Intracytoplasmic Sperm Injection Indications: How Rigorous?

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1 283 Intracytoplasmic Sperm Injection Indications: How Rigorous? Samir N. Babayev, MD 1 Chan Woo Park, MD 1,2 Orhan Bukulmez, MD 1 1 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 2 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Cheil General Hospital & Women s Healthcare Center, Kwandong University College of Medicine, Seoul, Korea Semin Reprod Med 2014;32: Abstract Keywords intracytoplasmic sperm injection ICSI in vitro fertilization IVF conventional IVF assisted reproductive technology ART indications Intracytoplasmic sperm injection (ICSI) was introduced in 1992 by Palermo et al for the treatment of infertile couples with severe male factor and unexplained total fertilization failure (TFF). 1,2 Soon after its introduction to human assisted reproductive technologies (ARTs), ICSI received wide acceptance even before any reassuring human data were available. Since ICSI requires only one good spermatozoon, it has been perceived to be much more efficient than in vitro fertilization (IVF) with conventional insemination which requires many Address for correspondence Orhan Bukulmez, MD, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX ( Orhan.Bukulmez@UTSouthwestern.edu). Up to 15% of all couples of reproductive age are diagnosed with subfertility and about one-third of those will have male factor infertility as a contributing factor. Intracytoplasmic sperm injection (ICSI) has proven to be invaluable for couples with severely compromised semen parameters. Since its introduction into the clinical practice in 1992, the indications for ICSI were dramatically expanded to include various patient populations with normal or mildly abnormal semen parameters. Moreover, some fertility programs choose to perform ICSI for all of their patients needing assisted reproductive technologies. By all means, the male factor indications for ICSI are not well defined, apart from its absolute utility with surgically obtained spermatozoa in the presence of low motility, or in cases of severe defects with sperm concentration and motility. Based on current evidence, ICSI is not indicated for routine use. Its adoption for previous history of total fertilization failure, in vitro oocyte maturation, cryopreserved oocytes, polyploidy prevention, poor-quality oocytes, diminished ovarian reserve, and advanced reproductive age are not supported by current evidence, albeit further research with well-designed studies is warranted. Finally, from a biological standpoint ICSI is considered to be more invasive, more energy consuming for the oocyte itself and its adverse genetic and epigenetic effects cannot be ruled out. Although more studies are needed to clarify definitive indications for ICSI, many of its current applications can be deemed empiric at this time. more spermatozoa to achieve fertilization. We intend to review the widely proposed indications of ICSI to assess if in fact they are well supported by the published literature or not. Male Factor Indications for Intracytoplasmic Sperm Injection ICSI involves manual selection of the spermatozoa and its subsequent injection into the oocyte, following the stripping Issue Theme Common Practices in Reproductive Endocrinology and Infertility Supported by Weak or No Evidence; Guest Editor, Orhan Bukulmez, MD Copyright 2014 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) DOI /s ISSN

2 284 Intracytoplasmic Sperm Injection Indications Babayev et al. of the cumulus cells, using micromanipulation techniques. Therefore, ICSI quickly became the treatment of choice when only surgically obtained testicular or epididymal sperm is available, in cases of acrosomal aplasia and for patients with immotile, but viable spermatozoa. 3 It has been challenging to establish a strict definition for male factor infertility requiring treatment with ICSI, whereas subfertile men may also benefit from less costly and less invasive method of intrauterine insemination (IUI). Over the past 25 years, the World Health Organization revised its criteria aimed at defining reference values for semen characteristics three consecutive times after its first introduction in Over time, normal values for quantitative and qualitative parameters of sperm had been decreased down to the semen volume of 1.5 ml; sperm count of 15 million per ml; total motility of 40%; and morphologically normal forms of 4.0%. 4 These values represent semen characteristics associated with achieving pregnancy within 12 months of unprotected sexual intercourse. Unfortunately, these cutoff values cannot be used for accurate identification of fertile and infertile men, but the trend toward decreasing lower reference limits demonstrates evolution of our understanding of how much and what quality spermatozoa are necessary for natural conception. There are no randomized clinical trials comparing ICSI with conventional IVF in couples with moderate to severe abnormalities in semen parameters. It is generally accepted that ICSI is the only treatment of choice for those cases. However, it is important to note that certain types of male factor can be successfully treated with IUI, which is less invasive and less costly than either conventional IVF or ICSI. 3 For instance, a meta-analysis, which included 17 prospectively designed studies showed increased pregnancy rates with IUI compared with timed intercourse in couples with male factor infertility. 5 One prior study also indicates that inseminating motile count of 1 million spermatozoa may be enough for successful treatment with IUI and even lower counts may lead to pregnancy with IUI if a 4% strict morphology criterion has been met. 6 Proceeding directly with ICSI in men with only one abnormal semen parameter or resorting to IVF/ICSI, early in the process of evaluation of male subfertility may be questionable in terms of its cost-effectiveness. 7 The decision for choosing ICSI over conventional IVF in male factor infertility is generally arbitrary with some authors proposing the use of total inseminating motile count cutoff values of below 0.5 to 1 million to recommend that the couple should be offered ICSI over conventional IVF. 8,9 Kruger et al in 1986 proposed their strict sperm morphology criteria and correlated these with probability of fertilization by conventional IVF. 10 Later other investigators adopted and refined the system concluding that at least 5% morphologically normal sperm is necessary to optimize the fertilization rate. 11,12 Initially high insemination concentration and then, ICSI was recommended for treatment of teratozoospermia (< 5% morphologically normal sperm) Yet, other studies questioned whether teratozoospermia has any negative effects on fertilization, pregnancy, and live birth rates Therefore, well-designed studies are still needed to make a definitive conclusion on the impact of strict sperm morphology on pregnancy outcomes, as well as the utility of different techniques used to overcome the suggested adverse effects of teratozoospermia. Recently introduced technique of intracytoplasmic morphologically selected sperm injection (IMSI), using ultrahigh magnification of 6,000 to 10,000 (compared with regular ICSI with ) has been proposed as a method enabling selection of motile spermatozoa free of any structural defects to optimize ICSI outcome for couples with male factor infertility. 20 Reports of success of IMSI are conflicting with some studies showing advantage over ICSI and others failed to show clinically significant difference. 21 Again definitive criteria to resort to IMSI do not exist. It has been suggested that there is a relationship between direct antisperm antibody (ASA) levels in semen and pregnancy rate after ART but reports in the literature have been conflicting. A meta-analysis that included 16 studies (10 IVF and 6 ICSI) indicates that the presence of ASA does not influence pregnancy rates after IVF or ICSI. 22 Hence, the presence of ASA should not drive the decision of using ICSI versus IVF. In summary, male factor indications for ICSI are not scrupulous apart from its use with surgically obtained spermatozoa, certain sperm abnormalities, and severe male factor defined as inseminating motile count of < 0.5 million. All According to 2011 Society for Assisted Reproductive Technology registry, ICSI was used in two-thirds of all ART cycles in the United States. 23 It would be naïve to think that ICSI is being used only when male factor infertility is present. The same database shows that in only 35% of the patients male component was a factor. The routine use of ICSI regardless of cause of infertility has been proposed by different authors. 24 Probably the best study evaluating this issue is a multicenter trial from the United Kingdom which randomized 415 couples with non male factor infertility to ICSI versus conventional IVF. 25 Primary outcome of implantation rate was actually higher with conventional IVF than that was achieved with ICSI (30 vs. 22% with p < 0.05). Of note, fertilization rates per oocyte retrieved and the pregnancy rate were also higher in IVF group, although the difference in the pregnancy rates did not reach statistical significance (58 vs. 47% with p < and 33 vs. 26% with p ¼ 0.11, respectively). ICSI resulted in a higher fertilization rate per oocyte injected, whereas this could be due to a better detection of the oocyte maturational status after cumulus cells were removed. Authors concluded that ICSI requires significantly higher resource utilization without offering any clinical advantage over conventional IVF for couples with non male factor infertility. Failed Fertilization with IVF An outcome of TFF following conventional IVF is a frustrating experience for patients as well as clinicians. In normospermic

3 Intracytoplasmic Sperm Injection Indications Babayev et al. 285 patients undergoing conventional IVF, the rates of TFF and low fertilization (defined as < 25% fertilization rate) were reported as 5 to 15 and 20%, respectively. 26,27 When TFF occurs during conventional IVF, most clinicians would proceed directly with ICSI in subsequent cycles. Some authors have proposed splitting the sibling oocytes into ICSI and conventional IVF in subsequent cycles. 28 The chance of recurrence of TFF in a subsequent conventional IVF cycle is reported to be between 30 and 50% rather than an expected rate of 100%. Therefore, it is suspected that some factors other than the insemination method used may be responsible for TFF and the condition may not be repetitive. Hence, it may be reasonable to undertake additional conventional IVF attempts before reverting to ICSI. One retrospective analysis of 555 couples showed that 87% of the patients (mean age with standard deviation [SD] ) were able to achieve fertilization in subsequent cycles using conventional IVF with delivery rates being 44% per patient, 25% per embryo transfer (ET), which was comparable to the delivery rates observed in ICSI group (23% per patient and 23% per ET). 29 Another interesting retrospective report compared the fertilization, implantation, and pregnancy rates after ICSI, among 65 non male factor patients with history of failed/low fertilization with conventional IVF, and in 219 patients with primary male factor. Despite the comparable fertilization and cleavage rates and similar number and quality of embryos transferred, patients with previous failed/low fertilization with conventional IVF had significantly lower clinical pregnancy rate following subsequent ICSI than those with primary male factor (19.6 vs. 33.5%, respectively). 30 These studies suggest that conventional IVF may be reattempted in subsequent cycles considering the encouraging fertilization and pregnancy rates with this more conservative approach. To our knowledge, there is only one published prospective study reporting that ICSI may be better than conventional IVF. 28 In this study, sibling oocytes were subjected to ICSI and IVF after the first IVF attempt resulted in TFF and low fertilization. In TFF group, fertilization rate per oocyte retrieved was 48% for ICSI and 11% for conventional IVF. The same rates for ICSI and conventional IVF in prior low fertilization group were 60 and 22%, respectively, favoring ICSI. It is noteworthy that this study included only 38 couples and the pregnancy rates were difficult to assess since some patients received both ICSI and IVF fertilized embryos. Thus, reaching adefinitive conclusion on this matter is somewhat difficult. Even American Society for Reproductive Medicine (ASRM) concluded that although subsequent total failed fertilization may be related to the IVF stimulation, utilizing IVF/ICSI may decrease the risk of subsequent fertilization failure, without mentioning about any potential improvement in pregnancy rates. 31 Rescue Intracytoplasmic Sperm Injection Rescue ICSI has been proposed as a method for overcoming fertilization failure of oocytes the next day following conventional IVF. 32 To rescue the IVF cycle, reinsemination of the unfertilized oocytes 15 to 18 hours after the initial insemination has been performed with ICSI. 33 However, the reports indicated that normal fertilization rates after rescue ICSI remained relatively low and the generated embryos achieved low pregnancy rates. 33,34 It is thought that oocyte quality diminishes 24 hours after retrieval, and although some oocytes still may be fertilized, the embryos derived from rescue ICSI procedures often arrest in early stages of development. Furthermore, high rates of polyploidy were reported in embryos fertilized by rescue ICSI. 33 Considering the very low pregnancy rates in addition to the potential genetic risks associated with the aging oocytes in culture, some authors suggest that rescue ICSI should not be attempted. 33 Unexplained Infertility Up to 30% of couples seeking fertility treatment are ultimately diagnosed with unexplained infertility. 35 Although the underlying cause of infertility is unknown, it is has been hypothesized that the mechanisms may include fertilization defects even when the semen parameters are normal. In general, IVF has been shown to be an effective therapy for couples with unexplained infertility. However, in 5 to 25% of these couples, TFF may occur with the use of conventional IVF. 14,36 Since it is difficult to predict which couples with unexplained infertility may experience TFF, some physicians resort to the practice of split ICSI and IVF, to minimize the risk of TFF. A recent meta-analysis examined the fertilization rates per retrieved oocyte of couples with unexplained infertility in 11 randomized controlled studies. 37 In five of these studies, sibling oocytes were specifically stated to be assigned to ICSI or conventional IVF before assessment of maturity while no relevant information was presented in others. To avoid significant heterogeneity one of these five trials were not included in relative risk (RR) calculation. The likelihood of fertilization per allocated oocyte was significantly higher for ICSI when compared with conventional IVF. The pooled RR of fertilization per oocyte before randomization was higher with ICSI than with conventional IVF (RR, 1.43; 95% confidence interval [CI], ). The pooled RR of TFF for all 11 trials included in the metaanalysis was significantly higher with conventional IVF than with ICSI (RR, 8.22; 95% CI, ). Importantly, the number of ICSI procedures needed to prevent one case of TFF was five. In these trials, each patient served as her own control, thereby reducing the impact of potentially important confounders, such as maternal age, ovarian stimulation, oocyte and sperm quality, and laboratory conditions. It is important to note that the oocytes allocated to ICSI are routinely assessed for nuclear maturity, while those fertilized by conventional IVF are not. This selection bias may contribute to a higher fertilization rate per oocyte compared with unselected oocytes undergoing conventional IVF at least in some of these trials. Above all, the comparison of pregnancy and live birth rates between the

4 286 Intracytoplasmic Sperm Injection Indications Babayev et al. two groups could not be made, since only few studies reported pregnancy-related outcomes with widely different embryo selection criteria. Furthermore, the pooling of embryos obtained from ICSI and conventional IVF for ET and relatively few numbers of transfers rendered pregnancy outcome assessment impossible. Although there are clues that ICSI may benefit some couples with unexplained infertility, more studies reporting pregnancy outcomes are needed. Poor- Quality Oocytes It has been hypothesized that ICSI might be a better method of fertilization than conventional IVF for patients with poor-quality oocytes, which is assessed mainly by morphological features. To our knowledge, there is no published study assessing whether the use of ICSI actually improves the pregnancy outcome in morphologically abnormal oocytes. Poor Responders ICSI is commonly used in poor responders to ovarian stimulation with the idea of improving fertilization rates in the few oocytes that are available for fertilization. One prospective trial randomized 96 non male infertility cases with six or fewer retrieved oocytes to conventional IVF and ICSI. 38 ICSI and conventional IVF resulted in similar fertilization rates per retrieved oocyte (56.5 vs. 58.8%, respectively). Furthermore, comparable quantity and quality of embryos, implantation rates, and pregnancy rates (17.3 vs. 21.1%, with ICSI and conventional IVF, respectively) were obtained. Few retrospective reports also confirmed that ICSI did not improve outcomes in patients with poor response to stimulation when semen parameters were normal As it was concluded in recently published committee opinion of the ASRM, based on current evidence, the use of ICSI for low oocyte yield does not significantly improve fertilization rates, embryo number and quality, or pregnancy rates. 31 Prevention of Triploidy The incidence of triploidy in human embryos after conventional IVF has been reported as anywhere from 2 to 10% with dispermy, that is, the fertilization of a haploid oocyte with two haploid sperm, being the most common cause. 42 A retrospective analysis of 95 couples with 20% incidence of tripronucleated (3PN) zygotes in their initial conventional IVF cycles followed by the use of ICSI in a subsequent attempt showed that after ICSI, the rate of two pronucleated (2PN) zygotes was higher (65 vs. 34.1%) and the rate of 3PN ones was lower (5.0 vs. 33.9%) than the prior conventional IVF procedures. Eventually, more 2PN embryos were obtained with ICSI as compared with conventional IVF ( vs [mean SD], respectively). There was no difference in embryo quality between ICSI and conventional IVF in this study. 43 This study did not use any additional genetic methods to assess if the 3PN embryos observed were in fact triploid or not. Surprisingly, one published report claimed that the percentage of multipronucleated zygote formation may in fact be a good prognostic sign for conception because significantly higher 2PN fertilization and improved pregnancy rates were found in these cases. 44 Of note, the embryos developed from multinucleated zygotes were deemed to be untransferrable. A possible explanation for this correlation could be that the multipronucleated zygote formation was associated with higher number of retrieved oocytes and better sperm motility parameters after swim-up. Although it might be possible to prevent polyploidy caused by dispermy utilizing ICSI, the development of multinucleated zygotes can also be caused by oocyte-related factors such as the fertilization of a diploid oocyte by the haploid sperm, suggesting that ICSI may not prevent all cases of polyploidy. Although it is possible to decrease the number of ARTassociated polyploidy cases utilizing ICSI, further studies are needed to assess if it is actually beneficial in terms of achieving more healthy live births. Prevention of Male-to-Female HIV and HCV Transmission ICSI has been suggested to be instrumental for prevention of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) transmission from infected men in serodiscordant couples based on the idea that using single spermatozoon per inseminated oocyte would be safer. 45,46 Contrary to this idea, the Dutch Society of Obstetrics and Gynecology recommends against using ICSI in HIV serodiscordant couples, justifying it with the concern that the injection of a spermatozoon that might carry an HIV-1 particle, directly into the oocyte may lead to incorporation of the viral genetic material into the future embryo with unknown but potentially disastrous consequences. 47 To our knowledge, there are no welldesigned studies to address this issue. Since most serodiscordant couples are not infertile, ICSI may not be used routinely. Although being much cheaper and less invasive than ICSI, sperm gradient separations in combination with IUI have been used for prevention of male-to-female viral transmission with more than 20 years of clinical experience. 48 The efficacy of gradient techniques to reduce viral loads in sperm preparations is well established, and it may be improved by incorporation of polymerase chain reaction based techniques for viral detection. 48,49 It is important to note that the decrease in viral load does not translate into complete prevention of viral transmission. Nevertheless, in the absence of strong supporting evidence, there is no consensus about the relative merits of ICSI to prevent a horizontal transmission of HIV and HCV from male-to-female partner. 50 Finding the right evidence is especially challenging in HCV transmission research, since it was shown to be a rare event. 51

5 Intracytoplasmic Sperm Injection Indications Babayev et al. 287 Preimplantation Genetic Testing Irrespective of what approach is taken for preimplantation genetic testing (PGT), accurate diagnosis is of utmost importance. Although the testing errors can arise from many different problems in a PGT procedure, the most common and worrisome ones have been quoted to be the issues related to sample contamination. Utilizing ICSI rather than the conventional IVF in PGT cycles is recommended to prevent paternal contamination from excess sperm introduced into the zona pellucida, or maternal contamination from granulosa cells especially with PGT performed for single gene defects. 52 Although literature is lacking well-designed trials, in light of biological plausibility, ICSI was recommended for PGT when DNA amplification techniques are used. But again by using more advanced technologies in proper identification of maternal and paternal DNA as well as the embryonic DNA, these concerns can be relieved in the near future. 53,54 Oocytes Matured In Vitro For in vitro maturation (IVM) cases, due to the potential changes in the characteristics of the oocytes and zona pellucida potentially caused by prolonged exposure of immature oocytes to the culture media, ICSI has been advocated as the preferred method of fertilization. The outcomes from one randomized trial suggest that in the presence of normal semen parameters, conventional IVF would be a valid fertilization technique for oocytes retrieved from women with polycystic ovary syndrome and matured in vitro. 55 The lack of a significant difference in fertilization rates between the ICSI and conventional IVF groups in this study indicates that IVF can be used to achieve acceptable fertilization rates after IVM. The similarity in cleavage and blastocyst development rates suggests that the oocytes inseminated by conventional IVF are just as capable of producing viable embryos as ICSI fertilized oocytes. Although in another retrospective analysis, fertilization rates appear to be increased by utilizing ICSI after IVM, the better fertilization rates did not translate into better outcomes. In this study, the fertilization rates in oocytes subjected to IVM were 37.7% (229/608) with conventional IVF as compared with 69.3% (318/459) when ICSI was used. Despite the lower fertilization rate, the implantation rate was significantly higher in embryos derived from oocytes fertilized with conventional IVF as compared with ICSI (24.2 vs. 14.8%; p < 0.05). Furthermore, the clinical pregnancy rate per ET was significantly higher with conventional IVF as well (34.5 vs. 20.0%; p < 0.05). 56 With the improvements in IVM culture conditions, conventional IVF can achieve viable embryos with improved implantation and pregnancy rates while being a more costeffective and less invasive technique. Oocyte Cryopreservation and Intracytoplasmic Sperm Injection ICSI has currently been adopted as a method for fertilization of cryopreserved oocytes. It is thought that the removal of cumulus cells and the cryopreservation process itself might decrease the chances of conventional IVF to fertilize the oocyte after it is thawed. Evidence examining this hypothesis is conflicting with some studies reporting comparable fertilization rates in cryopreserved oocytes between conventional IVF and ICSI and yet others proclaiming ICSI as the superior method in that respect Again, there is lack of evidence from prospective randomized studies to suggest that ICSI is superior to conventional IVF, although ICSI has been the method of choice in majority of the clinical studies on cryopreserved oocytes. 60 Risks of Intracytoplasmic Sperm Injection One must be aware of the differences between the fertilization processes with conventional IVF and ICSI. In biological terms, ICSI is quite an invasive procedure. ICSI bypasses the natural selection barriers of the oocyte with a potential of introducing a genetically defective material. Moreover, ICSI also involves the immobilization of spermatozoon before injection, which is commonly achieved by crushing its tail with subsequent exposure of its internals to potentially damaging medium components. Another important difference between ICSI and conventional IVF is that during ICSI, the entire spermatozoon is introduced into the ooplasm including the acrosome with its hydrolyzing enzymes, sperm plasma membrane, tail, and mitochondria, whereas these components are largely avoided during conventional IVF and the oocyte will not require extra energy and material for their intracytoplasmic elimination. 61 We may not rule out the potentially harmful effects of acrosomal enzymes on embryo development with injection of acrosome-intact spermatozoa as well. It seems that ICSI is more energy consuming for the oocyte itself than conventional IVF, which may have implications for its routine application for oocytes retrieved from women with advanced reproductive age. 61 Pregnancies achieved by ART are considered as high-risk pregnancies with increased risks of hypertensive disorders, preeclampsia, intrauterine growth restriction, premature birth, and intrauterine fetal demise. 62 These risks are assumed to be the complications of multiple pregnancies associated with ART and also associated with other specifics of infertile couples and not the methods used for their treatment. Although, earlier reports did not show any significant increase in the congenital malformations after ICSI, the quality of these studies has been questioned Even in studies where ICSI has been shown to be associated with an increased risk of adverse outcome, this risk was attributed to the underlying male factors as the indications for the use of ICSI. Recent studies are indicating slight increase in the congenital malformation risk in children born after ICSI. One prospective, multicenter study from Germany included 3,372 children and fetuses beyond 16 weeks of gestation

6 288 Intracytoplasmic Sperm Injection Indications Babayev et al. conceived via ICSI, mainly indicated for severe male factor infertility and fertilization failure in prior IVF cycles. Compared with 8,016 children and fetuses conceived naturally, the RR for major malformation in ICSI cohort was 1.44 (95% CI, ). After adjustment for confounding factors, ICSI cohort still showed increased odds ratio (1.24; 95% CI, ). 69 With two-thirds of ART pregnancies in the United States achieved by ICSI, clearly this technique is used not only for male factor infertility. Unfortunately, there are no studies evaluating safety of ICSI in non male factor infertility and hence the literature still suggests that the perceived increase in birth defect rates with ICSI as compared with conventional IVF might be related to the genetic or epigenetic factors associated with severe male factor. 70 Conclusion In the field of ART, it is common to see widespread adoption of new technology with ever expanding indications but without sufficient evidence to support these uses unequivocally. The application of the new technologies tends to be empirical, and the available evidence may be largely ignored or dismissed in favor of the new intervention. ICSI is used as a robust method for achieving fertilization by many IVF centers around the world, whereas the reproductive specialists may also consider offering the most cost-effective and the least invasive techniques while maximizing the likelihood of a healthy live birth. The current scientific evidence does not support the widespread use of ICSI without well-established indications. There is paucity of good-quality data from prospective studies to further clarify the indications for ICSI. There is also lack of comparative studies between ICSI and conventional IVF reproducing the previously reported findings. Therefore, currently, the indications for ICSI are not rigorous and ICSI is applied for any perceived reason that conventional IVF may not work in a very subjective fashion, at times to convince the couples that most advanced techniques have been used in their particular case. ICSI indications need to be revisited by designing more prospective studies investigating meaningful clinical outcomes. References 1 Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340(8810): Van Steirteghem AC, Nagy Z, Joris H, et al. 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