Intracytoplasmic Sperm Injection and Other Micromanipulation Techniques for Assisted Reproduction

Microsurgical techniques have been used in studies of living cells for over a century, with the first documented microdissection of a protozoan dating back to 1835, some 200 years after the microscope became a useful tool for scientific investigations.^{1, 2} These techniques are usually performed on single cells or a small group of cells under a microscope to alter their structure or composition, and are collectively referred to as micromanipulation. In the past few decades micromanipulation techniques have aided many scientific advancements in numerous disciplines. For example, cloning and genetic engineering of animals for research were made possible by micromanipulation of mammalian eggs and embryos. In the 1980s, micromanipulation of human gametes and embryos began to improve the efficacy of assisted reproductive technologies (ART) to treat infertility, and has since become an integral part of ART. Micromanipulation is an indispensable tool in other medical procedures derived from ART, such as genetic testing of in vitro generated embryos. This chapter reviews established and new applications of micromanipulation techniques in ART, with an emphasis on our understanding of the benefits and risks of these techniques. More comprehensive, historical reviews of this subject can be found elsewhere.^{3, 4, 5, 6, 7}

In the conventional in vitro fertilization (IVF) procedure, oocytes are coincubated with motile sperm to achieve fertilization. This conventional method usually produces a fertilization rate averaging between 50% and 70%. In the case of infertility caused by a male factor, fertilization can be severely compromised, if it occurs at all. Improving fertilization for male infertility was probably the first application of micromanipulation in ART.

Human oocytes, similar to oocytes from most mammalian species, are covered by a transparent glycoprotein coat called the zona pellucida (ZP). In order to gain access to the oocyte plasma membrane (oolemma) for fertilization, a sperm has to be able to negotiate its way through this barrier. Microassisted fertilization techniques have been designed to circumvent this ZP barrier. The first technique reported for human IVF, known as zona drilling, involved “driling” a hole in the ZP by applying an acidic solution (e.g., Tyrode’s solution with pH adjusted to 2–3) to a localized area with a micropipette.⁸ Partial zona dissection (PZD) refers to a micromanipulation procedure whereby the ZP is sliced open by a microneedle.⁹ Zona opening can also be accomplished by laser ablation.¹⁰ An alternative to zona opening is to place sperm directly next to the oolemma in the perivitelline space using a micropipette, which is named SUZI (subzonal insemination).¹¹ The efficacy of these assisted fertilization techniques is compromised by two problems. First, not all sperm can effectively interact with the oolemma for the subsequent fertilization events to occur, even after they have bypassed the ZP barrier. Second, the block to polyspermic fertilization is not effective at the oolemma level in the human. Thus, multiple sperm could enter the oocyte resulting in polyploidy. Consequently, these techniques have produced relatively low and inconsistent fertilization results as treatment for male infertility. However, these techniques have the advantages of being relatively atraumatic to the oocyte and allowing, to some extent, natural selection of the fertilizing sperm.

The injection of a single sperm into the cytoplasm of the oocyte, or intracytoplasmic sperm injection (ICSI), provided a satisfactory solution to the problems of the assisted fertilization techniques developed earlier.¹² In this procedure, a single sperm is first immobilized by touching the sperm tail with an injection pipette (inner diameter 5–7 μm). The injection pipette picks up the immobilized sperm, pierces the ZP and oolemma, and delivers the sperm inside the oocyte cytoplasm (Fig. 1). In 1976 using hamsters as a model, Uehara and Yanagimachi¹³ were probably the first to report the injection of sperm into oocyte cytoplasm (ooplasm). It was later attempted on rabbit¹⁴ and human oocytes,¹⁵ although the first successful human pregnancy was not reported until 1992 by the Free University of Brussels’ group in Belgium.¹² During the natural fertilization process, the human sperm undergoes acrosome reaction on binding to ZP sperm receptor. The acrosome reaction enables the sperm to penetrate the ZP and to come into contact with the oolemma. The sperm cell membrane fuses with the oolemma and the sperm cell is incorporated into the ooplasm. The fusion activates the oocyte. Only after activation can an oocyte proceed to complete meiosis with the extrusion of the second polar body and allow sperm nucleus decondensation.¹⁶ In comparison with this natural process, ICSI does not require the sperm to complete the acrosome reaction. Fusion between sperm and oocyte membranes does not take place. Instead, the dislodging of the ooplasm by the ICSI pipette serves as a trigger for oocyte activation.^{17, 18, 19} The breakage of cell membrane on the sperm tail during immobilization facilitates the release of a putative oocyte-activation factor from the sperm that is also essential for activating the oocyte.^{20, 21}

Because ICSI is much more effective in fertilizing oocytes than other assisted fertilization techniques, it has become the ART of choice for male infertility.^{5, 22, 23} Indications for ICSI encompass most types of male infertility. Satisfactory fertilization can be achieved by ICSI when any of the following is diagnosed: low sperm count or motility, poor sperm morphology, presence of antisperm antibodies in the male (auto-) or female (allo-), or prior experience of failed or suboptimal fertilization with conventional IVF. In case of azoospermia or necrospermia, viable sperm can be extracted surgically from the epididymis or testis under many circumstances, and such surgically collected sperm can effectively fertilize oocytes with the aid of ICSI.^{24, 25, 26} Cryopreservation does not significantly reduce the fertilizing ability of surgically collected sperm,^{27, 28, 29} which negates having to schedule the urology surgery and oocyte retrieval on the same day. Although immature spermatozoa have been successfully used for fertilization by ICSI,³⁰ treatment outcome after using spermatids has been disappointing, largely because of the difficulty in correctly identifying round spermatids from somatic cells.^{31, 32} For semen sample with poor sperm motility, fertilization may be further enhanced by ICSI after treating the sperm with phosphodiesterase inhibitors, such as pentoxifylline.^{33, 34} 

The applications of ICSI have expanded beyond treating male infertility. ICSI can be used when suboptimal fertilization is anticipated because of the adverse effects of some in vitro conditions. For example, mammalian oocytes matured in vitro tend to develop a hardened ZP (resistance to proteinase digestion) that may hinder fertilization.^{35, 36} Optimal fertilization of in vitro matured oocytes has been obtained by ICSI.^{37, 38, 39} Similarly, cryopreservation also causes zona hardening,^{40, 41} and fertilization of the cryopreserved human oocytes can be optimized by the use of ICSI procedure.^{42, 43} The combination of ovarian tissue cryopreservation followed by in vitro oocyte maturation has become one of the potential avenues for fertility preservation prior to cancer therapy in women of reproductive age.⁴⁴ Fertilization of such oocytes is ensured by ICSI.⁴⁵ In the conventional IVF method, oocytes are coincubated with a suspension of 50,000–100,000 sperm for fertilization. In comparison, oocytes are exposed to individual sperm during ICSI. Thus, IVF by ICSI can greatly reduce the likelihood of transmitting microorganisms from the male to the female partner. Successful attempts have been reported for using ICSI as a procreation method to prevent transmission of human immune deficiency viruses between couples with discordant serotypes.^{46, 47} However, this practice has been cautioned against by the findings from rhesus monkey studies in which foreign DNA attached to sperm was introduced to oocytes via ICSI and the DNA was subsequently incorporated as functional genes into the embryo genome.^{48, 49} Taken together, these studies suggest that ICSI may minimize, but does not eliminate, the chance of transmitting infectious diseases between partners. The procedure may facilitate the introduction of foreign DNA and other pathogens to the oocytes, potentially leading to greater health complications. In some ART centers, the conventional IVF method has been replaced by ICSI not only for male infertility but also for infertility without an identified cause (unexplained infertility) in order to ensure that fertilization occurs. This is because fertilization usually fails to occur in 2–5% of IVF attempts even though all semen parameters are within normal ranges. The use of ICSI may prevent such complete failures.⁵⁰ However, fertilization failure may still occur even when ICSI is used.⁵¹ Therefore, taking into account the added expenses and the potential risks of the procedure, it remains debatable whether ICSI should be used exclusively in place of the conventional IVF method for all patients.⁵²

As ICSI becomes an established ART procedure, many concerns have been raised over its potential detrimental effects on the resultant embryos and children. One of the main concerns arises from the fact that ICSI bypasses the natural selection of sperm for fertilization. Sperm with defects that would have prevented them from fertilizing ooyctes may do so with the aid of ICSI and may pass the defects on to the next generation. Submicroscopic deletions (microdeletions) in the long arm of the Y chromosome have been found in approximately one fifth of men with azoospermia or severe oligospermia who would be prime candidates for ICSI.^{53, 54, 55} These microdeletions are probably de novo events and may be transmitted to the male offspring.⁵⁶ In some men the microdeletions are caused by mitotic errors postfertilization, resulting in mosaicism in the affected individual. For these men, microdeletions may not be transmitted to all male offspring.⁵⁷ Furthermore, because a direct cause relation has not been established between these chromosomal anomalies and fertility, it is uncertain whether the transmission of Y chromosome microdeletions will necessarily render all the male offspring infertile. Sperm from infertile men have a higher prevalence of aneuploidy than those from fertile controls.^{58, 59} This may explain, at least partially, the small increase in the incidence of chromosomal anomalies in ICSI-generated embryos.^{60, 61, 62}

Apart from facilitating the transmission of genetic defects, the process of sperm injection may inevitably cause physical damages to the oocyte and interfere with subsequent embryo development. In comparison with embryos from the conventional IVF method, ICSI-generated embryos have been found to be less likely to attain the blastocyst stage in vitro and more likely to develop fragments.^{63, 64, 65} However, such differences were not observed in other studies.^{66, 67, 68} The discrepancy may be partially the result of technical differences or operator variations, which are known to influence fertilization and embryo development.^{69, 70} Because the position of the second meiotic spindle is variable and is not always beneath the first polar body,^{71, 72} the insertion of ICSI pipette could damage the meiotic spindle even if the area adjacent to the first polar body is avoided. Wang and coworkers⁷³ reported a polarizing optical system that allows the visualization of the meiotic spindle without destroying the oocyte. Under the guidance of this system, damage to the spindle by the ICSI pipette can be avoided, thereby reducing the incidence of chromosomal anomalies or other cellular damages in ICSI-generated embryos. To prepare for the ICSI procedure, motile sperm are usually placed in a high-viscosity medium containing 10% (w/v) of a synthetic polymer, polyvinylpyrrolidone (PVP). A small amount of PVP is inevitably injected into the oocyte together with the sperm. To date, there is no evidence that PVP has any detrimental effect on oocytes or embryos. Nevertheless, the replacement of PVP with a natural material, hyaluronic acid, has been reported for preparing sperm for ICSI but its benefit remains to be confirmed.⁷⁴

Despite the observed and potential detrimental effects on the embryos at the genetic and cellular levels, ICSI has not been associated with increased incidence of birth defects.^{75, 76, 77, 78} Recently, public concerns over the safety of ART were raised again by a report showing a higher incidence of birth defects in IVF babies.⁷⁹ In that report, however, the incidence of birth defects was not further increased by ICSI. It is possible that the damage done by ICSI, if any, is restricted to early stages of embryo development. Embryos that survive this early stage do not have further disadvantages compared to embryos resulting from conventional IVF during their subsequent development. The limited information available suggests that ICSI children may have a small delay in mental development,^{80, 81} although it is unknown if this mental impairment is caused by the ICSI procedure or by factors inherent to the patients who require ICSI in the first place. Concerns were raised when imprinting disorders were found in a small number of children born from ICSI procedures.^{82, 83} However, closer examination of large numbers of children born after fertility treatments, including ICSI, dimissed any direct association between ICSI and the risk of imprinting defects.^{84, 85}

Obviously further studies of the long-term effects of ICSI on the offspring involving multiple centers with well-controlled study designs are needed to minimize confounding variables, such as operator/technical variations and population variations. Until conclusive data become available, patients should be counseled carefully before ICSI is offered as an ART treatment.

Embryo implantation has long been recognized as the bottleneck limiting the success of ART treatments. Unfortunately, little is known of the pathophysiology underlying implantation failures after ART. Scientific research in this area has been hampered not only by technical difficulties but also by ethical complications because scientific research would inevitably have to involve experimentation on human embryos. Despite the lack of knowledge of the fundamentals of embryo implantation, new technologies have been developed in the past decade to improve the implantation potential of in vitro-produced embryos.

Assisted hatching

Assisted hatching is a laboratory procedure developed to improve the implantation potential of in vitro-produced embryos by creating an opening in the ZP.^{86, 87} The ZP persists after fertilization until the embryo is ready to implant in the uterus. Under in vivo conditions, hatching is a misnomer for most mammalian species. The ZP of mammalian embryos is not cracked open from the inside by the embryo, rather, it is dissolved from outside by proteolytic agents in the uterine fluid.^{88, 89, 90, 91, 92} Only under in vitro conditions is the embryo observed to generate an opening in the ZP through which the embryo escapes, leaving behind an empty zona. If assisted hatching can benefit embryo implantation, it probably does so primarily by assisting the uterus, rather than the embryo, in removing the ZP. However, this is not to imply that the embryo has nothing to do with the zona loss in vivo. The loss of ZP is most likely a result of embryo-uterine interactions. For example, the embryo may secrete a protease activator that activates a latent protease in the uterine fluid, and the activated uterine protease then acts to digest the ZP.⁹³ Moreover, certain zona characteristics, such as thickness and resistance to enzyme digestion, have been found to correlate with embryo quality and implantation potential.^{94, 95, 96, 97}

Assisted hatching can be accomplished by the same methods as those developed initially for assisted fertilization, such as zona drilling, PZD, and zona ablation with laser.^{8, 9, 10} During the zona drilling procedure, the same micropipette that delivers the acid can be used to remove fragments from the embryo. The presence of fragments in IVF-generated human embryo is not uncommon and may interfere with cell-cell interactions within the embryo. Therefore, the removal of fragments can presumably further improve embryo development. When using acid for assisted hatching, it is important to avoid acid contact with embryonic cells. Once a hole is made, excessive acid solution near the hatched site should be removed by the hatching pipette and the embryo should be rinsed in fresh media immediately following the hatching procedure (Fig. 2). In PZD, cross slits have been shown to be more effective than the originally described two-dimensional slicing method.⁹⁸ To mimic more closely the in vivo events of zona loss, the ZP can be globally thinned by laser or completely dissolved enzymetically or chemically.^{99, 100, 101, 102} A study evaluated four different assisted hatching methods (zona drilling, PZD, laser ablation, and enzymatic thinning) and found that these different methods produced similar implantation and pregnancy rates.¹⁰³

Although assisted hatching has been practiced in many ART centers for some time, it remains inconclusive whether assisted hatching can improve ART treatment outcome. Some studies support the initial observation by Cohen and colleagues¹⁰⁴ that assisted hatching improves implantation and pregnancy rates;^{105, 106, 107, 108} while other studies disagree.^{109, 110, 111, 112} These conflicting observations are difficult to reconcile because of many important differences between the studies, such as study design, patient population, sample size, and assisted hatching techniques or skills. It is likely that assisted hatching only benefits a selective group of patients, provided that the procedure is performed correctly by an experienced operator. In the Practice Committee Report in 2000, the American Society for Reproductive Medicine suggests that “assisted hatching may be clinically useful” and recommends that “individual ART programs should evaluate their own patient populations in order to determine which subgroups may benefit from the procedure. The routine or universal performance of assisted hatching in the treatment of all IVF patients appears, at this point, to be unwarranted.”¹¹³ In spite of its uncertain benefits, assisted hatching has not been associated with impaired embryo development or major birth defects. Assisted hatching and assisted fertilization techniques have been implicated in increasing the incidence of monozygotic twinning.^{114, 116, 117} However, other studies attributed this problem to other aspects of ART, such as the gonadotropin treatment or the embryo culture conditions.^{118, 119, 120} Nevertheless, this phenomenon warrants further investigation because monozygotic twinning is more likely to result in miscarriage or other obstetrical complications.

Cytoplasm/germinal vesicle transfers

To remedy repeated implantation failures after ART treatments, another laboratory procedure has been described in which a small amount of cytoplasm is transferred from donor oocytes into the patients’ own oocytes.^{121, 122} The hypothesis underlying this procedure is that in some patients, certain defects in ooplasm may prevent the resultant embryo from normal development and implantation, and such defects may be corrected by infusion of cytoplasm from oocytes of fertile women. The importance of cytoplasmic factors in embryo development has been recognized for some time.¹²³ Under certain culture conditions, embryos from outbred mice cannot develop beyond the two-cell stage (two-cell block). This block could be overcome after the embryos were injected with cytoplasm from inbred mouse embryos without the two-cell block. Mitochondria have been implicated as one of the candidates for the cytoplasmic regulator(s) of embryo development.^{124, 125, 126} Although successful pregnancies have resulted from cytoplasm transfers, there are no controlled studies to verify that it can improve the developmental potential of IVF-generated embryos. One study found this procedure ineffective in improving pregnancy rates in patients with diminished ovarian reserve.¹²⁷ The caveat of this study, similar to many others evaluating the efficacy of ART, is that its study subjects were not randomized.

Based on the same hypothesis, an even bolder procedure has been proposed whereby the germinal vesicle from immature oocytes of infertile patients is removed and transplanted into enucleated oocyte from fertile donors.^{128, 129} To the best of our knowledge, no successful human pregnancy has been produced from this procedure. Both cytoplasm transfer and germinal vesicle transfer procedures can allow the patient’s genetic inheritance to be passed on to the offspring. However, clinical application of these techniques is not anticipated in the forseeable future because of ethical complications and safety concerns regarding these procedures.

The establishment of successful human pregnancies after preimplantation genetic diagnosis (PGD) was reported in 1990.¹³⁰ Preimplantation genetic diagnosis became possible as a result of the coexistence of expertise in ART techniques and techniques for single-cell genetic analysis. PGD was initially developed to assist those carriers of severe genetic disorders who wish to have a healthy child without having to terminate an affected pregnancy. Indications for PGD continue to expand.¹³¹ By the year 2002, more than 5000 PGD cycles had been performed worldwide, resulting in the births of over 1000 infants. In the latest report of the European Society of Human Reproduction and Embryology PGD Consortium of 39 centers for the year 2005 alone, 3488 cycles of PGD were performed with 670 babies born. However, less than one third of these PGD cycles were performed for patients with genetic disorders and most of the cycles were performed to screen embryos for aneuloidy in an attempt to improve implantation rates.

born because of this new technology. The complete sequencing of the human genome and availability of more sensitive instruments will further accelerate technological advancement of PGD.^{132, 133} It can be anticipated that as public awareness of the genetic information grows, so will the demand for PGD.^{134, 135}

Microbiopsy and embryologic work

Equipment and related facilities for biopsy are the same as for the use for ICSI and other embryo micromanipulative procedures. Methods for opening the ZP are the same as for assisted hatching, except that the opening may be slightly larger (approximately 30–40 μm). Strictly speaking, PGD can be performed either on oocytes prior to fertilization, also known as preconception diagnosis, or on embryos prior to the initiation of implantation. Procedures for polar body biopsy have been described by Verlinsky and colleagues.¹³⁶ One of the advantages of preconception diagnosis is that polar bodies are not involved in subsequent embryo development and their removal, therefore, would cause little harm on embryonic development. Preconception diagnosis is advantageous also because the oocyte is not regarded as an individual and, therefore, manipulation on oocytes is more acceptable from religious or ethical points of view. However, this procedure is cumbersome because it often requires sequential biopsies of the first and second polar body to avoid misdiagnosis because of crossing-over.¹³⁷ Furthermore, genetic disorders carried by the male partner cannot be detected by testing polar bodies of the oocyte.

Biopsy of cleavage-stage embryos is probably the most common approach for PGD, wherein one or two cells (blastomeres) are removed from the embryo (Fig. 3). Blastomere biopsy should preferably be done no later than day 3 postfertilization because embryos start to compact around that time.¹³⁸ The adhesion between blastomeres in a compacted embryo makes biopsy difficult. Compaction is mainly a function of cell-cell junctional proteins and is calcium/magnesium dependent.^{139, 140} By briefly exposing embryos to a calcium-magnesium-free medium, embryos can be decompacted, thus allowing the removal of one or two blastomeres without damaging the embryo.¹⁴¹ Decompaction can be reversed without harmful effect if the embryos are transferred back to the regular medium within 10 minutes. Embryo development is not significantly compromised by blastomere biopsy.¹⁴² Van de Velde and colleagues¹⁴³ evaluated 188 PGD cycles and found that embryo survival was not different after removing one or two blastomeres from embryos with seven or more cells. The removal of two blastomeres for genetic analysis is preferred because it will help to produce a more accurate and reliable diagnosis.¹⁴⁴ Frozen-thawed embryos, if they have survived well, can be used for biopsy. However, the combination of biopsy and cryopreservation can substantially reduce the embryo survival.^{145, 146} Therefore, “banking” embryos for future biopsy should be avoided.

In humans, the embryo usually reaches the blastocyst stage around day 5 postfertilization. Embryo culture media can now support the in vitro development of IVF-generated embryos to the blastocyst stage,¹⁴⁷ making blastocyst biopsy feasible. Blastocyst biopsy has the potential to provide more cells for genetic analysis. It appears to cause less damage to the embryo.^{148, 149} The removal of trophoblast cells (the peripheral cells surrounding the blastocele cavity) may cause the blastocyst to collapse but the embryo can usually recover after the opening of blastocele cavity is resealed. Two factors limit the use of blastocyst biopsy for PGD. First, blastocyst biopsy can only be performed on day 5, which leaves little time for genetic analysis before the embryo must be transferred to the uterus. Second, mosaicism in trophectoderm is common, which will make diagnosis inaccurate.¹⁵⁰ Blastocyst biopsy may become an option for PGD in the future when a fast and accurate genetic testing method emerges.

Chromosome analysis

For most chromosomal analyses, the biopsied cell must be properly processed to remove as much cytoplasm as possible. One fixation method was first described by Tarkowsky¹⁵¹ and later modified by Griffin and colleagues¹⁵² and Munne and colleagues.¹⁵³ In this method, the cell is swelled in a hypotonic solution and placed on a glass slide. The fixative (acetic acid and methanol) is then added drop by drop onto the cell. The impact of fixative drops helps to spread the cell and remove the cytoplasm. Another approach was reported by Coonen and colleagues¹⁴¹ and Harper and colleagues.¹⁵⁴ In this method, the removal of cytoplasm is accomplished by exposing the cell to a hypotonic denaturing solution containing a detergent. Xu and coworkers¹⁵⁵ compared the two methods, and found that both worked well with adequate training.¹⁵⁵ More recently, Dozortsev and McGinnis¹⁵⁶ suggested that a combination of the two methods works even better. Fixed nuclei are exposed to fluorescent-labeled DNA probes to allow the probes to hybridize with complimentary DNA sequences on the target chromosome, a process known as fluorescence in situ hybridization (FISH). Specific fluorescent signals can be visualized under a microscope with filters of appropriate wavelength. Up to nine chromosomes, for example, X, Y, 13, 14, 16, 18, 21, 22, can now be tested after two sequential FISH analyses in a single cell (Fig. 4).^{157, 158} Detection and elimination of chromosome aberrations in preimplantation-stage embryos first became possible in the early 1990s with the advent of the fast, sensitive, and accurate FISH technology¹⁵² and the availability of directly labeled probes. Indeed, FISH may be the easiest and most accurate way of determining the gender of an embryo, and it has the advantage of including additional DNA probes to screen other chromosomes.

Aneuploidy occurs in 10–30% of human conceptuses, mostly as a result of errors occurring during female meiosis. Aneuploidy accounts for at least one third of miscarriages and is the leading genetic cause of developmental disabilities and mental retardation.¹⁵⁹ The frequency of aneuploidy in embryos increases with maternal age. An embryo’s morphologic appearance does not always reflect its genetic makeup because embryos with various kinds of aneuploidy and unbalanced chromosomes could well survive beyond the blastocyst stage in vitro.^{160, 161} Therefore, it seems desirable to screen for aneuploidy in preimplantation embryos so that only embryos with euploidy will be used to establish a pregnancy. Because aneuploidy is mainly attributed to female meiotic errors, screening polar bodies is a reasonable option.^{162, 163} The effectiveness of aneuploidy screening has been confounded with several difficulties. First, most candidates for aneuploidy screening often suffer diminished ovarian reserve because of advanced age. The decline in ovarian reserve begins in a woman in her late 20s.¹⁶⁴ Consequently, these women do not respond well to ovarian stimulation, and often produce a limited number of oocytes and embryos for aneuploidy screening. Second, not all the cells in the early cleavage-stage embryo are carrying exact the same number of chromosomes. It is estimated that up to 40% of human embryos are mosaic,¹⁶⁵ which will adversely affect the accuracy of PGD. Current aneuploidy screening methods also suffer from the limited number of chromosomes that can be examined on a single cell. Polymerase chain reaction (PCR)-based aneuploidy analyses, such as multiplex, fluorescent polymerase chain reaction (MF-PCR),¹⁶⁶ and comparative genome hybridization (CGH)^{167, 168} can potentially screen all chromosomes in a single cell. However, their complexity and the lengthy time required to complete a test severely limit their routine use for PGD.^{169, 170}

A retrospective, multicenter report indicates that preimplantation screening for aneuploidy can reduce the incidence of miscarriage but does not increase pregnancy rates after ART treatments.¹⁷¹ This finding was later confirmed in our randomized, controlled clinical trial.¹⁷² Taken together, screening for aneuploidy in preimplantation embryos may be beneficial under certain circumstances, but the effectiveness does not appear to justify its routine use for all patients. The decision for doing aneuploidy analysis, indeed, is a complex one. A risk-benefit evaluation should be performed to take into consideration multiple factors, such as the woman’s age, previous fertility history, response to stimulation, number and quality of embryos available for analysis, patient attitude toward miscarriages and abortion, etc. Perhaps, the single most important factor seems to be whether or not there is a selective advantage, that is, whether the number of embryos available for transfer exceeds the number of embryos acceptable for transfer without risking a higher-order multiple gestation. Aneuploidy screening may be recommended to those patients who have previous IVF failures with morphologically normal embryos, or those who have miscarriages because of known chromosomal anomalies.

In addition to aneuploidy, structural anomalies in the chromosome can also be detected by PGD. Structural chromosomal rearrangement may affect human reproduction. Schreurs and associates¹⁷³ found that the prevalence of autosomal reciprocal balanced translocation was seven times higher in patients (1.14%) who cannot establish or maintain pregnancies than in the general population (0.16%). Prolonged culture of embryos to the blastocyst stage may be selective against embryos with balanced translocations because embryos carrying this type of translocations cannot develop normally in vitro.^{160, 174} However, blastocyst culture will not eliminate embryos with unbalanced translocations or other anomalies. First reports of PGD for chromosome translocation were published in the late 1990s.^{162, 175} Several strategies for chromosomal rearrangements have been attempted. Polar body biopsy and fluorescent whole-chromosome painting probes allow a direct assessment of chromosomal structure, thereby predicting the normalcy of the embryos. However, polar body chromosomes may easily clump together or degenerate, and thus not all polar bodies are informative. It is not an option if the male parent carries a chromosomal rearrangement. For the detection of chromosomal rearrangement, various types of DNA probes have been used. Breakpoint specific probes may provide the most informative diagnostic value,¹⁷⁶ but these probes have to be made for each individual patient because they are carrier-specific. Therefore, it is less practical as the cost alone will prevent its wide application. Another more sophisticated approach is the so-called interphase-metaphase conversion, and its clinical application has been reported.^{177, 178} Telomeric probes are made from the DNA sequences located at the tip of each chromosome arm. Such probes have been used successfully for PGD.^{179, 180, 181} If the breakpoints are so close to the tip, for example, in the case of cryptic translocation, the only useful probes will be the telomeric ones. The commercial availability of telomeric probes for all human chromosomes has provided a convenient source of testing reagents. One of the limitations of telomeric probes is that they will not distinguish the balanced status from the normal ones.

Once the precise diagnosis of a genetic disorder is made at the DNA level, it can be detected by PGD techniques. It is estimated that PGD attempts for more than 50 autosomal recessive conditions and autosomal dominant disorders have been reported from various PGD centers around the world, including cystic fibrosis,¹⁸² Huntington’s disease,¹⁸³ sickle cell anemia,¹⁸⁴ and early-onset Alzheimer disease,¹⁸⁵ to name a few. Reports from two international working groups provide a glimpse of the activities in the area,^{134, 135} but they are quickly outdated. Knowledge of human genetic diseases has been accumulated astonishingly fast in the last several years as the Human Genome Project nears completion and this trend will certainly continue in the coming decade.^{132, 133}

Compared to established prenatal diagnosis procedures, PGD has additional technical challenges because only a minute amount of DNA (approximately 6 pg of DNA per cell) is available for analysis. The advent of PCR technology made single-cell DNA analysis possible.¹⁸⁶ Unlike chromosomal analysis, biopsied cells are simply placed individually into small test tubes for DNA analysis by PCR. Usually, a nested PCR (two sets of DNA primers and two rounds of PCR) is preferred in most centers because it will increase sensitivity. Although single-cell PCR usually is efficient, as high as 18% of the cells might not produce amplification signals.¹⁸⁷ If multiple mutation sites are involved in a disorder, either a multiplex PCR or whole-genome amplification can be applied so that multiple loci can be analyzed simultaneously.^{188, 189, 190} One of the major challenges of this procedure is to prevent foreign DNA contamination, which can produce in a false result. Allele dropout (ADO) in PCR is another common cause for false results. In a situation of compound mutations, or when an autosomal dominant mutation is involved, ADO could lead to misdiagnosis with an affected pregnancy. In a very recent report on PCR for γ-globin gene, 8.6% of ADO was detected.¹⁹¹ To reduce the risk of ADO, linkage markers should be included in the PCR.¹⁴⁴

After DNA amplification with PCR, a classic and effective method to detect mutation is restriction enzyme analysis. Because many alterations in DNA sequence result in loss or gain of a restriction site, DNA from homozygous normal, heterozygous carrier or homozygous affected embryos can be easily distinguished after restriction digestion and electrophoresis of the PCR amplification product (Fig. 5). Alternatively, PCR primers can be designed so that embryonic DNA can be amplified selectively for or against the mutated sequence, and the presence or absence of the amplification product will indicate whether the sample is from an affected embryo. This approach is particularly useful for the detection of point mutations that do not affect a restriction site. A variety of other new modifications of PCR methods have been introduced. Fluorescent PCR is sensitive and has become popular. It can be used for detecting deletions (e.g., CF ΔF508 mutation) and insertions (for example TSD 1278ins4 mutation), replacing an old method called heteroduplex formation (Fig. 6).¹⁹² Fluorescent PCR also has been used for dealing with complex systems involving many loci.¹⁶⁵ Real-time PCR refers to PCR systems that allow continuous measurement of the accumulation of a specific amplification product, and is useful in PGD because the electrophoretic analysis of PCR product is eliminated.¹⁹³ Liquid chromatography can be used to sequence PCR product directly, which is particularly advantageous when the mutation does not involve a gain or loss of a restriction site.¹⁹⁴

The list of indications for PGD has been grown rapidly¹³¹ and is no longer restricted to lethal genetic disorders. Mutations that predispose patients to certain diseases, such as cancer, have been screened for by PGD, even though a direct causal-effect relation has not been established between the mutations and the disease.^{195, 196} In one case, a healthy infant was born after a successful PGD for retinoblastoma (K. Xu and colleagues, April 2002, unpublished data). PGD for Alhzeimer’s disease is another indication that has been successfully carried out.¹⁸⁵ Verlinsky and coworkers¹⁹⁷ reported the first PGD case for avoiding Fanconi’s anemia, while at the same time selecting a HLA matching sibling donor for stem cell transplantation. This development is beneficial to many affected families, but at the same time has caused public debate. One cannot expect that there will be a uniform acceptance by the society for this type of new technology. However, it is certain that more couples will benefit from PGD in the future.

Micromanipulation techniques were originally developed to facilitate scientific investigations. Scientific breakthroughs have been made, and continue to be made with the aid of micromanipulation technologies. Some of the new technologies may lead to cures for many human diseases. However, whether they will have a place in future ART is dependent not only on further technical improvement but also on public acceptance. Medical professionals should be keenly aware of their professional as well as social and ethical responsibilities in the pursuit of technical advancement.

Cloning

Cloning is making a copy of an individual. The simplest approach is to split a cleavage-stage embryo and allow individual cells to grow into individual embryos. A sheep embryo was split and the two resulting halves of the embryo were transferred and gave rise to two identical sheep.¹⁹⁸ Cloning can also be accomplished by transplanting the nucleus from a somatic cell into an enucleated oocyte, also known as somatic cell nuclear transfer (SCNT). The famous sheep, Dolly, was born as a result of SCNT.¹⁹⁹ Cloning by SCNT offers great potential for scientific investigations of cell biology. It serves as an indispensable model for studying cytoplasm-nuclear interactions. Therapeutic cloning refers to SCNT technologies that can generate cells, tissues, or even organs to cure diseases. Using SCNT for reproductive purposes is not acceptable ethically by the majority of the public and is not safe, given the observations of many health problems in the cloned animals.²⁰⁰

Stem cell technology

Until a certain stage in development (day 3.5 in mice), embryonic cells are considered totipotent, that is, they have the potential to differentiate into any cell type found in the body. Such cells can be genetically engineered and used to generate tissues or organs with new genetic characteristics.^{201, 202} These cells can also be injected into a blastocyst and the resulting offspring could have cells or tissues derived from the genetically engineered stem cells. If the germ cells are derived from these cells, the unique genetic trait will become inheritable. Thus, the injection of stem cells with a specifically mutated gene can create a line of animals lacking the function of the gene, a technology called targeted mutagenesis or gene knockout.²⁰³ This technology has produced many animal models for studying various human diseases, ranging from cancer, to neurologic and immunologic disorders.^{204, 205, 206} Stem cells have been generated from human preimplantation embryos,²⁰⁷ paving the way for medical applications of the stem cell technologies. However, the generation of stem cells requires the destruction of live embryos, to which many in society strongly object on ethical or religious grounds.

Gene therapy

Foreign genes can be injected into the pronuclei of a newly fertilized embryo (zygote), and the injected genes can be incorporated into the embryo’s genome and be expressed.²⁰⁸ This technology is referred to as transgenesis. The generation of transgenic embryos or live animals provides a useful model system for studying an individual gene’s function and interaction with other genes. A specific trait of medical or economic value can be introduced to animals by transgenic technology. For example, a transgenic goat has been created to produce human plasminogen activator in its milk, which is a convenient source for isolating the therapeutic agent for treating stroke and heart attacks.²⁰⁹ However, the insertion of a foreign gene is poorly controlled, and as a result, the injected genes are not always functional or the insertion of the gene could interrupt the normal function of host genome. Until these problems are resolved, no attempt of transgenic technology should be made in humans.

Since the birth of the first IVF baby more than 30 years ago, ART has quickly become one of the most dynamic disciplines of medicine. Micromanipulation techniques have been, and will continue to be, an important catalyst for the rapid advancement in this area. Further development in this area will be dependent on improved understanding of human reproduction. More studies are also needed to evaluate the long-term benefits and risks of the current technologies.

We would like to thank Randall Barnes, MD, and Karen Horan, MSc, for their comments on the manuscript. We also wish to thank our respective colleagues at Northwestern University and Weil Cornell Medical College IVF programs for their assistance and support during the preparation of this manuscript.