaps this is not surprising as apart from the physical damage that may occur during and/or after injection (Dumoulin et al., 2001Go), there are clear differences in the synchrony of fertilization events in ICSI compared with IVF e.g. changes in the pattern of Ca2+ induced transients (Tesarik, 1998Go) and decondensation of the spermatozoon which may specifically lead to abnormal development. For example, in both rhesus monkeys (Hewitson et al., 1999Go) and humans (Bourgain et al., 1998Go; Terada et al., 2000Go) there is atypical decondensation of the nucleus and delayed replication of the male genome. In addition, the non-random positioning of the chromosomes in the nucleus, combined with the atypical nuclear decondensation, may lead to higher levels of aneuploidy (Luetjens et al., 1999Go; Terada et al., 2000Go). Thus, the ICSI procedure itself may make a contribution to the poorer embryo development in ICSI embryos as compared with IVF. Clearly, more comprehensive studies are required to address this specific issue. These must include, where possible, follow up data including conception rates, as one randomized controlled study which compared ICSI with IVF in non male-factor cases concluded that implantation and pregnancy rates were not different (Aboulghar et al., 1996Go).

Economic analysis based on live birth rates
In the reporting period between 1998 and 1999, the HFEA showed an overall live birth rate per fresh treatment cycle for ICSI of 22.6% (4082/18042) significantly higher (P = 0.01) than the rate of 21.6% (5969/27617) for IVF.

However, ICSI is substantially more expensive than IVF. In our clinic, as in many parts of the UK, the cost difference is about £600 per fresh cycle completed (Philips et al., 2000Go). In the UK 25% of treatments are funded by the National Health Service (NHS) (Kerr et al., 1999Go). We suggest that recommending the use of ICSI for all those needing IVF is unlikely to be considered a judicious use of scarce resources. To illustrate this, we used the HFEA 1998/99 data, assuming that the advantage of ICSI was sustained even when performed on the population of couples who would have had IVF for female or unexplained factors. We used the data comparing live birth rates per fresh cycle to derive the absolute treatment effect (ATE = 0.01), and NNT (NNT = 100) (see Table IIGo). Cost benefit analyses show that each live birth produced by ICSI costs £2000 extra. The main cost implication however is the incremental cost effectiveness or cost needed to treat (CNT). This shows that £60 000 will be needed to gain one additional live birth when ICSI is advocated for all patients requiring IVF. Budget impact analysis (BIA) shows that CNT (£60 000) can treat an extra 29 cycles of conventional IVF.


View this table:
[in this window]
[in a new window]
 
Table II. Cost analysis comparing cost benefit and cost effectiveness of ICSI with IVF using HFEA 1998–1999 UK national data
 
Safety
Several reports suggests that our initial fears about an increased incidence of major congenital malformations and possible imprinting disorders in the offspring following ICSI are unfounded (Bonduelle et al., 1999Go; Loft et al., 1999Go; Manning et al., 2000Go; Wennerholm et al., 2000Go). However, it is important to remember that we still do not know the long-term effects of the ICSI procedure and that many of the putative follow-up studies contain insufficient numbers of patients and often have a relatively high incidence of patients lost to follow up (Hawkins and Barratt, 1999Go; Hawkins et al., 1999Go). Clearly more comprehensive, long-term and possibly national studies are necessary.

It is probable that the increases in congenital abnormalities observed in some ICSI children, such as sex chromosome abnormalities, are due to the use of sub-optimal male gametes. However, the technique itself may play a role in the formation of these abnormalities. In addition, there is experimental evidence that provides caution against the widespread use of ICSI, for example, the incorporation of exogenous DNA into spermatozoa and subsequent transmission to the offspring (Perry et al., 1999Go; Chan et al., 2000Go). Experiments in mice have shown an enhancement of a genetic defect (sperm morphology) through ICSI (Akutsu et al., 2001Go). The incorporation of foreign DNA combined with the possible enhancement of defects by bypassing the natural selection mechanisms now needs rigorous experimentation in the human.

The difficult question
The arguments above do not support the routine use of ICSI in all IVF treatments. However, there is a clear group of patients, e.g. those with normal but dysfunctional spermatozoa, that have zero or significantly reduced fertilization success at IVF (Barratt and Publicover, 2001Go). Such patients can be successfully treated by ICSI. Whilst it is possible, using sophisticated sperm function assays such as zona binding, to predict which men may have reduced success at IVF, such assays are impossible to use on a routine basis (Whitmarsh et al., 1996Go). The question that all clinics therefore face is: at what IVF fertilization rate does ICSI become a more effective treatment than IVF? The answer is not clear-cut. We have attempted to address this by using the HFEA data (Templeton and Morris, 1998Go; Human Fertilisation and Embryology Authority, 2000Go). Table IIIGo illustrates three scenarios where the IVF fertilization rate and number of embryos created, which have significant effects on live birth rate (LBR) varies. In these examples an IVF fertilization rate of <40% would only result in a maximum 20% LBR with no embryos available for freezing. Thus, the use of ICSI will result in a significantly higher LBR per fresh cycle and allow the possibility of 2–3 embryos for transfer in a subsequent cycle. Under such circumstances, it would be better to advise ICSI. However, the situation becomes less obvious when the fertilization rate is >=50% as the LBR for fresh transfers are comparable with ICSI (28%) and, depending on the exact fertilization rate, a number of embryos are available for freezing. Of course, in these examples we have assumed that the spermatozoa look normal but are defective thus, embryos and pregnancies can be achieved by ICSI whereas conventional IVF would have been unsuccessful (Liu and Baker, 2000Go). This is not always the case and our examples in Table IIIGo only apply to such cases where ICSI can circumnavigate the use of defective male gametes.


View this table:
[in this window]
[in a new window]
 
Table III. Live birth rate (LBR) depending on fertilization rate and number of embryos available for transfer with conventional IVF in comparison with ICSI
 

    Conclusion
 Top
 Abstract
 Introduction
 Conclusion
 Acknowledgements
 References
 
Using the currently available clinical, scientific and economic data, there appears to be no advantage to using ICSI instead of IVF for all patients requiring IVF. However, there are some situations where ICSI may be of benefit. These are generally where there is fertilization failure or significantly reduced fertilization success (see Table IIIGo). In clinics where these two factors are prominent, and cannot be corrected by other means, then the use of ICSI represents an effective tool. However, the real challenge is to identify, prospectively, which cases (men) are likely to give poor success at IVF.

In summary, from both the safety and scientific viewpoints, ICSI should only be used in cases where success at IVF is regarded as unlikely.


    Acknowledgements
 Top
 Abstract
 Introduction
 Conclusion
 Acknowledgements
 References
 
This work is supported by a programme grant from the NHS. We are grateful for constructive comments from Dr D.Sakkas.


    Notes
 
1 To whom correspondence should be addressed. E-mail: c.l.barratt{at}bham.ac.uk Back

Note added at proof

After completion of this manuscript Bhattacharya and colleagues reported on a trial comparing ICSI and IVF in non-male factor and mild male factor infertility. Although live birth rates were not reported, implantation rates were similar between the two groups (Battacharya et al., 2001Go).


    References
 Top
 Abstract
 Introduction
 Conclusion
 Acknowledgements
 References
 
Aboulghar, M.A., Mansour, R.T. and Serour, G.I. (1996) Prospective controlled randomised study of in vitro fertilization versus intracytoplasmic sperm injection in the treatment of tubal factor infertility with normal semen parameters. Fertil. Steril., 66, 753–756.[ISI][Medline]

Akutsu, H., Tres, L.L., Tateno, H. et al. (2001) Offspring from normal mouse oocytes injected with sperm heads from the azh/azh mouse display more severe sperm tail abnormalities than the original mutant. Biol. Reprod. 64, 249–256.[Abstract/Free Full Text]

Aytoz, A., Van den, A.E., Bonduelle, M. et al. (1999) Obstetric outcome of pregnancies after the transfer of cryopreserved and fresh embryos obtained by conventional in-vitro fertilization and intracytoplasmic sperm injection. Hum. Reprod., 14, 2619–2624.[Abstract/Free Full Text]

Barratt, C.L.R. and Publicover, S.J. (2001) Interaction between sperm and zona pellucida in lae infertility. Lancet, in press.

Battacharya, S., Hamilton, M.P.R, Shaaban, M. et al. (2001) Conventional in-vitro fertilization versus intracytoplasmic sperm injection for the treatment of non-male factor infertility: a randomized controlled trial. Lancet, 357, 2075–2079.[ISI][Medline]

Bernardini, L., Martini, E., Geraedts, J.P. et al. (1997) Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in-situ hybridisation. Mol. Hum. Reprod. 3, 431–438.[Abstract]

Bonduelle, M., Camus, M., De Vos, A. et al. (1999) Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum. Reprod., 14, (Suppl. 1), 243–264.[ISI][Medline]

Bourgain, C., Nagy, Z.P., De Zutter, H. et al. (1998) Ultrastructure of gametes after intracytoplasmic sperm injection. Hum Reprod., 13, (Suppl. 1) 107–116.[Medline]

Chan, A.W., Luetjens, C.M., Dominko, T. et al. (2000) Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live rhesus monkey births. Mol. Hum. Reprod., 6, 26–33.[Abstract/Free Full Text]

Damario, M.A., Hammitt, D.G., Galanits, T.M. et al. (1999) Pronuclear stage cryopreservation after intracytoplasmic sperm injection and conventional IVF: implications for timing of the freeze. Fertil. Steril., 72, 1049–1054. Erratum, Fertil. Steril., 73, 874.[ISI][Medline]

Dumoulin, J.C.M., Coonen, E, Bras, M. et al. (2000) Comparison of in-vitro development of embryos originating from either conventional in-vitro fertilization or intracytoplasmic sperm injection. Hum. Reprod., 15, 402–409.[Abstract/Free Full Text]

Dumoulin, J.C.M., Coonen, E., Bras, M. et al. (2001) Embryo development and chromosomal abnormalities after ICSI: effects of the injection procedure. Hum. Reprod., 16, 306–312.[Abstract/Free Full Text]

EIM/ESHRE (2001) The European IVF-monitoring programme for ESHRE. Assisted reproductive technology in Europe, 1997. Results generated from European registers by ESHRE. Hum. Reprod., 16, 384–391.[Abstract/Free Full Text]

Fishel, S., Aslam, I., Lisi, F. et al (2000) Should ICSI be the treatment of choice for all cases of in-vitro conception? Hum. Reprod., 15, 1278–1283.[Abstract/Free Full Text]

Griffiths, T.A., Murdoch, A.P. and Herbert, M. (2000) Embryonic development in vitro is compromised by the ICSI procedure. Hum. Reprod., 15, 1592–1596.[Abstract/Free Full Text]

Hawkins, M.M. and Barratt, C.L.R. (1999) Intracytoplasmic sperm injection. Science, 286, 51–52.[ISI][Medline]

Hawkins, M.M., Barratt, C.L.R., Sutcliffe, A.G. and Cooke, I.D. (1999) Male infertility and increased risk of diseases in future generations. Lancet, 354, 1906–1907.[ISI][Medline]

Hewitson, L., Dominko, T., Takahashi, D. et al., (1999) Unique checkpoints during the first cell cycle of fertilisation after intracytoplasmic sperm injection in rhesus monkeys Nat. Med., 5, 1–33.[ISI][Medline]

Horne, G., Atkinson, A., Brison, D.R. et al. (2001) Achieving pregnancy against the odds: successful implantation of frozen-thawed embryos generated by ICSI using spermatozoa banked prior to chemo/radiotherapy for Hodgkinapos;s disease and acute leukaemia. Hum. Reprod., 16, 107–109.[Abstract/Free Full Text]

Hu, Y., Maxson, W.S., Hoffman, D.I. et al. (1999) A comparison of post-thaw results between cryopreserved embryos derived from intracytoplasmic sperm injection and those from conventional IVF. Fertil. Steril., 72, 1045–1048.[ISI][Medline]

Human Fertilisation and Embryology Authority (2000) Patientsapos; information and guide. URL http://www.hfea.gov.uk/frame2.htm

Kastrop, P.M., Weima, S.M., Van Kooij, R.J. et al. (1999) Comparison between intracytoplasmic sperm injection and in-vitro fertilization (IVF) with high insemination concentration after total fertilization failure in a previous IVF attempt. Hum. Reprod., 14, 65–69.[Abstract/Free Full Text]

Kerr, J., Brown, C., Balen, A.H. et al. (1999) The experiences of couples who have had infertility treatment in the United Kingdom: results of a survey performed in 1997. Hum. Reprod., 14, 934–938.[Abstract/Free Full Text]

Liu, D.Y. and Baker, H.W.G. (2000) Defective sperm-zona pellucida interaction: a major cause of failure of fertilization in clinical in-vitro fertilization. Hum. Reprod., 15, 702–708.[Abstract/Free Full Text]

Loft, A., Petersen, K., Erb, K. et al. (1999) A Danish national cohort of 730 infants born after intracytoplasmic sperm injection (ICSI) 1994–1997. Hum. Reprod ., 14, 2143–2148.[Abstract/Free Full Text]

Luetjens, C.M., Payne, C. and Schatten, G.S. (1999) Non-random chromosome positioning in human sperm and sex chromosome anomalies following intracytoplasmic sperm injection. Lancet, 353, 1240.[ISI][Medline]

Manning, M., Lissens, W., Bonduelle, M. et al. (2000) Study of DNA-methylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no imprinting defects. Mol. Hum. Reprod., 6, 1049–1053.[Abstract/Free Full Text]

Naysmith, T.E., Blake, D.A., Harvey, V.J. et al (1998) Do men undergoing sterilizing cancer treatments have a fertile future? Hum. Reprod., 13, 3250–3255.[Abstract]

NHS Centre for Reviews and Dissemination (1999) Getting evidence into practice.Effective Health Care, 5, 1–16.

Perry, A.C., Wakayama, T., Kishikawa, H. et al. (1999) Mammalian transgenesis by intracytoplasmic sperm injection. Science, 284, 1180–1183.[Abstract/Free Full Text]

Philips, Z., Barraza-Llorens, M. and Posnett, J. (2000) Evaluation of the relative cost effectiveness of treatments for infertility in the UK. Hum. Reprod., 15, 95–100.[Abstract/Free Full Text]

Ruiz, A., Remohi, J., Minguez, Y. et al. (1997) The role of in vitro fertilisation and intracytoplasmic sperm injection in couples with unexplained infertility after failed intrauterine insemination. Fertil. Steril., 68, 171–173.[ISI][Medline]

Sakkas, D. Mariethoz, E., Manicardi, G. et al.. (1999) Origin of DNA damage in ejaculated human spermatozoa. Rev. Reprod. 4, 31–37.[Abstract/Free Full Text]

Shoukir, Y., Chardonnens, D., Campana, A. et al. (1998) Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence? Hum. Reprod., 13, 1632–1637.[Abstract]

Staessen, C., Camus, M., Clasen, K. et al. (1999) Conventional in-vitro fertilization versus intracytoplasmic sperm injection in sibling oocytes from couples with tubal infertility and normozoospermic semen. Hum. Reprod., 14, 2474–2479.[Abstract/Free Full Text]

Templeton, A.A. and Morris, J.K. (1998) Reducing the risk of multiple births by transfer of two embryos after in vitro fertilisation. N.E.J.M.., 339, 573–577.[Abstract/Free Full Text]

Terada, Y. Luetjens, C.M., Sutovsky, P. et al. (2000) Atypical decondensation of the sperm nucleus, delayed replication of the male genome, and sex chromosome positioning following intracytoplasmic human sperm injection (ICSI) into golden hamster eggs: does ICSI itself introduce chromosome anomalies. Fertil. Steril., 74, 454–460.[ISI][Medline]

Tesarik, J. (1998) Oocyte activation after intracytoplasmic injection of mature and immature sperm cells. Hum. Reprod., 13, (Suppl. 1), 117–127.

Whitmarsh, A.J., Woolnough, M.J. Moore, H.D.M. et al. (1996) Biological activity of recombinant human ZP3 produced in vitro:potential for a sperm function test. Mol. Hum. Reprod., 2, 911–919.[Abstract]

Wennerholm, U.B., Bergh, C., Hamberger, L. et al. (2000) Incidence of congenital malformations in children born after ICSI. Hum. Reprod., 15, 944–948.[Abstract/Free Full Text]

Yang, D., Shahata, M.A., al Bader, M. et al. (1996) Intracytoplasmic sperm injection improving embryo quality: comparison of the sibling oocytes of non-male-factor couples. J. Assist. Reprod. Genet., 13, 351–355.[ISI][Medline]