The Heart and Soul of ART……is in the Laboratory The Inside Story Prof Prakash Trivedi, Priti P Trivedi
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1The Art of Art: Perspectives2

ART: Embryologist's ViewpointCHAPTER 1

Jayant G Mehta
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INTRODUCTION
Just like me, most clinical embryologist worldwide had to adapt and contribute to complex embryological methods introduced into assisted conception post in vitro fertilization (IVF),1,2 period. Increased knowledge about the embryo, and the use of micromanipulation has enabled oocytes and embryos to be surgically examined and modified. Third party reproduction has been introduced and in more extreme cases using assisted reproductive techniques (ART), devastating genetic disorders have been avoided for couples using genetic techniques such as Preimplantation Genetic Diagnosis (PGD). Although in the early days of ART the success rates were low, today clinics report up to 50 percent clinical pregnancy per transfer. This increase success has come through a combination of clinical and laboratory improvements, which include improved drug regimes for ovarian stimulation, as well as simplified and more accurate methods of predicting follicle and oocyte maturity and oocyte harvesting. In the laboratory, the improvements have been primarily at the level of media, allowing development of embryos in vitro and selection at a more advanced stage.313 The purpose of this chapter is to discuss how an embryologist has incorporated numerous technical developments in the laboratory protocols to integrate various infertility treatment alternatives available for both women and men. A brief discussion on part and emerging ethical issues perceived by the embryologist is also presented.4
 
IN VITRO FERTILIZATION AND ITS ASSOCIATED TREATMENTS
 
In Vitro Fertilization
The last two decades has seen the emergence of IVF as the standard treatment for irreparable tubal diseases, failed IUI, male factor infertility, endometriosis, cervical and immunological factors as well as unexplained infertility unresponsive to IUI. The steps involved in IVF treatment include ovulation induction, the monitoring of the follicle and endometrial development, oocyte harvesting, in vitro fertilization of a mature ovum by a spermatozoa and embryo transfer is carried out by highly skilled embryologist using state-of-the-art equipment in most appropriate laboratory environment have cultured these oocytes and embryos.
 
Metaphase II Stage Oocyte
Numerous reports have been made relating the morphological appearance of the oocyte to treatment outcome. Embryologists use the quantity and expansion of the cumulus cells as an indicator of maturity, but this is inexact and may depend on the type of ovarian stimulation used. In routine IVF it is difficult to assess oocytes as the cumulus cells are left intact. However, during ICSI procedures, the cumulus cells are stripped of, allowing visualization of the oocyte. Assessment of the oocytes by the embryologists has resulted in a body of literature indicating that parameters such as the presence of vacuoles, cytoplasmic pitting and particles in the perivitelline space are associated with decreased pregnancy outcome.
During maturation of the oocyte, the metaphase spindle is formed, meiosis proceeds, and the polar body is extruded. Formation of the first polar body results from a highly asynchronous cell division at the end of the first meiotic division. The meiotic spindle forms near the center of the oocyte and then tracks along its long axis to the oocyte periphery. This process is controlled by c-mos (cellular viral mos oncogene) and MAPK pathways.14 The tracking of the spindle to the periphery ensures that very little cytoplasm is lost when the first reduction division occurs, since the first polar body plays no further role in development.15 After extrusion, the polar body – which is complete by about 40 hr after hCG – undergoes timed, programmed disintegration, which is again controlled by c-mos and MAPK.14 This is a natural phenomenon, and in the human oocyte is complete by approximately 20 hr after extrusion.16 This could be a strategy to prevent sperm binding and entry to the polar body, as the chromosome complement in the polar body is normal. In fact, the first polar body can be successfully used in nuclear transfer experiments to form viable offspring.17 Any oocytes presenting with very large first polar bodies are most likely abnormal, at the level of c-mos and MAPK expression or function. As this also directly relates to the spindle, where sister chromatid exchange occurs and is the most likely point of non-disjunction or aneuploidy formation, abnormal polar body size and appearance can indicate oocyte quality.
A further selection criterion for that is being used in the laboratory for oocytes grading is the morphology of the polar body that can be visualized, especially if it is in the state of disintegration. As it is initially intact and begins to disintegrate with time, a fragmenting polar body at the time of ICSI (40-42 hr post-hCG) could indicate an early or abnormal extrusion of the polar body or a disruption of the c-mos-MAPK pathways, either of which could indicate aneuploidy or subsequent abnormal development.
Few reports have been made which link first polar body abnormalities (primarily the appearance of fragmentation or polar body disintegration) with lowered fertilization and reduced day 3 and/or day 5 development and implantation.1819 Thus, the state or morphology of the first polar body can provide a good insight to the molecular events that have occurred in its formation and which are very tightly linked with spindle formation and the most likely point of aneuploidy formation. This is a simple, non-invasive screening technique, which is easily applied in ICSI cases but not in IVF. Further oocyte screening tools are needed for IVF oocytes.
A newer technique available to embryologist is to screen metaphase II oocytes by visualization of the 5metaphase spindle. This has been accomplished non-invasively through a form of differential polarized light microscopy, all the light generally passes through an object and is gathered by the condenser such that an image is seen. Light passing through an object travels at different speeds depending on the path it takes, or how much it is scattered. Light passing down an ordered structure will travel at a very different speed to that passing through a disorganized or chaotic structure. The difference in light speeds passing through an object produces birefringence, which is quantified using a polarizing microscope, to produce an image of the ordered structure20. Polarizing light microscopy makes use of the ordered form of the spindle. As it is merely light microscopy but with the light passing through the embryo being gathered differently, it is no more invasive than routine microscopy.
Spindles are formed from microtubules, which arrange in an array with an attachment site at each end (bipolar). Chromosomes attach to this array, through kinetochores, to short microtubules in the equatorial region of the long array. This attachment and alignment is controlled by checkpoints, which essentially prevent incomplete or incorrect events (such as faulty chromosome attachments) occurring in the cell21. If there is damage or incorrect alignment or assembly of the spindle, then checkpoints will prevent further progression of meiosis until the correct spindle architecture is obtained.22 Disruptions to the checkpoint mechanism could allow meiosis to proceed with incorrect or incomplete spindle assembly. This ordered array of the spindle (and potential disruptions to it) could be detected using polarized microscopy. Non-bipolar or abnormal spindles can be distinguished from normal bipolar ones. If the spindle array is completely disorganized or disrupted, then the birefringent image will not be seen.
When polarizing light microscopy is applied clinically, oocytes without a birefringent spindle have lowered fertilization and slower rates of development in vitro.2324 It has also been shown that oocytes without birefringent spindles survive freeze-thawing less well than those with spindles, and that much of the damage that occurs in oocytes during freezing occurs at the spindle level. However, oocytes with birefringent spindle can progress, even to the blastocyst stage,23 but will have little to no developmental capacity. Thus, observation of the spindle structure by these non-invasive techniques could provide an early selection point against embryos with little developmental capability, even if they form morphologically normal blastocyts.
 
Embryo Transfers
The soaring technological advances of microscopes, laboratory equipment and culture media have allowed the pregnancy rates to rise from 4 percent upto 50 percent per attempt in the past several years. However, the reports of high clinical pregnancy rates has come at an extreme cost to the patient and society, in that these are achieved by the use of multiple embryo transfer, leading to a worldwide escalating incidence of both twin and high order multiple pregnancies.25 As a result some countries have introduced mandates while others have issued guidelines and recommendations for reductions. In developing third world countries like India, it may not be possible to implement these guidelines, as 90 percent of patients attending ART programmes may only have single opportunity at this very expensive treatment, and would want to capitalize on their chances of success being greater with multiple embryo transfer.
Further, clinical evidence accumulated over two decades of human IVF and embryo transfer26 shows that between 25 and 50 percent of the cytoplasmic volume of an early embryo must be represented in normal cells as a condition for full embryo viability. However, a substantial proposition of embryos produced in the course of routine IVF show abnormal cleavage and lose cells and cytoplasmic volume through cell fragmentation, degeneration and mitotic arrest. Extensive multinuclear cytogenetic studies have demonstrated a high frequency of chromosomal anomalies in such embryos.27 As a consequence roughly 60 percent of human embryos do no meet basic viability criteria and in most clinics they are 6simply discarded at the end of treatment cycles.28 Such embryos often do contain one or more surviving, apparently normal blastomeres. Two key observations of: (i) a proportion of individual cells from highly fragmented embryos undergo division and cavitation when cultured in isolation, and (ii) simple removal of fragments from some fragmented embryos leads to reorganization of the intact blastomere and facilities compaction,28 require a wider investigation to assess the implications of abnormal cleavage and degenerative changes that occur in early developing human embryos. Embryologist today faces a challenge to select one embryo with maximum implantation potential, truly a very difficult task.
Embryos are primarily selected on morphological and development criteria. These have included pronuclear oocyte morphology29,30 and the early entry into the first mitotic division.31 As embryos cleave, the form or evenness of cell division32 the fragmenting patterns and the rate of development33 have an impact on implantation. Embryos are on a continuum of development from the time of initiation of oocyte growth in the primordial follicle through implantation and fetal development. Many of the methods of selection rely on a static observation of the embryos, where as the embryo itself, after fertilization, is on a very strict set of clocks governing division and initiation of key events from gene activation through compaction and blastulation.34 This would imply that to evaluate an embryo's potential reliably would need a sequence of reference points as it makes the necessary transitions from oocyte, through fertilization, initiation of RNA synthesis, switch from maternal to embryonic genome and the initiation of growth through protein synthesis and differentiation.35
 
Embryo Polarity
Recent studies36 indicate that traditional ideas on the onset of polarity during early mammalian development need to be reconsidered. As with lower-order animals, mammalian embryos have been shown to have polarity and ordered directional development, from fertilization through compaction to the blastocyst stage. Although blastomeres from early embryos are said to be totipotent, and desegregated cells can form small blastocyst structures in vitro, they have little to no developmental potential. Increasing evidence is pointing to a very ordered and polarized progression from the oocyte to the fetus in mammalian development.36 In humans, it has been suggested that at the 4-cell stage, only one of the blastomeres continues to develop into the clone of cells that produces hCG.37
This would imply that the removal of cells from the early embryo could disrupt this order and even result in an embryo with decreased implantation potential due to the removal of a vital functional part. It has long been evident that mammalian embryos have plasticity in that they can develop after losing a few cells, but there must be a limit to how many cells they can lose without compromising viability. The later the cells are removed, the less likely it will cause harm. However, the more cells the embryo has, the more the chances of selecting a normal versus abnormal cell in aneuploid mosaic cases.
 
Blastocyst Transfers
The recent availability of commercially manufactured sequential media and blastocyst culture media has enabled the cleavage of embryos to the blastocyst stage in essentially all clinical programs, permitting the selection of higher quality embryos and implantation rates, even though the first blastocyst, transfer resulting in a clinical pregnancy was reported in 1976.1 By transferring fewer embryos of superior quality and considering day 3 versus day 5 transfers, an attempt has been made at reducing multiple pregnancy rates worldwide. On the other hand, about half of the fertilized eggs fail to produce blastocyst; as a result it is possible that some patients will not have viable embryos available for transfer. For these reasons and because it is a new technology the full value of blastocyst culture has not been fully determined. Although its potential value in reducing multiple gestation in women at risk is nearly certain, although, there are not enough satisfactory data on the resulting cryopreservation at the blastocyst stage.7
Blastocysts formation and morphology have also been related to implantation. The use of blastocyst transfers has been proposed as a means of selecting viable embryos that had made all of these transitions, leading to increased implantation potential.38 The clinical use of blastocyst transfer has indeed shown higher implantation rates compared with day 3 results in selected groups of patients,39 including older patients.40 In these studies, only patients with an adequate cohort of embryos with good day 1, 2 or 3 morphology were allowed to proceed to day 5 transfers. There are few controlled randomized studies comparing day 3 and 5 transfers and these show that, in good prognosis patients, the pregnancy rates are equivalent and that what differs is the implantation rate.41 This is a start of solving the problem. However, the reported rates of blastocyst formation in vitro, using the culture systems that are currently available, range from 20 to 50 percent from both stimulated and unstimulated cycles. This means that there is great attrition from day 3 to 5 of embryos that may appear morphologically normal on day 3. There are no data available to show that these embryos could not implant if transferred at an earlier stage.
 
GAMETE INTRAFALLOPIAN TRANSFER (GIFT)
Prior to the undesired use of ICSI and due to inferior in vitro culture media systems (1984-1994), GIFT enjoyed popularity among infertility specialists worldwide. The relative merits of intratubal transfer of gametes, such as fertilization and embryo development in the natural tubal microenvironment, avoid possible hazards of the in vitro culture, especially if optimal laboratory conditions are not available. Whether endometrial trauma caused by the intrauterine embryo replacement is avoided is still under debate.
GIFT has been employed for the treatment of failed IUI attempts, previous difficulties with transcervical embryo transfers, minimal and mild endometriosis, adhesions amenable to surgical endoscopic treatment and by the occasion of a diagnostic laparoscopy. The gametes may be transferred to the fallopian tubes via the fimbria and under laparoscopic guidance or inter-cervically under ultrasound guided control.
The issue of GIFT versus IVF as far as the procedure of choice for unexplained and mild male infertility is concerned is still debatable in view of the retrospective nature of the reports and the lack of properly designed, prospective randomized studies. However, the necessity of laparoscopy even with the accompanying risks and the recent availability of superior in vitro culture media has favored IVF as the first choice.
As an embryologist, I feel that GIFT should not be completely abandoned. It may still be the best choice in cases of very difficult transfers and poor laboratory conditions caused by unexpected emergencies. It may help when shortage of media occurs because of difficulties in the importation of foreign supplies as observed from time to time in less developed countries. In addition, it still remains the only advanced assisted reproduction technique allowed for some religious orthodox groups.
 
ZYGOTE INTRAFALLOPIAN TRANSFER (ZIFT) AND TUBAL EMBRYO TRANSFER (TET)
In 1986, ZIFT/TET was successfully used in a patient with antisperm antibodies. Several centers worldwide have employed this procedure for severe male factor, female antisperm antibodies, frozen thawed embryo transfer and previously failed GIFT cycles. ZIFT involves harvesting the oocyte, inseminating them with sperm first as in IVF and transferring 3 to 4 fertilized egg (zygotes) 24 hours postaspiration. Advantage of TET on the other hand has been the documentation of normal oocyte fertilization and embryonic development.
Depending on the degree of tubal pathology, the incidence of ectopic pregnancy is higher for women undergoing tubal transfer of both gametes and embryos. ZIFT/TET has now become a seldom-used alternative for patients with a history of difficult trans-cervical transfers, a selective group of women over the age of 40 and for those transferring frozen thawed pre-embryos, 8as it demands the use of laparoscopic transfers soon after follicular aspiration, which can make it expensive for patients.
 
OOCYTE AND EMBRYO MANIPULATIONS
 
Intracytoplasmic Injection of a Single Spermatozoon into an Ooctye (ICSI)
ICSI is arguably the most significant advance in assisted reproductive technology since the successful birth of Louise Brown over two decades ago. Clinical pregnancy rates rose from 31.6 to 36.2 percent and birth rates increased from 25.7 to 29.5 percent. As with any new technique, possible risks such as the short-and long-term health consequences of the offspring must be considered. Thus far, the outcome data regarding ICSI offspring have been reassuring. Many practitioners worldwide recommend ICSI to all IVF cycles, bypassing some natural selection. Also, few data exist comparing IVF and ICSI in non-male factor cases. One obvious advantage of ICSI is the near avoidance of unexplained fertilization failure, particularly when used in conjunction with preimplantation genetic diagnosis and polar body biopsy. The embryologist may gain additional information regarding oocyte maturity and morphology when preparing for the ICSI procedure. While ICSI has unarguably revolutionized the treatment of male factor infertility, critics question its potentially negative impact on the genetic make-up of future generations, since ICSI enhances the reproductive potential of mutant male genotypes with the resulting possibility of altering the genetic make-up of future human populations.
The incidence of chromosomal abnormalities in infertile men varies between 5.8 and 13.7 percent compared with 0.38 percent in phenotypically normal male newborns. Furthermore, concerns related to the ICSI technique itself and with the use of spermatozoa from males with abnormal chromosomes could have long-term impacts on the offspring. The large follow-up studies conducted by the Belgium group and at Cornell University (Ithaca, New York, USA) including over 4000 children produced by ICSI, revealed a similar incidence of major and minor malformations between IVF and general population babies.
Microdeletions of the Y chromosome have emerged as a significant factor in causing severe oligozoospermia. The existence of a gene (or a gene complex) associated with normal spermatogenesis has been suggested since mid-70's. Microdeletions of the distal part of the Y chromosome (interval 6 of the Yq 11) occur in about one in eight men with azoospermia or severe oligozoospermia. To date, three genes have been cloned from interval 6 (RBM-RNA Binding Motif: DAZ – Deleted in Azoospermia and SPGY). These genes are expressed specifically in the testes and encode the presumed RNA-binding proteins. Microdeletions outside interval 6 may play an important role in the regulation of spermatogenesis. Microdeletions of the Y chromosome have been identified in upto 18 percent of severely oligospermic and azoospermic males and these microdeletions can be transmitted from father to son. Furthermore, cytogenetic studies on oligospermic and azoospermic men disclosed chromosomal abnormalities in 4.6 and 13.7 percent of these groups, respectively. Congenital absence of the vas deferens is caused by a mutation of the cystic fibrosis transmembrane regulator gene.
Despite reassuring data regarding the outcome of ICSI, potential long-term risks of the ICSI procedure are still to be determined and the universal use of ICSI to all IVF patients calls for caution and clinical judgment. Only ICSI trained embryologist should carry out ICSI.
 
EMBRYO CRYOPRESERVATION
The first successful pregnancy from a frozen human embryo occurred in 1983. Embryo cryopreservation enhances the cumulative pregnancy potential after single oocyte retrieval, decreases the risk of multiple gestations, and reduces the risk of severe ovarian hyperstimulation syndrome. A predominant number of studies on cryopreservation have shown that human embryos survive and are implanted at a higher rate, when frozen during the pronuclear stage, as opposed to the cleavage stage. Obviously, this 9method does not rely on embryo preselection. Propanediol with 0.1 mol of sucrose used as a cryoprotectant and a slow, computer programmed freezing process have rendered the most consistent results over time.
A recent summary on the outcome of 103 frozen-thawed two-pronucleate embryos from the Mayo Clinic (Rochester, Minnesota, USA) revealed that 90.2 percent of them survived the thawing process.42 Of the 398 embryos thawed, 90% cleaved and 354 (88.9%) were transferred. The implantation rate was 21.5 percent; the overall pregnancy rates were 41.8 percent clinical and 36.9 percent delivered. Although a trend towards a decline in delivery rates per frozen transfer was associated with increasing female age, the cumulative chance for a live born per oocyte retrieval for the entire program was 56.3 percent. In addition, multiple gestation rates for the frozen transfer compared favorably with those obtained by fresh transfer (52.5% and 45.4%, respectively).
Disadvantages, such as the length of time required for freezing, an experienced technician to monitor the process and expensive equipment, make the use of ultrarapid freezing and vitrification a somewhat sensible alternative. Experience with vitrification is emerging; with ultrarapid freezing 80 percent of the embryos survive thawing, and the clinical pregnancy rate is below 16 percent. It is becoming more apparent that cryopreserved pronucleate oocytes survive freezing, thawing, and progress thorough syngamy while still achieving similar potential for implantation and pregnancy when compared with fresh embryos. The outcomes obtained with embryo preselection and extended cultures should be prospectively compared with those obtained from the strategy where there is no embryo preselection for the fresh transfer, and which includes pronucleate oocyte freezing-thawing. Furthermore, patient convenience and the lower overall risks of not going through a stimulated cycle makes pronucleate cryopreservation preferable. Further studies need to be carried out on embryos freezing which will determine the method of choice for cyropreservation.
 
ASSISTED ZONA HATCHING
Successful hatching of the embryo from the zona pellucida is a prerequisite for implantation in the uterus. Assisted hatching was introduced in IVF program to breach the zona pellucida and promote the natural process of hatching. In a prospective randomized study using acid Tyrode's solution for zona drilling of 3-day-old embryos, Cohen et al (1992).43 showed that this approach could improve the implantation rate in poor prognosis patients. Several other techniques to facilitate the hatching process have been reported, including partial zona dissection using a sharp pipette and laser technology. Recently a technique where complete zona removal of day 3 embryos using acid Tyrode's solution has been reported as an option to improve pregnancy rates after ICSI in women with a poor prognosis for conception.44
Results of published studies vary, but different models and patient selection criteria, often with low numbers of subjects, make any generalized interpretation of the findings difficult. Assisted hatching in women with poor prognosis because of old age, previous IVF/ICSI cycle failures, and thawed embryos has been shown to be beneficial.
 
PREIMPLANTATION GENETIC DIAGNOSIS (PGD)
The use of preimplantation genetic screening or diagnosis to select against embryos carrying some form of aneuploidy has been proposed as a means of increasing implantation in older women or those with repeated ART failure.45 These have been found in morphologically normal appearing embryos or from embryos with an obvious morphological abnormality.32,46 The abnormalities are persistent through to the blastocyst stage in many instances47. Embryos can present with uniformly normal, uniformly abnormal, mosaic or chaotic chromosome constitutions. The mosaic type is of concern as it may take more than one cell in a biopsy to identify this condition.48 Further, it is not a non-invasive strategy since the embryo is subjected to manipulation and cell removal, 10which could have severe consequences in later development when embryo polarity is considered.
However, the data available do not point to a great increase in the take-home-baby rate when this technique is applied in a clinical setting.49 The data from the two largest published studies show no dramatic improvement in the implantation or live delivery rates in older women or in the group of repetitive ART failures.50
The first clinical application of PGD using IVF, cleavage-stage embryo biopsy, and single-cell genetic analysis to identify unaffected female embryos in couples at risk for X-linked disease was about a decade ago. Since then the number of centers offering PGD has increased slowly, mainly because of embryo ploidy can be assessed using PGD. The relatively low viability of human embryos is fundamentally determined by the large proportion of chromosomal abnormalities that have been frequently and consis-tently reported in embryos examined by karyotype analyses. The use of fluorescence in situ hybridization (FISH) to identify the number of chromosomes in the interphase nuclei of embryos has confirmed a high level of aneuploidy. Actually, upto 63 percent of aneuploidy embryos were identified in three groups of women with poor prognosis for pregnancy in IVF (aged over 38 years, IVF failure in more than two trails, women exhibiting mosaicism in the karyotype as 46XX/45X). Diagnosis of the aneuploidies enables the transfer of an increased number of chromosomally normal embryos and increases the consequent implantation rate. As the diagnosis of aneuploidy involves a degree of error (at least 6%) patients should be advised to confirm preimplantation diagnosis through a more conventional prenatal diagnosis.
Single gene disorders have been identified in increasing numbers. Specific polymerase chain reaction (PCR) diagnoses have been performed for the evaluation of Duchenne and Becker's muscular dystrophy as well as fragile X mental retardation. PCR technology was also applied to investigate several other autosomal dominant and recessive disorders, as well as sex-linked genes. Fluorescence PCR has also been introduced for mutation detection and linkage analysis.
In programs offering preimplantation genetic diagnosis, the role of counselors is paramount. Obtaining detailed patient and family histories discussing the genetic, clinical and molecular diagnosis along with the preparation of patient literature and irregular follow-ups would greatly benefit the patient.
 
ETHICS OF ASSISTED CONCEPTION: PAST AND PRESENT
I have briefly traced the advances in laboratory methods designed to alleviate human infertility. Stable or very slow moving over centuries, huge changes in practice have been introduced in the last 25 years. Further changes are inevitable. As the new methods described above were introduced, they were accompanied by a constant crescendo of ethical and public attention. This is not to suggest that ethics has ever been far away from this field of human endeavor.
Of course, the greatest ethical challenges have emerged from the large-scale introduction of embryology and genetics into human conception. The birth of Louise Brown signified the opening of a new era of dynamic change in outlook and practice. Indeed, the scale and breadth of the scientific advances are beyond the scope of this brief article. IVF itself was met with fierce opposition, some of it theological, as before 1978 human conception was combined with spiritual factors. This situation appears to have changed worldwide as IVF has spread from country to country. There were scientific objections—that all IVF babies would be abnormal, that the infertiles were being misled because of scientists simply wished to experiment on human embryos and that risks of cloning were too great. These particular criticisms were always beyond reality. Others were patiently wrong, e.g. that IVF did not correct infertility because the women were still infertile after delivery of their IVF baby. All these issues, except cloning, are scarcely discussed today, perhaps because scientist, clinicians and lawyers initiated the ethical debate.11
ICSI, ZIFT, GIFT, TET and many other derivatives attracted little approbation. Yet there were no animal studies before ICSI was introduced clinically, and today there are still critics who are fearful of its genetic consequences. GIFT was accepted in some quarters if a bubble of air separated oocytes and spermatozoa in the injection catheter. Preimplantation genetic diagnosis is questioned in Germany, but increasingly practised even though numbers of births are still small. Recently publicity on designer babies, and the diagnosis of the p53 gene reveal a glimpse of an astonished future.
Diagnosing age onset conditions in an embryo, e.g. Huntington's chorea can be very bad news for one of the parents who also carries the specific disease gene. Embryo cryopreservation is still queried, but is practised in most countries. Its benefits are great, although many embryos accumulate in clinics, forgotten deliberately or otherwise by the parents. A limit has to be set to ensure that a clinic must store them perpetuity.
Today's trends in unusual forms of assisted reproduction were largely foreseen before the first IVF baby was conceived. Yet when they arrive, they can have an immense impact on society. Surrogate pregnancy can spell one of the highest aspects of love between mother and daughter. It can equally involve high finance or the deliberate retention or rejection of a surrogate birth. Oocyte and embryo donation again raise questions about the problems faced by some parents about telling a child about its genetic origins.
Assisted conception has compelled governments worldwide to pass legislation over its regulation, changed outlooks on fertilization and embryology beyond recognition and promises to compel even more changes in outlook. In this chapter, cloning, embryo stem cells and cord blood cryopreservation for newborn babies, cytoplasmic transfers, nuclear transfer, somatic cell haplodisation to help in later life have been barely mentioned as they are all still in experimental stages. It is surely certain that the role of the clinical embryologist will further evolve in the new century, which will witness many novel changes.
 
SUMMARY
As a clinical embryologist, I have briefly expressed my opinion on the changing approaches and methods of assisted reproduction throughout recent centuries. This has been combined with detailed analyses of the salient considerations with respect to treatment options in the arena of infertility. Each treatment has its own merits, and others continue to develop. It has long been clear that the treatment of choice will always depend on the specific context and needs of each patient. Patient choice has never been wider than today. Clearly the primary goal today remains the need to develop and to explore rapidly changing technologies to ensure the widest number of options for each individual case.
REFERENCES
  1. Steptoe PC, Edwards RG: Reimplantation of a human embryo with subsequent tubal pregnancy. Lancet 1:880–82, 1976.
  1. Edwards RG, Steptoe PC, Purday JM: Establishing full term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynecol 87:737–59, 1980.
  1. Bavister BD: Environmental factors important for in vitro fertilization in the hamster. J Reprod Fertil 18:544–45, 1969.
  1. Goddard MJ, Pratt HPM: Control of events during early cleavage of the mouse embryo: an analysis of 2-cell block. J Embryol Exp Morphol 73: 111–33, 1983.
  1. Chatot CL, Ziomeck CA, Bavister BD et al: An improved culture medium supports the development of random-bred 1-cell mouse 2 embryos. In vitro J Reprod Fertil 86: 679–88, 1989.
  1. Leese HJ: Energy metabolism of the blastocyst and uterus at implantation. In Yoshinaga K (Ed): Blastocyst Implantation. Adam Publishing group  Boston MA, 39–44, 1989.
  1. Leese HJ: The environment of the preimplantation embryo. In Edwards RG (Ed): Establishing a Successful Human Pregnancy. Serono symposium. Oxford UK 143–54, 1990.
  1. Leese HJ: The metabolism of the preimplantation mammalian embryo. In Mulligan SR (Eds): Oxford Reviews of Reproductive Biology. Oxford University Press.  Oxford  35–72, 1991.
  1. Leese HJ: Metabolic control during preimplantation mammalian development. Hum Reprod Update 1:63–72, 1995.
  1. Lawitts JA, Biggers JD: Culture of preimplantation embryos. Methods Enzymol 225:153–64, 1993.

  1. 12 Leese HJ, Conaghan J, Martin KL, et al: Early human embryo metabolism. Bioassays 15:259–64, 1993.
  1. Quinn P, Moinipanah R, Steinberg JM et al: Successful human in vitro fertilization using a modified human tubal fluid medium lacking glucose and phosphate ions. Fertil Steril 63: 922–24, 1998.
  1. Gardner DK, Lane M: Culture and selection of viable blastocysts a feasible proposition for human IVF?. Hum Reprod Update 3:367–82, 1997.
  1. Verlhac M, Lefebvre C, Guilland P et al: Asymmetric division in mouse oocytes with or without Mos. Curr Biol 10: 1303–06, 2000.
  1. Araki K, Naito K, Haraguchi S et al: Meiotic abnormalities of C-mos knock-out mouse oocytes: activation after the first meiosis or entrance into third meiotic metaphase. Biol Reprod 55: 1311–24, 1996.
  1. Ortiz M, Lucero P, Croxatto H: Postovulatory aging of human ova. spontaneous division of the first polar body. Garnete Res 7:269–76, 1983.
  1. Wakayama T, Yanagimach R: The first polar body can be used for production of normal offspring in mice. Biol Reprod 59:100–04, 1998.
  1. Ebner T, Moser M, Yaman C et al: Elective transfer of embryos selected on the basis of first polar body morphology is associated with increased rates of implantation and pregnancy. Fertil Steril F2, 599–603, 1999.
  1. Ebner T, Yaman C, Moser M et al: Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection. Hum Reprod I5:427–30, 2000.
  1. Katoh K, Hammar K, Smith P et al: Birefringence imaging directly reveals, architectural dynamics of filamentous action in living growth cones. Mol Biol Cell 10:197–210, 1999.
  1. Gorbsky G: Cell cycle check points: arresting progress in mitosis. Bioessays 19: 193–97, 1997.
  1. Steuerwald N, Cohen J, Herrera R et al: Association between spindle assembly check point expression and maternal age in human oocytes. Mol Hum Reprod 7:49–55, 2001.
  1. Wang W, Meng L, Hackett R et al: Development ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination. Hum Reprod 16: 1464–68, 2001.
  1. Wang W, Meng L, Hackett R et al: The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril 75:348–53, 2001.
  1. Racowsky C: High rates of embryonic loss, yet high incidence of multiple births in human ART: is this paradoxical?. Theriogenology 57:87–96, 2002.
  1. Alikani M, Willadsen S: Human blastocysts from aggregated mononucleated cells of two or more non-viable zygote-derived embryos. RBM online 5(1):56–58, 2000.
  1. Alikani M, Calderon G, Tomkin G, et al: Cleavage anomalies in early human embryos and survival after prolonged culture. Hum Reprod 15:2634–43, 2000.
  1. Alikani M: Cytoplasmic fragments in human embryos in vitro: implications and the relevance of fragment removal. In Gardner D. Weissman A, Howles C, Shoham Z (Eds): Textbook of Assisted Reproductive Techniques. Laboratory and Clinical Perspectives. Martin Dumitz.  United Kingdom 169–82, 2001.
  1. Balaban B, Urman B, Isklar A et al: The effects of pronuclear morphology on embryo quality parameters and blastocyst transfer outcome. Hum Reprod 16:2357–61, 2001.
  1. Zollner U, Zollner KP, Hartl G et al: The use of a detailed zygote score after IVF/ICSI to obtain good quality blastocyst: the German experience. Hum Reprod 17:1327–33, 2002.
  1. Fenwick J, Platteau P, Murdoch AP et al: Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro. Hum Reprod 17: 407–12, 2002.
  1. Hardarson T, Hanson C, Sjogren A, et al: Human embryos with unevenly sized blastomeres have lower pregnancy and implantation rates: indication for aneuplody and multinuclear. Hum Reprod 16:313–18, 2001.
  1. Check J, Wilson C, Summers-Chase D, et al: Pregnancy rates according to embryo cell number at time of embryo transfer (ET). Clin Exp Obstet Gynecol 28: 73–77, 2001.
  1. Johnson M, Day M: Egg timers: how is developmental time measured in the early vertabrate embryo?. Bioessays 22:57–63, 2000.
  1. Scott L: Essential IVF, basic science and clinical applications. In Van Blerkom J, Gregory L (Eds): Morphological Correlates of Oocytes and Embryo Competance-Identification. Kluwer Press,  2003.
  1. Gardner R: Flow of cells from polar to mural trophec-toderm is polarized in the mouse blastocyst. Hum Reprod 15: 694–701, 2001.
  1. Hansis C: Assessment of B-hCG, B-LH mRNA and ploidy in individual human blastomeres. Reprod Biomedicine Online 5: 156–61, 2002.
  1. Vidaeff AC, Racowsky C, Rayburn WF: Blastocyst transfer in human in vitro fertilization. A solution to the multiple pregnancy epidermic. J Reprod Med 45: 529–39, 2000.
  1. Kovacic B, Vlaisavljevic V, Reljic M et al: Clinical outcome of day2 versus day 5 transfers in cycles with one or two developed embryos. Fertil Steril 77:529–36, 2002.
  1. Shapiro B, Richter K, Harris D et al: Influence of patient age on the growth and transfer of blastocyst — stage embryos. Fertil Steril 77:700–705, 2002.
  1. Coskun S, Hollanders J, Al-Hassani S et al: Day 5 versus day 3 transfer: A controlled randomized trial. Hum Reprod 15:1947–52, 2000.

  1. 13 Damario MA, Hammitt DG, Session DR et al: Embryo cryopreservation at pronuclear stage and efficient embryo use optimizes the chance for a liveborn infant from a single oocyte retrieval. Fertil Steril 73:767–73, 2000.
  1. Cohen J, Alikani M, Trowbridge J et al: Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 7: 685–91, 1992.
  1. Mansour RT, Rhodes CA, Aboulghar MA, et al: Transfer of zona-free embryos improves outcome in poor prognosis patients; a prospective randomised controlled study. Hum Reprod 15:1061–64, 2000.
  1. Munne S, Wells D: Preimplantation genetic diagnosis. Curr Opin Obstet Gynecol 14: 239–44, 2002.
  1. Nass T, Escudero T, Marello E et al: Dissociation between embryo morphology and euploidy in IVF patients with poor prognosis. Ferti Steril 77:S11, 2002.
  1. Sandalinas M, Sadowy S, Alikani M, et al: Developmental ability of chromosomally abnormal human embryos to develop to the balstocyst stage. Hum Reprod 16:1954–58, 2001.
  1. Magli M, Jones G, Gras L et al: Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro. Hum Reprod 15: 1781–86, 2000.
  1. Egozcue J, Santalo JCG, Durbun M et al: Preimplantation genetic screening and human implantation. J Reprod Immunol 55:65–72, 2002.
  1. Gianaroli L, Magli M, Ferraretti A et al: Preimplementation diagnosis for aneuploidies in patients undergoing in vitro fertilisation with a poor prognosis: Identification of the categories for which it should be proposed. Fertil Steril 72:837–44, 1999.