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Microassisted fertilization in assisted conception: indications and patient impact
8 Microassisted fertilization in assisted conception: indications and patient impact COLIN D. MATTHEWS
Microassisted fertilization is poised at the exciting edge of current in vitro fertilization (IVF) technology and is set to traverse the interval between a research thrust confined to a small number of units and routine clinical practice. In the near future, reproductive programmes without access to microinjection will be offering only a limited service and failing to provide the proper range of options to infertile couples. This confidence in the place of microassisted fertilization stems from the markedly improved results of subzonal insemination (SUZI) and, more particularly, of intracytoplasmic sperm injection (ICSI) when compared to the older methodology of zona drilling and other zona breaching procedures. Fundamental to microassisted fertilization is that the human product is thus far indistinguishable from any other method of conception. The move to routine clinical medicine demands that more specific attention be paid to the clinical indications for the procedure and the general impact of the technology on patients. Given the still expanding role for conventional IVF, the demand for microassisted fertilization has been curtailed thus far only by the limited success of the procedure. Now that the results of microassisted fertilization, particularly ICSI, are so impressive, the demand is unlikely to be easily satisfied given the large contribution that infertile males make to the problem of infertility. Nevertheless, microassisted fertilization demands considerable specialized scientific skills, added to a consistent and experienced IVF system. It must always be remembered that the infertile couple is a vulnerable couple, willing to consider a variety of uncomfortable measures in their quest for a child, and must not be exploited by false hopes. A considerate professional unit will tailor the entry to microassisted fertilization to the results being achieved-with improving results widening the indications for treatment. Our own experience with zona drilling with the mouse model (Depypere et al, 1988) led us to believe that this was the optimal technique for microassisted fertilization in the human. Two techniques, zona drilling (ZD) (Gordon and Talansky, 1986) and zona cutting (ZC) (Tsunoda et al, 1986), were investigated to assess the effect of decreasing concentrations of normal epididymal sperm on fertilization rates, blastocyst formation and the establishment of pregnancy in the mouse. While both ZD and AC achieved Bailli~re"s Clinical Obstetrics and Gynaecology--
Vot. 8, No. 1, Marcht994 ISBN0-7020--1844-9
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fertilization at sperm concentrations associated with lack of fertilization of control (non-ZD or non-ZC) oocytes, ZD provided better results. Extrapolating these results to the human was, however, unrewarding. Twenty-seven patients undertook 40 cycles (249 oocytes) of ZD. The fertilization rate (24.5 %) was clearly increased over controls (8.3 %) but no pregnancies were achieved from the 21 transfers (mean 2.1 embryos). Evidence was apparent that the quality of the embryos was less than that in conventional IVF (Payne et al, 1991a) and ZD was abandoned in favour of ZC. In the first trial of zona cutting (ZC1), 22 patients undertook 23 cycles (147 oocytes), 47 (32%) of the oocytes fertilized and 15 patients received an embryo transfer (mean 2.2 embryos), but again no pregnancies were achieved. Once again, ZC was associated with relatively low numbers of quality embryos (6% grade 1), and a second trial (ZC2) was commenced which included sucrose shrinkage of the oocyte prior to micromanipulation together with immunosuppression and antibiotic regimens over the transfer period, following the report of Cohen et al (1990). Thirty-six patients undertook 36 cycles (233 oocytes), 27% of the oocytes fertilized and 19 women received an embryo transfer. One patient (5.3%) became pregnant, representing an implantation rate per transferred embryo of 2.6%. The rate of polyspermia was considerably higher (19%) than previously (6%). Because of the possibility that blastomeres were being extruded from the replaced embryos, a third trial (ZC3) was instituted and embryo transfer was delayed until day 3 to allow better compaction of the embryo. In this trial, 35 patients undertook 35 cycles (282 embryos) with a fertilization rate of 34% and 18 women receiving an embryo transfer (mean 2.3 embryos). Three patients (16.7) became pregnant, providing an implantation rate per embryo of 7.3%. Combining the results of these trials, 120 couples had undertaken 134 cycles of micromanipulation and 73 couples had an embryo transfer involving 157 embryos for four pregnancies, two of which ended in miscarriage (Table 1). In 1991, SUZI (Ng et al, 1988; Fishel et al, 1990a,b; 1991, 1992) was introduced to our programme with the injection of 3-12 spermatozoa depending on the general semen characteristics. In this trial 78 patients undertook 84 cycles (776 oocytes) with 34.4% of the oocytes fertilized. Fifty-two women received an embryo transfer (mean 2.0 embryos) and 16 (30.8 %) became pregnant, representing an implantation rate per embryo of 18.4%. Put another way, 5.4 SUZI embryos were replaced to achieve one implantation; this figure compared more than favourably with routine IVF in our own hands, when 10.4 transferred embryos are required to achieve each pregnancy. It must be recognized, however, that general IVF contains a wide spectrum of clinical conditions including older women and women who have repeatedly failed cycles. Clearly, a major clinical barrier has been broken with the advent of SUZI and the impressive aspect is the implantation potential of the transferred embryos. It is to be anticipated that, with the additional sophistication of ICSI to improve fertilization results without detracting from the implantation potential (Van Steirteghem et al, 1993a), clinical results can be anticipated rapidly to improve further. The downside of SUZI is very important. With few sperm injected,
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MICROASSISTED FERTILIZATION IN ASSISTED CONCEIrI'ION Table 1. Results of microassisted fertilization 1989-1993. ZD Number Number Number Number Number
(%)
couples cycles oocytes fertilized (%) monospermic
Polyspermic (%) Number couples with ET (%) Number embryos transferred Number embryos transferred Number pregnancies (% ET) Implantation rate per embryo transferred
ZC1
ZC2
ZC3
SUZI
ICSI
27 22 36 35 78 42 40 23 36 35 84 42 249 147 233 282 776 413 62 (24,5) 47 (32.0) 63 (27.0) 97 (34.3) 267 (34.4) 268 (64.9) 60 (97) 44 (94) 51 (81) 70 (72) 147 (55) 248 (60.0) 2 (3.2) 21
3 (6.4) 15
12 (19.0) 19
27 (27.8) 120 (44.9) *20 (7.5) 18 52 39 (92.9)
44
33
39
41
2.1
2.2
2.0
2.3
103 2.0
Nil
Nil
1 (5.3)
3 (16.6)
16 (30.8)
Nil
Nil
2.6
7.3
18.4
104 2.7 10 (25.6) N/A
(%)
ZD = zona drilling, ZC = zona cutting, SUZI = sub zonal sperm insemination, ICSI = intra cytoplasmic sperm injection 1, 2, and 3 refer to trials (see text). * 3PN were seen in 20 eggs despite 1 sperm only being injected into the egg. The 3rd PN is probably a result of failure to extrude the second polar body.
poorer rates of fertilization can be expected; if too many sperm are injected then the rate of polyspermic fertilization is increased dramatically. Both of these aspects reduce the embryos available for transfer. Even more important is the unpredictability of the SUZI procedure. While several attempts have been made to rationalize the number of sperm to be injected by morphological, motility and acrosome assessments of the spermatozoa to be injected, the ability to predict the result remains elusive (Cohen et at, 1992a). The initial cycle remains a trial with little correlation between fertilization and the semen characteristics. How can differences between ZC and SUZI be explained? It is very unlikely that the clinical spectrum of patients offers any significant explanation. Both ZC and SUZI are performed principally for male factor infertility and there is little to suggest that differences in the fertility of the female partner between series are marked. It should, however, be recognized that these clinical cases may represent 'pure' male infertility and the female side may be 'unadulterated' by unknown factors curtailing fertility. In other words, the female side may be even more normal than in 'unexplained' infertility, and perhaps this factor at least partially explains differences between microassisted fertilized embryos and conventional IVF. Clearly, there is a difference in the size of the zonal breach between ZC and SUZI, and it has been suggested that toxic elements from the uterus, including infective agents and leukocytes, can adversely affect embryo development after transfer. The larger the zonal defect, presumably the more opportunity there is for a more detrimental effect. It is also possible
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that some toxic elements may be operable during culture conditions of the early oocyte-embryo before transfer. The nature and size of the zona breach of the SUZI embryo may be more optimal once the embryo is replaced in the uterus compared with ZC, which may be associated with blastomere loss through the defect. Interestingly, our experience with blastocyst hatching following ZD and blastomere biopsy in the mouse suggests that hatching often takes place well away from the site of zona breach (Cui et al, 1993). A further factor related to the zonal breach is the possibility that cervical mucus collected inadvertently in the transfer catheter causes disruption of the embryo and the herniation of blastomeres through the zona during embryo transfer (Cohen et al, 1992b). One major difference between ZC (and conventional IVF) and SUZI is that ZC embryos are normally inseminated and cultured. It has been suggested that spermatozoa may be responsible for detrimental products gaining access to the embryo through the zonal disruption to cause adverse effects that are not obvious even by day 3 of embryo culture. Of the seminal products, reactive oxygen species are current agents under suspicion (Aitken et al, 1992). This idea urgently requires testing as there are considerable implications for conventional IVF. There are other differences to consider between microassisted fertilized embryos and those derived from conventional IVF, including earlier cumulus removal and exposure to hyaluronidase of the microassisted fertilized oocyte, which also may be important (Palermo et al, 1993). The welcome results with SUZI are already dated by the exceptional results being achieved from ICSI. Intracytoplasmic injection of sperm has a considerable history in non-human species (Markert, 1983) but until recently has always been associated with unacceptably high rates of damage to the oocyte. Improvements to the instrumental side and skills gained with micromanipulation have now reduced the damage rate of ICSI to less than 10% of oocytes. Recent results indicate that when a single spermatozoa is injected to a metaphase II oocyte (n = 1409) a 64.9% monospermic fertilization rate (uninfluenced by semen characteristics) can be obtained with 71.2% of fertilized oocytes reaching the cleavage stages. Ninety percent of couples (n = 135) received an embryo transfer (75% triple embryo transfer) with a clinical pregnancy rate of 39.2% per embryo transfer (n = 53). In addition, 237 supernumerary embryos were cryopreserved (Van Steirteghem et al, 1993b). Such results make a huge impact on the clinical indications for microassisted fertilization. Our own initial experience with ICSI has also demonstrated the exciting potential of this technique (Table 1). CLINICAL INDICATIONS FOR MICROASSISTED FERTILIZATION Failed fertilization
At present, microassisted fertilization is logically offered to couples who have had a total or near total failure of fertilization at conventional IVF.
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This group is usefully divided into couples with a recognizable semen defect and those with more normal semen values. Failure of fertilization, particularly in the normospermic couple, may be due to oocyte factors that have been increasingly better recognized over the past few years. IVF has long been a very important option for male infertility (for review see Avery, 1992), One difficulty has been the inappropriateness of the World Health Organization (1987) criteria for semen analysis when IVF is being considered. The distinction between unexplained infertility and the male factor patient with the better semen analysis is blurred. However, very poor sperm analysis (fewer than 10 million sperm per ml, less than 10% motility and morphology parameters) is a common clinical problem and the likelihood of poor fertilization is much more predictable (Kruger et al, 1988). Until recently, the criteria for entry to conventional IVF in our unit has been the isolation of about 200 000 motile spermatozoa from pathological semen, usually after discontinuous Percol procedures (Ord et al, 1990; Kirby et al, 1991). This number of sperm will allow for the optimal insemination of the 6-8 oocytes expected to be recovered. In the absence of valid tests of sperm function, clearly, any arbitrary cut-off excludes semen samples that might achieve fertilization and includes samples that would be associated with failure of fertilization. With the availability of microassisted fertilization it would be advantageous to be able to recognize the semen sample that qualifies for conventional IVF but is destined to fail to fertilize oocytes. Many approaches, including the recognition of the state of the acrosome, objective assessment of motility, hamster oocyte penetration, acrosin activity, zona binding properties and conventional semen analysis (for review see Liu and Baker, 1992), have been examined in order to predict efficient fertilization. Unfortunately, no test thus far has gained sufficient clinical confidence to preclude the advice to attempt IVF. In an effort to refine our advice, we have recently examined the conventional semen parameters (World Health Organization, 1987) associated with 294 initial cycles of IVF performed for tubal infertility (to avoid unrecognized female factors) where at least four oocytes were available for fertilization (Duncan et al, 1993). The study included only those couples eligible for IVF (> 200 000 motile sperm after swim-up). Multiple regression analysis has allowed a distinction to be drawn between poor fertilization (<33%) and adequate fertilization (>33%) rates. The most helpful parameters were the combined indices of the percentage of progressive motile sperm in the isolated fraction and the percentage normal morphology of the fresh sample. Semen samples displaying <20% motility or < 20% normal morphology can be predicted to fail fertilization at conventional IVF with an 80% confidence. This prediction has also been tested prospectively with similar good results. A two-dimensional chart has been constructed (Figure 1) which may be helpful for programmes to assist the counselling of couples with the expected outcome of IVF. This ability to predict with some confidence allows for some flexibility of approach, particularly to the borderline semen sample in the presence of multiple oocytes. A combination of microassisted fertilization and conventional IVF may offer both improved diagnostic and therapeutic chances, even at the initial IVF procedure.
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relationship between the percentage normal morphology in fresh semen, the percentage motility in the isolated motile fraction and the fertilization rate. Cycles with fertilization rates < 35% (closed symbols) or ~>35% (open symbols) are indicated by different symbols. The curved line was predicted by the regression model; most cycles with a fertilization rate < 35% are below the line, whereas most cycles with a rate I> 35% are above it.
Failure of fertilization at conventional IVF may occur in the presence of 'normal' semen values. We analysed 303 unselected couples with normal semen values undertaking an initial single IVF cycle where at least 4 oocytes (median 5.4) were recovered. Nineteen subjects (6.3%) failed to fertilize oocytes. Of these 19, 13 undertook a further cycle of IVF and three again demonstrated failed fertilization (Matthews, 1990). Molloy et al (1991) and Lipitz et al (1993) have made similar observations. Thus, for the normospermic male the expectation of sporadic failed fertilization at conventional IVF is of the order of 6%, and about 1% of such normospermic men may consistently (at least two cycles) fail to achieve fertilization. The reason for failure is commonly related to the poor zona binding and penetration of sperm (Bedford and Kim, 1993), and ultrastructural anomalies of sperm not evident to light microscopy have been described (Carlon et al, 1992). Microassisted fertilization is dearly indicated for this group of normospermic males, but the outcome thus far from ZC and SUZI requires to be further evaluated before being proven effective. Alikani and Cohen (1992) have reported a reduced pregnancy rate from microassisted fertilization when performed for couples with normal semen. Failure of fertilization may be due to oocyte factors that may operate associated with poor semen values, but perhaps more often associated with normal semen. When the oocyte has poor cytoplasmic-cumulus quality, a poor expectation of fertilization is likely (Laufer et al, 1984; Mahadevan et al, 1987). This failure may be due to general immaturity (germinal vesicle or
MICROASSISTED FERTILIZATION IN ASSISTED CONCEPTION
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metaphase-I oocytes) (Gwatkin et al, 1989) or as a consequence of abnormalities of the zona structure (Tesarik et al, 1988). Bedford and Kim (1993) have drawn attention to the fact that cytoplasmic abnormalities are more common in cases of failure of fertilization in the presence of normal semen values, compared with oocytes failing to become fertilized with defective semen. We have also tried to estimate the contribution that oocyte factors make to fertilization failure and have described different morphological abnormalities of oocytes that have failed to fertilize, including thickened and thinned zona, vaculated, granular and fragmented cytoplasm, or fertilization anomalies. In a study of 503 unfertilized oocytes examined by Nomarski optics, 39% were judged abnormal. More than one half (57%) of the oocytes had a zona pellucida disorder, 31% had cytoplasmic disorders, 7% had fertilization abnormalities and only 7% were immature. When semen abnormalities were taken into account, the incidence of oocyte abnormalities was judged to be 7% in the presence of abnormal semen and 61% in normospermic conditions. Since disorders of the zona constituted the largest grouping, microassisted fertilization techniques would seem to offer considerable possibilities (Payne et al, 1991b). The recognition of the contribution of oocyte abnormalities to total or partial fertilization failure is important as microassisted fertilization may not achieve such favourable results with defective oocytes as in true semen factor infertility with normal oocytes. Finally, it should be mentioned that a place for microassisted fertilization during the second day of culture has been suggested for failed conventional fertilization in order to rescue oocytes. No large series has yet been described. Clearly, there is a need for a precise scientific assessment of cycles associated with failed fertilization. The number, maturity and morphological appearance of the oocytes require to be documented, together with the outcome of the procedure including rates of unfertilized, monospermic and polyspermic oocytes-embryos plus some assessment of sperm binding. This full documentation may be vital to the arrangements for the next cycle. Failure to qualify for conventional IVF
In addition to the group of patients who have attempted IVF, there are those couples whose semen values fail to qualify for conventional IVF. There are a large group of men with unexplained defective semen, usually with normal or slightly raised follicle stimulating hormone (FSH) levels. Commonly there is no history of relevance, and physical examination is not contributory. Many semen samples fail to qualify for IVF because it is not possible to isolate an adequate number of motile sperm, and a common clinical finding is sperm apparent only after centrifugation of the semen. The requirement for so many sperm to fertilize the oocyte under in vitro conditions remains somewhat unclear, although numbers of sperm appear to be required to allow passage through the egg vestments. Included in this group of men with poor semen are males who have undergone cytotoxic treatment, often during pubertal years, for haematological malignancies and whose spermatogenic processes are now suboptimal.
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These men usually have raised FSH levels with minimal semen activity. Not too dissimilar are men who have had radiotherapy and cytotoxic treatment for malignant tumours of the testis. Coincidently, microassisted fertilization brings a different attitude towards the cryopreservation of semen from similar men with testicular cancer, Hodgkin's disease and other forms of cancer. It is common to reject semen samples from such men because of the poor post-thaw results. IVF has already played an important role by achieving pregnancies for such men who not uncommonly have poor semen (Sanger et al, 1992) but microassisted fertilization offers even more. The recovery of only a few sperm from the post-thaw sample may be the difference between sterility and fertility. Another condition that would preclude admission to an IVF programme is globozoospermia--complete absence of the acrosome. Although such cases apparently fail to achieve fertilization after SUZI, there is the possibility of procuring conception by ICSI. The lack of the appropriate fusion proteins, which might occur in globozoospermia, might be circumvented by ICSI. Finally, there are many men, often in an older age group, who have had reversal of vasectomy performed with poor results. This may be related to the reparative surgery itself or to epididymal pathology as a consequence of long-term vasectomy (Silber, 1979). The challenge of these men is now being addressed by SUZI and ICSI, given the need of those procedures for very few sperm. Most of these couples currently constitute the demand for donor semen, and this group contains couples for whom microassisted fertilization is set to make the most dramatic impact as pregnancies can now be anticipated from any ejaculate that contains any live sperm. It will, however, require considerable collective experience to determine whether spermatozoa derived from such seriously defective ejaculates have an equal potential for fertilization, pregnancy and normal children as from the normospermic sample. While it would seem feasible to expect pregnancies from immotile but live sperm (Bongso et al, 1989), it is not yet clear whether dead sperm have DNA in an appropriate state that pregnancies can also be achieved. It is to be noted that spermatozoa contain no reparative DNA repair enzymes (Munne and Estop, 1993).
Azoospermic and anejaculate males There are several other significant groups of subjects who appear to be on the verge of qualifying for microassisted fertilization. These include some azoospermic males and men who are unable to produce an ejaculate, often from post-surgical or neurological causes. These categories are now beginning to come into focus for treatment. Not uncommonly do azoospermic males have normal spermatogenesis within the testis but with obstruction within the epididymis as a result of previous infection or the after-effects of biochemical malfunction due to genetic defects (commonly mutations of the cystic fibrosis gene) or associated with bronchiectasis (Young, 1970). In addition, at least 5% of azoospermic males have congenital absence of the whole or part of the vas.
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This latter syndrome has been shown to be amenable to synchronous epididymal sperm recovery and IVF-GIFT techniques (Silber et al, 1990), but the results have not been easily reduplicated. The combination of microassisted fertilization with epididymal aspiration would seem to offer real prospects for such couples. In addition, there now exists the possibility for cryopreservation of epididymal spermatozoa. The application of conventional glycerol-based cryopreservation techniques shown useful for ejaculated sperm (Polge et al, 1949) is not generally useful for epididymal sperm. However, if only a few sperm are now required, or perhaps only preserved DNA, then the prospect for avoiding repetitive epididymal surgical procedures may be real. Not least are those males who have ejaculatory dysfunction associated with retrograde ejaculation. We have already achieved a pregnancy for a couple using several spermatozoa injected subzonally following retrograde ejaculation and recovery from urine. Such cases illustrate the revolution that microassisted fertilization can bring to otherwise very exceptionally difficult cases. Even though microassisted fertilization is a very young scientific initiative, the above traditional ways of considering clinical indications should now be re-examined in the light of the exceptional results of ICSI. If the results of ICSI can be shown to be repeatable in many other units, the pertinent question is which males do not qualify for the procedure? Perhaps the only males not able to be considered are those with absent or severe defects of spermatogenesis itself. The ICSI results, particularly the implantation potential, also pose questions for the performance of conventional IVF as it is practised today. PATIENT IMPACT With the exception of certain hereditary diseases, a child of one's own genetic material is always the first quest for couples, and microassisted fertilization now offers reasonable opportunities for pregnancy where none existed previously. What is this quest for one's own genes? The perception and influence of genes project not only forward to future generations but backwards from grandparents. The features and mannerisms of children are often easily recognizable as one's own or one's partner by extended family and close friends. This visual continuity is often comforting and a source of pride. Commonly, marriage and partnership bring an unexplainable sense of wanting to create children, not just to promote a family but a family as part of one's life partner. There is a strong innate female sense of wishing to provide 'fatherhood' for her mate rather than simply building a family. What then is this fatherhood? Perhaps it is a sense of being responsible for, whatever the product. A willingness to stand up for and to accept. Not simply an opportunity to nurture and provide an environment for a child to develop its gifts, but a chance to have played a role and to have left something of one's own individuality in the world when long departed. A child of one's genes may be at less risk of harm or disinterest than a child
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derived from a donor, but this risk will require years of study to prove or disprove. For the male, microassisted fertilization must offer the chance of a 'cure' for a problem until now regarded as 'hopeless', a fulfilling of the sexual relationship and the chance to remove the cloud of having to consider a donor. The total number of couples who consider and reject the use of donor sperm is not easily acquired but most couples who reject donor insemination will be concerned with their attitudes toward a donor child or because of the unknown risks of transmissible agents heightened by the recent controversies of HIV and Creutzfeldt-Jakob disease. Very few will reject donor insemination because of the medical technology involved. What proportion of infertile couples due to the male will be attracted by microassisted fertilization remains to be seen, and will be greatly influenced by the success rates of the procedure. Countries and states with reproductive legislation will commonly demand appropriate ethical approval before microassisted fertilization is instituted. In our experience, the principal ethical concerns have been the prospect for success, the waste of oocytes and embryos through technical damage and polyspermia, and the risk of congenital abnormality. Microassisted fertilization makes considerable demands on clinical time to explain risks, consent forms, chances of success, expense and general support and explanation at failure. Nevertheless, the clinician is often surprised how easily microassisted fertilization is accepted, such is the anxiety of the infertile couple to succeed. Fertilization is where the action is. Fertilization becomes the real focus of concern and the inconveniences of the IVF programme are relegated to secondary consideration. Almost irrespective of conservative counselling about the chances of success, microassisted fertilization is one situation where optimism reigns. The hopes are high but almost all patients are devastated by fertilization failure and describe themselves as 'useless' for several days. It is a severe grief, another failure, another end of the road and another prod of their infertility. 'Not even one (fertilized)' is a common response. One consequence of this focus on fertilization is a more direct relationship between the scientists responsible for microassisted fertilization and the couple. Effectively, this means that the scientists are more personally involved with couples and more immediately share the joys and disappointments. Microassisted fertilization must raise more hopes and more initial attempts at IVF. Currently, about 20% of IVF cycles are performed because of male factor infertility and this translates into about 4000 cycles per year in Australia alone. Most units offering microassisted fertilization find that about 25% of cycles are devoted to the procedure within a short period of time. This, together with the assessment of failed fertilization, places substantial burdens on laboratory staff and quickly demands appropriate divisions of labour. One consequence of the success of microassisted fertilization will be the push to recover more oocytes to improve the chances of embryo transfer. The danger lies in the percentage of couples requiring management for ovarian hyperstimulation, which is a significant morbidity. If the pregnancy
MICROASSISTED FERTILIZATION IN ASSISTED CONCEPTION
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potential of SUZI and ICSI is truly realized, this thrust will be quelled and may be replaced with reduced ovarian stimulation and a lessened number of treatment cycles required per couple. How results are obtained is another issue that impacts importantly on patients. Several units have utilized the tubal transfer of microassisted fertilized oocytes (McLachlan et al, 1994; Imoedemi et al, 1993). This is theoretically a hazardous procedure because of the risk of the development of polyspermic embryos, particularly with SUZI, after the injection of several sperm; however, nature's protective mechanisms may reduce the risk. In our hands using laparoscopy, conventional gamete intrafallopian transfer (GIFT) offers substantially improved chances of pregnancy per oocyte-embryo replaced compared with IVF (Wang et al, 1993a), but other studies have not shown any difference for male-factor couples (Tournaye et al, 1992). On the whole, GIFT appears unnecessary to obtain acceptable results with microassisted fertilization. Cryopreservation of embryos subjected to microassisted fertilization has proven to be possible (Wilton et al, 1989; Depypere et al, 1991). Cryopreservation of SUZI or ICSI embryos will have a considerable impact on patients, as it has had with conventional IVF. In our experience, couples with frozen embryos double the pregnancy rate for conventional IVF and enhance GIFT results by 15%. Almost 90% of the embryos are transferred within 3 years of a failed IVF cycle (non-ongoing pregnancy) or following the delivery of an IVF child (Wang et al, 1994). Countries with regulations limiting embryos to less than 10 years' storage will therefore impose unnecessary restrictions on the prospects for pregnancy from microassisted fertilization. The issue of congenital abnormality is significant to microassisted fertilization, but little evidence is yet available to suggest that rates are higher than normal. Since the frequency of birth defects in most advanced countries is 1-2%, the true incidence may be apparent (unless it is considerably increased) only with the delivery of several thousand babies. Only today, after a decade, can confidence be assured that conventional IVF is not hazardous. The issue of future morbidity or the transmission of defective genes from microassisted fertilization will have to await further studies. The rates of miscarriage will also be of interest, as it has recently been suggested that embryos derived from poor morphological sperm implant poorly irrespective of the method of microassisted fertilization (Palermo et al, 1993). It is almost impossible to discuss rationally the issue of expense. Clearly, the investment required for capital equipment, the training and skills necessary, and the time spent are considerable. Most considerate units will avoid charges until beneficial clinical practice can be claimed and then levy charges that allow fair and equal access of all patients to the programme. Costs in a practical sense are an important item for most couples and, yet, how does one place a value on a child? As previously noted, it is very important to avoid exploitation of the vulnerable couple with poor results. Finally, success with microassisted fertilization begins to reverse the need for programmes of donor sperm which must be at least theoretically
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beneficial given the complexities of achieving children from a donor source. This not inconsiderable impact will be welcomed by practitioners and counsellors, as well as by couples--the long-term adverse ramifications of not knowing or hiding biological origins will certainly be solved for successful couples. Microassisted fertilization is a major advance and has opened yet another door for infertile couples to succeed with their own gametes when little opportunity previously existed. It behoves the practitioner to evaluate carefully the process and the results as nature's method of working is surely being challenged.
SUMMARY Microassisted fertilization is fast becoming established as a routine clinical technique offering the chance of pregnancy to couples where none existed previously. The exceptional results of S U Z I and, more particularly, ICSI now raise the prospect of pregnancy with any sperm in the ejaculate and are bringing to treatment focus some azoospermic men and men without an antegrade ejaculate. The advance in clinical results from S U Z I and ICSI over earlier zona drilling and zona breaching techniques appear to be importantly related to the better implantation potential of the embryos. The implications for the way conventional I V F is performed still need to be digested but may be very important and may change our attitude to ovarian stimulation and insemination regimens.
Acknowledgements The author thanks Dianna Payne for excellent work and discussions and Carol Burford for typing the manuscript.
REFERENCES Aitken RJ, Buckingham D, West K et al (1992) Differential contribution of leukocytes and
spermatozoa to the generation of reactive oxygenspecies in ejaculates of oligozoospermic patients and fertile donors. Journal of Reproduction and Fertility 44: 451-462. Alikani M & Cohen J (1992) Advancesin clinical micromanipulationof gametes and embryos: assisted fertilization and hatching. Archives of Pathology and Laboratory Medicine 116: 373-378. Avery SM (1992) IVF and male infertility, Reproductive Medicine Review 1: 151-164. Bedford JM & Kim HH (1993) Sperm/eggbindingpatterns and oocyte culture in retrospective analysis of fertilization failure in vitro. Human Reproduction 8(3): 453-463. BongsoTA, Sathananthan AH, WongPC et al (1989) Human fertilisation by microinjectionof immotile spermatozoa. Human Reproduction 4" 175-179. Carlon N, Navarro A, Giorgettic & Roulier R (1992) Quantified ultrastructural study of spermatozoa in unexplained failure of in vitro fertilisation. Journal of Assisted Reproduction and Genetics 9(5): 475-481. Cohen J, Malter B, Elsner C et al (1990) Immunosuppressionsupports implantation of zona pellucida dissected human embryos. Fertility and Sterility 54: 662-665. Cohen J, Alikani M, Adler A et al (1992a) Microsurgical procedures: the absence of stringent criteria for patient selection. Journal of Assisted Reproduction and Genetics 9(3): 197-206.
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Cohen J, Malter HE & Talansky BE (1992b) Microsurgical fertilisation. In Brinsden P & Rainsbury PA (eds) In Vitro Fertilization and Assisted Reproduction, pp 205-226. Carnforth, Lancashire: Parthenon. Cui K-H, Verma PJ & Matthews CD (1993) Hatching rate: an optimal discriminator for the assessment of single blastomere biopsy. Journal of Assisted Reproduction and Genetics 10(2): 157-162. Depypere HT, McLaughlin K J, Seamark RF et al (1988) Comparison of zona cutting and zona drilling as techniques for assisted fertilization in the mouse. Journal of Reproduction and Fertility 84: 205-211. Depypere HT, Carroll J, Vanderkerckhove D & Matthews D (1991) Normal survival and in vitro development after cryopreservation of zona drilled embryos in mice. Human Reproduction 6(3): 432-435. Duncan WW, Glew MJ, Wang XJ et al (1993) Prediction of in vitro fertilization rates from semen variables. Fertility and Sterility 59(6): 1233--1235. Fishel S, Antinori S, Jackson P e t al (1990a) Twin birth after subzonal insemination. Lancet 335: 722-723. Fishel S, Jackson P, Antinori S et al (1990b) Subzonal insemination for the alleviation of infertility. Fertility and Sterility 54: 828-835. Fishel S, Antinori S, Jackson P e t al (1991) Presentation of six pregnancies established by sub-zonal insemination (SUZI). Human Reproduction 6: 124-130. Fishel S, Timson J, Lisi F & Rinaldi L (1992) Evaluation of 225 patients undergoing subzonal insemination for the procurement of fertilization in vitro. Fertility and Sterility 57:4): 840-849. Gordon JW & Talansky B E (1986) Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. Journal of Experimental Zoology 239: 347-354. Gwatkin RBL, Conover JC, Collins RL & Quigley MM (1989) Failed fertilization in human in vitro fertilization analysed with the deoxyribonucleic acid specific fluorochrome Hoechst 33342. American Journal of Obstetrics and Gynecology 160: 31-36. Imoedemi DAG, Sigue AB & Luciano EC (1993) Successful twin pregnancy and delivery after microinseminated oocyte fallopian transfer for male factor infertility. Fertility and Sterility 59(3): 662~63. Kirby CA, Flaherty SP, Godfrey BM et al (1991) A prospective trial of intrauterine insemination of motile spermatozoa versus timed intercourse. Fertility and Sterility 56(1): 102-107. Kruger TF, Acosta AA, Simmons KF et al (1988) Predictive value of abnormal semen morphology in in vitro fertilization. Fertility and Sterility 49: 112-117. Laufer N, Tarlatzis BC, DeCherney A H et al (1984) Asynchrony between human cumuluscorona cell complex and oocyte maturation after human menopause gonadotropin treatment for in vitro fertilisation. Fertility and Sterility 42: 366-372. Lipitz S, Ben-Rafael Z, Rabinovici J e t al (1993) Fertility and Sterility 59(2): 348-352. Liu DY & Baker HW (1992) Tests of human sperm function and fertilization in vitro. Fertility and Sterility 58(3): 465-483. McLachlan RI, Fuscaldo G, Calderon I e t al (1994) Microinjection and embryo/oocyte transfer. In Flaherty SP & Matthews CD (eds) The Infertile Male: Advanced Assisted Reproductive Technology. Melbourne: CSIRO. Mahadevan MM, Trounson AO, Wood C & Leeton JF (1987) Effect ofoocyte quality and sperm characteristics on the number of spermatozoa bound to the zona pellucida of human oocytes inseminated in vitro. Journal of ln Vitro Fertilization and Embryo Transfer 4: 223-227. Markert CL (1983) Fertilisation of mammalian eggs by sperm injection. Journal of Experimental Zoology 228: 195-201. Matthews CD (1990) The role of in vitro fertilization (1VF) in male infertility. Colloquium on Male Infertility, Canberra, 1989. Molloy D, Harrison K, Breen T & Hennessey J (1991) The predictive value of idiopathic failure to fertilise on the first in vitro fertilisation failure attempt. Fertility and Sterility 56: 285-289. Munne S & Estop AM (1993) Chromosomal analysis of human spermatozoa stored in vitro. Human Reproduction 8(4): 581-586. Ng SC, Bongso A, Ratnam SS et al (1988) Pregnancy after transfer of multiple sperm under the zona. Lancet ii: 790. Ord T, Patrizio P, Marello E et al (1990) A new method of semen preparation for IVF in severe male factor infertility. Human Reproduction 5: 987-989.
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Palermo G, Camus M, Joris H et al (1993) Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertility and Sterility 59(4): 826-835. Payne D, McLaughlin KJ, Depypere HT et al (1991a) Experience with zona drilling and zona cutting to improve fertilization rates of human oocytes in vitro. Human Reproduction 6(3): 423-431. Payne D, Warnes GM & Matthews CD (1991b) Oocyte abnormality as a factor contributing to fertilization failure in vitro. Proceedings of the Xth Annual Scientific Meeting of The Fertility Society of Australia, Lorne, Victoria, 113. Polge C, Smith A U & Parkes AS (1949) Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164: 666. Sanger WG, Olson JH & Sherman JK (1992) Semen cryobanking for men with cancer-criteria change. Fertility and Sterility 58(5): 1024-1027. Silber SJ (1979) Epididymal extravasation following vasectomy as a cause for failure of vasectomy reversal. Fertility and Sterility 30: 565-571. Silber SJ, Ord T, Balmaceda Jet al (1990) Congenital absence of the vas deferens: the fertilizing capacity of human epididymal sperm. New England Journal of Medicine 323: 1788-1792. Tesarik J, Pilka L & Travnik P (1988) Zona pellucida resistance of sperm penetration before the completion of human oocyte maturation. Journal of Reproduction and Fertility 83: 487495. Tournaye H, Devroey P, Camus M e t al (1992) Zygote intrafallopian transfer or in vitro fertilization and embryo transfer for the treatment of male factor infertility: a prospective randomised trial. Fertility and Sterility 58(2): 344-350. Tsunoda Y, Yasui T, Nakamura K et al (1986) Effect of cutting the zona pellucida in the pronuclear transplantation in the mouse. Journal of Experimental Zoology 240: 119--125. Van Steirteghem AC, Liu J, Joris H et al (1993a) Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. A report of a second series of 300 consecutive treatment cycles. Human Reproduction 8(8): 1055-1060. Van Steirteghem AC, Nagy Z, Joris H et al (1993b) High fertilization and implantation rates after intracytoplasmic sperm injection. Human Reproduction 8(8): 1061-1066. Wang XJ, Warnes GM, Kirby C et al (1993a) The outcome of GIFT following the elective or non-elective replacement of 2, 3 or 4 oocytes. Human Reproduction 8(8): 1231-1234. Wang XJ, Ledger W, Payne D et al (1994) The contribution of embryo cryopreservation to in vitro fertilization/gamete intra-fallopian transfer: 8 years experience. Human Reproduction 9(1): (in press). Wilton L, Shaw JM & Trounson AO (1989) Successful single cell biopsy and cryopreservation preimplantation mouse embryos. Fertility and Sterility 51: 513-517. World Health Organization (1987) WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Cambridge: Cambridge University Press. Young D (1970) Surgical treatment of male infertility. Journal of Reproduction and Fertility 23: 541-542.
Microassisted fertilization is poised at the exciting edge of current in vitro fertilization (IVF) technology and is set to traverse the interval between a research thrust confined to a small number of units and routine clinical practice. In the near future, reproductive programmes without access to microinjection will be offering only a limited service and failing to provide the proper range of options to infertile couples. This confidence in the place of microassisted fertilization stems from the markedly improved results of subzonal insemination (SUZI) and, more particularly, of intracytoplasmic sperm injection (ICSI) when compared to the older methodology of zona drilling and other zona breaching procedures. Fundamental to microassisted fertilization is that the human product is thus far indistinguishable from any other method of conception. The move to routine clinical medicine demands that more specific attention be paid to the clinical indications for the procedure and the general impact of the technology on patients. Given the still expanding role for conventional IVF, the demand for microassisted fertilization has been curtailed thus far only by the limited success of the procedure. Now that the results of microassisted fertilization, particularly ICSI, are so impressive, the demand is unlikely to be easily satisfied given the large contribution that infertile males make to the problem of infertility. Nevertheless, microassisted fertilization demands considerable specialized scientific skills, added to a consistent and experienced IVF system. It must always be remembered that the infertile couple is a vulnerable couple, willing to consider a variety of uncomfortable measures in their quest for a child, and must not be exploited by false hopes. A considerate professional unit will tailor the entry to microassisted fertilization to the results being achieved-with improving results widening the indications for treatment. Our own experience with zona drilling with the mouse model (Depypere et al, 1988) led us to believe that this was the optimal technique for microassisted fertilization in the human. Two techniques, zona drilling (ZD) (Gordon and Talansky, 1986) and zona cutting (ZC) (Tsunoda et al, 1986), were investigated to assess the effect of decreasing concentrations of normal epididymal sperm on fertilization rates, blastocyst formation and the establishment of pregnancy in the mouse. While both ZD and AC achieved Bailli~re"s Clinical Obstetrics and Gynaecology--
Vot. 8, No. 1, Marcht994 ISBN0-7020--1844-9
127 Copyright© 1994,by Bailli6reTindall All rights of reproductionin any formreserved
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fertilization at sperm concentrations associated with lack of fertilization of control (non-ZD or non-ZC) oocytes, ZD provided better results. Extrapolating these results to the human was, however, unrewarding. Twenty-seven patients undertook 40 cycles (249 oocytes) of ZD. The fertilization rate (24.5 %) was clearly increased over controls (8.3 %) but no pregnancies were achieved from the 21 transfers (mean 2.1 embryos). Evidence was apparent that the quality of the embryos was less than that in conventional IVF (Payne et al, 1991a) and ZD was abandoned in favour of ZC. In the first trial of zona cutting (ZC1), 22 patients undertook 23 cycles (147 oocytes), 47 (32%) of the oocytes fertilized and 15 patients received an embryo transfer (mean 2.2 embryos), but again no pregnancies were achieved. Once again, ZC was associated with relatively low numbers of quality embryos (6% grade 1), and a second trial (ZC2) was commenced which included sucrose shrinkage of the oocyte prior to micromanipulation together with immunosuppression and antibiotic regimens over the transfer period, following the report of Cohen et al (1990). Thirty-six patients undertook 36 cycles (233 oocytes), 27% of the oocytes fertilized and 19 women received an embryo transfer. One patient (5.3%) became pregnant, representing an implantation rate per transferred embryo of 2.6%. The rate of polyspermia was considerably higher (19%) than previously (6%). Because of the possibility that blastomeres were being extruded from the replaced embryos, a third trial (ZC3) was instituted and embryo transfer was delayed until day 3 to allow better compaction of the embryo. In this trial, 35 patients undertook 35 cycles (282 embryos) with a fertilization rate of 34% and 18 women receiving an embryo transfer (mean 2.3 embryos). Three patients (16.7) became pregnant, providing an implantation rate per embryo of 7.3%. Combining the results of these trials, 120 couples had undertaken 134 cycles of micromanipulation and 73 couples had an embryo transfer involving 157 embryos for four pregnancies, two of which ended in miscarriage (Table 1). In 1991, SUZI (Ng et al, 1988; Fishel et al, 1990a,b; 1991, 1992) was introduced to our programme with the injection of 3-12 spermatozoa depending on the general semen characteristics. In this trial 78 patients undertook 84 cycles (776 oocytes) with 34.4% of the oocytes fertilized. Fifty-two women received an embryo transfer (mean 2.0 embryos) and 16 (30.8 %) became pregnant, representing an implantation rate per embryo of 18.4%. Put another way, 5.4 SUZI embryos were replaced to achieve one implantation; this figure compared more than favourably with routine IVF in our own hands, when 10.4 transferred embryos are required to achieve each pregnancy. It must be recognized, however, that general IVF contains a wide spectrum of clinical conditions including older women and women who have repeatedly failed cycles. Clearly, a major clinical barrier has been broken with the advent of SUZI and the impressive aspect is the implantation potential of the transferred embryos. It is to be anticipated that, with the additional sophistication of ICSI to improve fertilization results without detracting from the implantation potential (Van Steirteghem et al, 1993a), clinical results can be anticipated rapidly to improve further. The downside of SUZI is very important. With few sperm injected,
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MICROASSISTED FERTILIZATION IN ASSISTED CONCEIrI'ION Table 1. Results of microassisted fertilization 1989-1993. ZD Number Number Number Number Number
(%)
couples cycles oocytes fertilized (%) monospermic
Polyspermic (%) Number couples with ET (%) Number embryos transferred Number embryos transferred Number pregnancies (% ET) Implantation rate per embryo transferred
ZC1
ZC2
ZC3
SUZI
ICSI
27 22 36 35 78 42 40 23 36 35 84 42 249 147 233 282 776 413 62 (24,5) 47 (32.0) 63 (27.0) 97 (34.3) 267 (34.4) 268 (64.9) 60 (97) 44 (94) 51 (81) 70 (72) 147 (55) 248 (60.0) 2 (3.2) 21
3 (6.4) 15
12 (19.0) 19
27 (27.8) 120 (44.9) *20 (7.5) 18 52 39 (92.9)
44
33
39
41
2.1
2.2
2.0
2.3
103 2.0
Nil
Nil
1 (5.3)
3 (16.6)
16 (30.8)
Nil
Nil
2.6
7.3
18.4
104 2.7 10 (25.6) N/A
(%)
ZD = zona drilling, ZC = zona cutting, SUZI = sub zonal sperm insemination, ICSI = intra cytoplasmic sperm injection 1, 2, and 3 refer to trials (see text). * 3PN were seen in 20 eggs despite 1 sperm only being injected into the egg. The 3rd PN is probably a result of failure to extrude the second polar body.
poorer rates of fertilization can be expected; if too many sperm are injected then the rate of polyspermic fertilization is increased dramatically. Both of these aspects reduce the embryos available for transfer. Even more important is the unpredictability of the SUZI procedure. While several attempts have been made to rationalize the number of sperm to be injected by morphological, motility and acrosome assessments of the spermatozoa to be injected, the ability to predict the result remains elusive (Cohen et at, 1992a). The initial cycle remains a trial with little correlation between fertilization and the semen characteristics. How can differences between ZC and SUZI be explained? It is very unlikely that the clinical spectrum of patients offers any significant explanation. Both ZC and SUZI are performed principally for male factor infertility and there is little to suggest that differences in the fertility of the female partner between series are marked. It should, however, be recognized that these clinical cases may represent 'pure' male infertility and the female side may be 'unadulterated' by unknown factors curtailing fertility. In other words, the female side may be even more normal than in 'unexplained' infertility, and perhaps this factor at least partially explains differences between microassisted fertilized embryos and conventional IVF. Clearly, there is a difference in the size of the zonal breach between ZC and SUZI, and it has been suggested that toxic elements from the uterus, including infective agents and leukocytes, can adversely affect embryo development after transfer. The larger the zonal defect, presumably the more opportunity there is for a more detrimental effect. It is also possible
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c.D. MATTHEWS
that some toxic elements may be operable during culture conditions of the early oocyte-embryo before transfer. The nature and size of the zona breach of the SUZI embryo may be more optimal once the embryo is replaced in the uterus compared with ZC, which may be associated with blastomere loss through the defect. Interestingly, our experience with blastocyst hatching following ZD and blastomere biopsy in the mouse suggests that hatching often takes place well away from the site of zona breach (Cui et al, 1993). A further factor related to the zonal breach is the possibility that cervical mucus collected inadvertently in the transfer catheter causes disruption of the embryo and the herniation of blastomeres through the zona during embryo transfer (Cohen et al, 1992b). One major difference between ZC (and conventional IVF) and SUZI is that ZC embryos are normally inseminated and cultured. It has been suggested that spermatozoa may be responsible for detrimental products gaining access to the embryo through the zonal disruption to cause adverse effects that are not obvious even by day 3 of embryo culture. Of the seminal products, reactive oxygen species are current agents under suspicion (Aitken et al, 1992). This idea urgently requires testing as there are considerable implications for conventional IVF. There are other differences to consider between microassisted fertilized embryos and those derived from conventional IVF, including earlier cumulus removal and exposure to hyaluronidase of the microassisted fertilized oocyte, which also may be important (Palermo et al, 1993). The welcome results with SUZI are already dated by the exceptional results being achieved from ICSI. Intracytoplasmic injection of sperm has a considerable history in non-human species (Markert, 1983) but until recently has always been associated with unacceptably high rates of damage to the oocyte. Improvements to the instrumental side and skills gained with micromanipulation have now reduced the damage rate of ICSI to less than 10% of oocytes. Recent results indicate that when a single spermatozoa is injected to a metaphase II oocyte (n = 1409) a 64.9% monospermic fertilization rate (uninfluenced by semen characteristics) can be obtained with 71.2% of fertilized oocytes reaching the cleavage stages. Ninety percent of couples (n = 135) received an embryo transfer (75% triple embryo transfer) with a clinical pregnancy rate of 39.2% per embryo transfer (n = 53). In addition, 237 supernumerary embryos were cryopreserved (Van Steirteghem et al, 1993b). Such results make a huge impact on the clinical indications for microassisted fertilization. Our own initial experience with ICSI has also demonstrated the exciting potential of this technique (Table 1). CLINICAL INDICATIONS FOR MICROASSISTED FERTILIZATION Failed fertilization
At present, microassisted fertilization is logically offered to couples who have had a total or near total failure of fertilization at conventional IVF.
MICROASSISTED FERTILIZATION IN ASSISTED CONCEPTION
131
This group is usefully divided into couples with a recognizable semen defect and those with more normal semen values. Failure of fertilization, particularly in the normospermic couple, may be due to oocyte factors that have been increasingly better recognized over the past few years. IVF has long been a very important option for male infertility (for review see Avery, 1992), One difficulty has been the inappropriateness of the World Health Organization (1987) criteria for semen analysis when IVF is being considered. The distinction between unexplained infertility and the male factor patient with the better semen analysis is blurred. However, very poor sperm analysis (fewer than 10 million sperm per ml, less than 10% motility and morphology parameters) is a common clinical problem and the likelihood of poor fertilization is much more predictable (Kruger et al, 1988). Until recently, the criteria for entry to conventional IVF in our unit has been the isolation of about 200 000 motile spermatozoa from pathological semen, usually after discontinuous Percol procedures (Ord et al, 1990; Kirby et al, 1991). This number of sperm will allow for the optimal insemination of the 6-8 oocytes expected to be recovered. In the absence of valid tests of sperm function, clearly, any arbitrary cut-off excludes semen samples that might achieve fertilization and includes samples that would be associated with failure of fertilization. With the availability of microassisted fertilization it would be advantageous to be able to recognize the semen sample that qualifies for conventional IVF but is destined to fail to fertilize oocytes. Many approaches, including the recognition of the state of the acrosome, objective assessment of motility, hamster oocyte penetration, acrosin activity, zona binding properties and conventional semen analysis (for review see Liu and Baker, 1992), have been examined in order to predict efficient fertilization. Unfortunately, no test thus far has gained sufficient clinical confidence to preclude the advice to attempt IVF. In an effort to refine our advice, we have recently examined the conventional semen parameters (World Health Organization, 1987) associated with 294 initial cycles of IVF performed for tubal infertility (to avoid unrecognized female factors) where at least four oocytes were available for fertilization (Duncan et al, 1993). The study included only those couples eligible for IVF (> 200 000 motile sperm after swim-up). Multiple regression analysis has allowed a distinction to be drawn between poor fertilization (<33%) and adequate fertilization (>33%) rates. The most helpful parameters were the combined indices of the percentage of progressive motile sperm in the isolated fraction and the percentage normal morphology of the fresh sample. Semen samples displaying <20% motility or < 20% normal morphology can be predicted to fail fertilization at conventional IVF with an 80% confidence. This prediction has also been tested prospectively with similar good results. A two-dimensional chart has been constructed (Figure 1) which may be helpful for programmes to assist the counselling of couples with the expected outcome of IVF. This ability to predict with some confidence allows for some flexibility of approach, particularly to the borderline semen sample in the presence of multiple oocytes. A combination of microassisted fertilization and conventional IVF may offer both improved diagnostic and therapeutic chances, even at the initial IVF procedure.
132
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relationship between the percentage normal morphology in fresh semen, the percentage motility in the isolated motile fraction and the fertilization rate. Cycles with fertilization rates < 35% (closed symbols) or ~>35% (open symbols) are indicated by different symbols. The curved line was predicted by the regression model; most cycles with a fertilization rate < 35% are below the line, whereas most cycles with a rate I> 35% are above it.
Failure of fertilization at conventional IVF may occur in the presence of 'normal' semen values. We analysed 303 unselected couples with normal semen values undertaking an initial single IVF cycle where at least 4 oocytes (median 5.4) were recovered. Nineteen subjects (6.3%) failed to fertilize oocytes. Of these 19, 13 undertook a further cycle of IVF and three again demonstrated failed fertilization (Matthews, 1990). Molloy et al (1991) and Lipitz et al (1993) have made similar observations. Thus, for the normospermic male the expectation of sporadic failed fertilization at conventional IVF is of the order of 6%, and about 1% of such normospermic men may consistently (at least two cycles) fail to achieve fertilization. The reason for failure is commonly related to the poor zona binding and penetration of sperm (Bedford and Kim, 1993), and ultrastructural anomalies of sperm not evident to light microscopy have been described (Carlon et al, 1992). Microassisted fertilization is dearly indicated for this group of normospermic males, but the outcome thus far from ZC and SUZI requires to be further evaluated before being proven effective. Alikani and Cohen (1992) have reported a reduced pregnancy rate from microassisted fertilization when performed for couples with normal semen. Failure of fertilization may be due to oocyte factors that may operate associated with poor semen values, but perhaps more often associated with normal semen. When the oocyte has poor cytoplasmic-cumulus quality, a poor expectation of fertilization is likely (Laufer et al, 1984; Mahadevan et al, 1987). This failure may be due to general immaturity (germinal vesicle or
MICROASSISTED FERTILIZATION IN ASSISTED CONCEPTION
133
metaphase-I oocytes) (Gwatkin et al, 1989) or as a consequence of abnormalities of the zona structure (Tesarik et al, 1988). Bedford and Kim (1993) have drawn attention to the fact that cytoplasmic abnormalities are more common in cases of failure of fertilization in the presence of normal semen values, compared with oocytes failing to become fertilized with defective semen. We have also tried to estimate the contribution that oocyte factors make to fertilization failure and have described different morphological abnormalities of oocytes that have failed to fertilize, including thickened and thinned zona, vaculated, granular and fragmented cytoplasm, or fertilization anomalies. In a study of 503 unfertilized oocytes examined by Nomarski optics, 39% were judged abnormal. More than one half (57%) of the oocytes had a zona pellucida disorder, 31% had cytoplasmic disorders, 7% had fertilization abnormalities and only 7% were immature. When semen abnormalities were taken into account, the incidence of oocyte abnormalities was judged to be 7% in the presence of abnormal semen and 61% in normospermic conditions. Since disorders of the zona constituted the largest grouping, microassisted fertilization techniques would seem to offer considerable possibilities (Payne et al, 1991b). The recognition of the contribution of oocyte abnormalities to total or partial fertilization failure is important as microassisted fertilization may not achieve such favourable results with defective oocytes as in true semen factor infertility with normal oocytes. Finally, it should be mentioned that a place for microassisted fertilization during the second day of culture has been suggested for failed conventional fertilization in order to rescue oocytes. No large series has yet been described. Clearly, there is a need for a precise scientific assessment of cycles associated with failed fertilization. The number, maturity and morphological appearance of the oocytes require to be documented, together with the outcome of the procedure including rates of unfertilized, monospermic and polyspermic oocytes-embryos plus some assessment of sperm binding. This full documentation may be vital to the arrangements for the next cycle. Failure to qualify for conventional IVF
In addition to the group of patients who have attempted IVF, there are those couples whose semen values fail to qualify for conventional IVF. There are a large group of men with unexplained defective semen, usually with normal or slightly raised follicle stimulating hormone (FSH) levels. Commonly there is no history of relevance, and physical examination is not contributory. Many semen samples fail to qualify for IVF because it is not possible to isolate an adequate number of motile sperm, and a common clinical finding is sperm apparent only after centrifugation of the semen. The requirement for so many sperm to fertilize the oocyte under in vitro conditions remains somewhat unclear, although numbers of sperm appear to be required to allow passage through the egg vestments. Included in this group of men with poor semen are males who have undergone cytotoxic treatment, often during pubertal years, for haematological malignancies and whose spermatogenic processes are now suboptimal.
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c.D. MA'ITHEWS
These men usually have raised FSH levels with minimal semen activity. Not too dissimilar are men who have had radiotherapy and cytotoxic treatment for malignant tumours of the testis. Coincidently, microassisted fertilization brings a different attitude towards the cryopreservation of semen from similar men with testicular cancer, Hodgkin's disease and other forms of cancer. It is common to reject semen samples from such men because of the poor post-thaw results. IVF has already played an important role by achieving pregnancies for such men who not uncommonly have poor semen (Sanger et al, 1992) but microassisted fertilization offers even more. The recovery of only a few sperm from the post-thaw sample may be the difference between sterility and fertility. Another condition that would preclude admission to an IVF programme is globozoospermia--complete absence of the acrosome. Although such cases apparently fail to achieve fertilization after SUZI, there is the possibility of procuring conception by ICSI. The lack of the appropriate fusion proteins, which might occur in globozoospermia, might be circumvented by ICSI. Finally, there are many men, often in an older age group, who have had reversal of vasectomy performed with poor results. This may be related to the reparative surgery itself or to epididymal pathology as a consequence of long-term vasectomy (Silber, 1979). The challenge of these men is now being addressed by SUZI and ICSI, given the need of those procedures for very few sperm. Most of these couples currently constitute the demand for donor semen, and this group contains couples for whom microassisted fertilization is set to make the most dramatic impact as pregnancies can now be anticipated from any ejaculate that contains any live sperm. It will, however, require considerable collective experience to determine whether spermatozoa derived from such seriously defective ejaculates have an equal potential for fertilization, pregnancy and normal children as from the normospermic sample. While it would seem feasible to expect pregnancies from immotile but live sperm (Bongso et al, 1989), it is not yet clear whether dead sperm have DNA in an appropriate state that pregnancies can also be achieved. It is to be noted that spermatozoa contain no reparative DNA repair enzymes (Munne and Estop, 1993).
Azoospermic and anejaculate males There are several other significant groups of subjects who appear to be on the verge of qualifying for microassisted fertilization. These include some azoospermic males and men who are unable to produce an ejaculate, often from post-surgical or neurological causes. These categories are now beginning to come into focus for treatment. Not uncommonly do azoospermic males have normal spermatogenesis within the testis but with obstruction within the epididymis as a result of previous infection or the after-effects of biochemical malfunction due to genetic defects (commonly mutations of the cystic fibrosis gene) or associated with bronchiectasis (Young, 1970). In addition, at least 5% of azoospermic males have congenital absence of the whole or part of the vas.
MICROASSISTED FERTILIZATION IN ASSISTED CONCEPTION
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This latter syndrome has been shown to be amenable to synchronous epididymal sperm recovery and IVF-GIFT techniques (Silber et al, 1990), but the results have not been easily reduplicated. The combination of microassisted fertilization with epididymal aspiration would seem to offer real prospects for such couples. In addition, there now exists the possibility for cryopreservation of epididymal spermatozoa. The application of conventional glycerol-based cryopreservation techniques shown useful for ejaculated sperm (Polge et al, 1949) is not generally useful for epididymal sperm. However, if only a few sperm are now required, or perhaps only preserved DNA, then the prospect for avoiding repetitive epididymal surgical procedures may be real. Not least are those males who have ejaculatory dysfunction associated with retrograde ejaculation. We have already achieved a pregnancy for a couple using several spermatozoa injected subzonally following retrograde ejaculation and recovery from urine. Such cases illustrate the revolution that microassisted fertilization can bring to otherwise very exceptionally difficult cases. Even though microassisted fertilization is a very young scientific initiative, the above traditional ways of considering clinical indications should now be re-examined in the light of the exceptional results of ICSI. If the results of ICSI can be shown to be repeatable in many other units, the pertinent question is which males do not qualify for the procedure? Perhaps the only males not able to be considered are those with absent or severe defects of spermatogenesis itself. The ICSI results, particularly the implantation potential, also pose questions for the performance of conventional IVF as it is practised today. PATIENT IMPACT With the exception of certain hereditary diseases, a child of one's own genetic material is always the first quest for couples, and microassisted fertilization now offers reasonable opportunities for pregnancy where none existed previously. What is this quest for one's own genes? The perception and influence of genes project not only forward to future generations but backwards from grandparents. The features and mannerisms of children are often easily recognizable as one's own or one's partner by extended family and close friends. This visual continuity is often comforting and a source of pride. Commonly, marriage and partnership bring an unexplainable sense of wanting to create children, not just to promote a family but a family as part of one's life partner. There is a strong innate female sense of wishing to provide 'fatherhood' for her mate rather than simply building a family. What then is this fatherhood? Perhaps it is a sense of being responsible for, whatever the product. A willingness to stand up for and to accept. Not simply an opportunity to nurture and provide an environment for a child to develop its gifts, but a chance to have played a role and to have left something of one's own individuality in the world when long departed. A child of one's genes may be at less risk of harm or disinterest than a child
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derived from a donor, but this risk will require years of study to prove or disprove. For the male, microassisted fertilization must offer the chance of a 'cure' for a problem until now regarded as 'hopeless', a fulfilling of the sexual relationship and the chance to remove the cloud of having to consider a donor. The total number of couples who consider and reject the use of donor sperm is not easily acquired but most couples who reject donor insemination will be concerned with their attitudes toward a donor child or because of the unknown risks of transmissible agents heightened by the recent controversies of HIV and Creutzfeldt-Jakob disease. Very few will reject donor insemination because of the medical technology involved. What proportion of infertile couples due to the male will be attracted by microassisted fertilization remains to be seen, and will be greatly influenced by the success rates of the procedure. Countries and states with reproductive legislation will commonly demand appropriate ethical approval before microassisted fertilization is instituted. In our experience, the principal ethical concerns have been the prospect for success, the waste of oocytes and embryos through technical damage and polyspermia, and the risk of congenital abnormality. Microassisted fertilization makes considerable demands on clinical time to explain risks, consent forms, chances of success, expense and general support and explanation at failure. Nevertheless, the clinician is often surprised how easily microassisted fertilization is accepted, such is the anxiety of the infertile couple to succeed. Fertilization is where the action is. Fertilization becomes the real focus of concern and the inconveniences of the IVF programme are relegated to secondary consideration. Almost irrespective of conservative counselling about the chances of success, microassisted fertilization is one situation where optimism reigns. The hopes are high but almost all patients are devastated by fertilization failure and describe themselves as 'useless' for several days. It is a severe grief, another failure, another end of the road and another prod of their infertility. 'Not even one (fertilized)' is a common response. One consequence of this focus on fertilization is a more direct relationship between the scientists responsible for microassisted fertilization and the couple. Effectively, this means that the scientists are more personally involved with couples and more immediately share the joys and disappointments. Microassisted fertilization must raise more hopes and more initial attempts at IVF. Currently, about 20% of IVF cycles are performed because of male factor infertility and this translates into about 4000 cycles per year in Australia alone. Most units offering microassisted fertilization find that about 25% of cycles are devoted to the procedure within a short period of time. This, together with the assessment of failed fertilization, places substantial burdens on laboratory staff and quickly demands appropriate divisions of labour. One consequence of the success of microassisted fertilization will be the push to recover more oocytes to improve the chances of embryo transfer. The danger lies in the percentage of couples requiring management for ovarian hyperstimulation, which is a significant morbidity. If the pregnancy
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potential of SUZI and ICSI is truly realized, this thrust will be quelled and may be replaced with reduced ovarian stimulation and a lessened number of treatment cycles required per couple. How results are obtained is another issue that impacts importantly on patients. Several units have utilized the tubal transfer of microassisted fertilized oocytes (McLachlan et al, 1994; Imoedemi et al, 1993). This is theoretically a hazardous procedure because of the risk of the development of polyspermic embryos, particularly with SUZI, after the injection of several sperm; however, nature's protective mechanisms may reduce the risk. In our hands using laparoscopy, conventional gamete intrafallopian transfer (GIFT) offers substantially improved chances of pregnancy per oocyte-embryo replaced compared with IVF (Wang et al, 1993a), but other studies have not shown any difference for male-factor couples (Tournaye et al, 1992). On the whole, GIFT appears unnecessary to obtain acceptable results with microassisted fertilization. Cryopreservation of embryos subjected to microassisted fertilization has proven to be possible (Wilton et al, 1989; Depypere et al, 1991). Cryopreservation of SUZI or ICSI embryos will have a considerable impact on patients, as it has had with conventional IVF. In our experience, couples with frozen embryos double the pregnancy rate for conventional IVF and enhance GIFT results by 15%. Almost 90% of the embryos are transferred within 3 years of a failed IVF cycle (non-ongoing pregnancy) or following the delivery of an IVF child (Wang et al, 1994). Countries with regulations limiting embryos to less than 10 years' storage will therefore impose unnecessary restrictions on the prospects for pregnancy from microassisted fertilization. The issue of congenital abnormality is significant to microassisted fertilization, but little evidence is yet available to suggest that rates are higher than normal. Since the frequency of birth defects in most advanced countries is 1-2%, the true incidence may be apparent (unless it is considerably increased) only with the delivery of several thousand babies. Only today, after a decade, can confidence be assured that conventional IVF is not hazardous. The issue of future morbidity or the transmission of defective genes from microassisted fertilization will have to await further studies. The rates of miscarriage will also be of interest, as it has recently been suggested that embryos derived from poor morphological sperm implant poorly irrespective of the method of microassisted fertilization (Palermo et al, 1993). It is almost impossible to discuss rationally the issue of expense. Clearly, the investment required for capital equipment, the training and skills necessary, and the time spent are considerable. Most considerate units will avoid charges until beneficial clinical practice can be claimed and then levy charges that allow fair and equal access of all patients to the programme. Costs in a practical sense are an important item for most couples and, yet, how does one place a value on a child? As previously noted, it is very important to avoid exploitation of the vulnerable couple with poor results. Finally, success with microassisted fertilization begins to reverse the need for programmes of donor sperm which must be at least theoretically
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beneficial given the complexities of achieving children from a donor source. This not inconsiderable impact will be welcomed by practitioners and counsellors, as well as by couples--the long-term adverse ramifications of not knowing or hiding biological origins will certainly be solved for successful couples. Microassisted fertilization is a major advance and has opened yet another door for infertile couples to succeed with their own gametes when little opportunity previously existed. It behoves the practitioner to evaluate carefully the process and the results as nature's method of working is surely being challenged.
SUMMARY Microassisted fertilization is fast becoming established as a routine clinical technique offering the chance of pregnancy to couples where none existed previously. The exceptional results of S U Z I and, more particularly, ICSI now raise the prospect of pregnancy with any sperm in the ejaculate and are bringing to treatment focus some azoospermic men and men without an antegrade ejaculate. The advance in clinical results from S U Z I and ICSI over earlier zona drilling and zona breaching techniques appear to be importantly related to the better implantation potential of the embryos. The implications for the way conventional I V F is performed still need to be digested but may be very important and may change our attitude to ovarian stimulation and insemination regimens.
Acknowledgements The author thanks Dianna Payne for excellent work and discussions and Carol Burford for typing the manuscript.
REFERENCES Aitken RJ, Buckingham D, West K et al (1992) Differential contribution of leukocytes and
spermatozoa to the generation of reactive oxygenspecies in ejaculates of oligozoospermic patients and fertile donors. Journal of Reproduction and Fertility 44: 451-462. Alikani M & Cohen J (1992) Advancesin clinical micromanipulationof gametes and embryos: assisted fertilization and hatching. Archives of Pathology and Laboratory Medicine 116: 373-378. Avery SM (1992) IVF and male infertility, Reproductive Medicine Review 1: 151-164. Bedford JM & Kim HH (1993) Sperm/eggbindingpatterns and oocyte culture in retrospective analysis of fertilization failure in vitro. Human Reproduction 8(3): 453-463. BongsoTA, Sathananthan AH, WongPC et al (1989) Human fertilisation by microinjectionof immotile spermatozoa. Human Reproduction 4" 175-179. Carlon N, Navarro A, Giorgettic & Roulier R (1992) Quantified ultrastructural study of spermatozoa in unexplained failure of in vitro fertilisation. Journal of Assisted Reproduction and Genetics 9(5): 475-481. Cohen J, Malter B, Elsner C et al (1990) Immunosuppressionsupports implantation of zona pellucida dissected human embryos. Fertility and Sterility 54: 662-665. Cohen J, Alikani M, Adler A et al (1992a) Microsurgical procedures: the absence of stringent criteria for patient selection. Journal of Assisted Reproduction and Genetics 9(3): 197-206.
MICROASSISTED FERTILIZATION IN ASSISTED CONCEPTION
139
Cohen J, Malter HE & Talansky BE (1992b) Microsurgical fertilisation. In Brinsden P & Rainsbury PA (eds) In Vitro Fertilization and Assisted Reproduction, pp 205-226. Carnforth, Lancashire: Parthenon. Cui K-H, Verma PJ & Matthews CD (1993) Hatching rate: an optimal discriminator for the assessment of single blastomere biopsy. Journal of Assisted Reproduction and Genetics 10(2): 157-162. Depypere HT, McLaughlin K J, Seamark RF et al (1988) Comparison of zona cutting and zona drilling as techniques for assisted fertilization in the mouse. Journal of Reproduction and Fertility 84: 205-211. Depypere HT, Carroll J, Vanderkerckhove D & Matthews D (1991) Normal survival and in vitro development after cryopreservation of zona drilled embryos in mice. Human Reproduction 6(3): 432-435. Duncan WW, Glew MJ, Wang XJ et al (1993) Prediction of in vitro fertilization rates from semen variables. Fertility and Sterility 59(6): 1233--1235. Fishel S, Antinori S, Jackson P e t al (1990a) Twin birth after subzonal insemination. Lancet 335: 722-723. Fishel S, Jackson P, Antinori S et al (1990b) Subzonal insemination for the alleviation of infertility. Fertility and Sterility 54: 828-835. Fishel S, Antinori S, Jackson P e t al (1991) Presentation of six pregnancies established by sub-zonal insemination (SUZI). Human Reproduction 6: 124-130. Fishel S, Timson J, Lisi F & Rinaldi L (1992) Evaluation of 225 patients undergoing subzonal insemination for the procurement of fertilization in vitro. Fertility and Sterility 57:4): 840-849. Gordon JW & Talansky B E (1986) Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. Journal of Experimental Zoology 239: 347-354. Gwatkin RBL, Conover JC, Collins RL & Quigley MM (1989) Failed fertilization in human in vitro fertilization analysed with the deoxyribonucleic acid specific fluorochrome Hoechst 33342. American Journal of Obstetrics and Gynecology 160: 31-36. Imoedemi DAG, Sigue AB & Luciano EC (1993) Successful twin pregnancy and delivery after microinseminated oocyte fallopian transfer for male factor infertility. Fertility and Sterility 59(3): 662~63. Kirby CA, Flaherty SP, Godfrey BM et al (1991) A prospective trial of intrauterine insemination of motile spermatozoa versus timed intercourse. Fertility and Sterility 56(1): 102-107. Kruger TF, Acosta AA, Simmons KF et al (1988) Predictive value of abnormal semen morphology in in vitro fertilization. Fertility and Sterility 49: 112-117. Laufer N, Tarlatzis BC, DeCherney A H et al (1984) Asynchrony between human cumuluscorona cell complex and oocyte maturation after human menopause gonadotropin treatment for in vitro fertilisation. Fertility and Sterility 42: 366-372. Lipitz S, Ben-Rafael Z, Rabinovici J e t al (1993) Fertility and Sterility 59(2): 348-352. Liu DY & Baker HW (1992) Tests of human sperm function and fertilization in vitro. Fertility and Sterility 58(3): 465-483. McLachlan RI, Fuscaldo G, Calderon I e t al (1994) Microinjection and embryo/oocyte transfer. In Flaherty SP & Matthews CD (eds) The Infertile Male: Advanced Assisted Reproductive Technology. Melbourne: CSIRO. Mahadevan MM, Trounson AO, Wood C & Leeton JF (1987) Effect ofoocyte quality and sperm characteristics on the number of spermatozoa bound to the zona pellucida of human oocytes inseminated in vitro. Journal of ln Vitro Fertilization and Embryo Transfer 4: 223-227. Markert CL (1983) Fertilisation of mammalian eggs by sperm injection. Journal of Experimental Zoology 228: 195-201. Matthews CD (1990) The role of in vitro fertilization (1VF) in male infertility. Colloquium on Male Infertility, Canberra, 1989. Molloy D, Harrison K, Breen T & Hennessey J (1991) The predictive value of idiopathic failure to fertilise on the first in vitro fertilisation failure attempt. Fertility and Sterility 56: 285-289. Munne S & Estop AM (1993) Chromosomal analysis of human spermatozoa stored in vitro. Human Reproduction 8(4): 581-586. Ng SC, Bongso A, Ratnam SS et al (1988) Pregnancy after transfer of multiple sperm under the zona. Lancet ii: 790. Ord T, Patrizio P, Marello E et al (1990) A new method of semen preparation for IVF in severe male factor infertility. Human Reproduction 5: 987-989.
140
c.D.
MATTHEWS
Palermo G, Camus M, Joris H et al (1993) Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertility and Sterility 59(4): 826-835. Payne D, McLaughlin KJ, Depypere HT et al (1991a) Experience with zona drilling and zona cutting to improve fertilization rates of human oocytes in vitro. Human Reproduction 6(3): 423-431. Payne D, Warnes GM & Matthews CD (1991b) Oocyte abnormality as a factor contributing to fertilization failure in vitro. Proceedings of the Xth Annual Scientific Meeting of The Fertility Society of Australia, Lorne, Victoria, 113. Polge C, Smith A U & Parkes AS (1949) Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164: 666. Sanger WG, Olson JH & Sherman JK (1992) Semen cryobanking for men with cancer-criteria change. Fertility and Sterility 58(5): 1024-1027. Silber SJ (1979) Epididymal extravasation following vasectomy as a cause for failure of vasectomy reversal. Fertility and Sterility 30: 565-571. Silber SJ, Ord T, Balmaceda Jet al (1990) Congenital absence of the vas deferens: the fertilizing capacity of human epididymal sperm. New England Journal of Medicine 323: 1788-1792. Tesarik J, Pilka L & Travnik P (1988) Zona pellucida resistance of sperm penetration before the completion of human oocyte maturation. Journal of Reproduction and Fertility 83: 487495. Tournaye H, Devroey P, Camus M e t al (1992) Zygote intrafallopian transfer or in vitro fertilization and embryo transfer for the treatment of male factor infertility: a prospective randomised trial. Fertility and Sterility 58(2): 344-350. Tsunoda Y, Yasui T, Nakamura K et al (1986) Effect of cutting the zona pellucida in the pronuclear transplantation in the mouse. Journal of Experimental Zoology 240: 119--125. Van Steirteghem AC, Liu J, Joris H et al (1993a) Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. A report of a second series of 300 consecutive treatment cycles. Human Reproduction 8(8): 1055-1060. Van Steirteghem AC, Nagy Z, Joris H et al (1993b) High fertilization and implantation rates after intracytoplasmic sperm injection. Human Reproduction 8(8): 1061-1066. Wang XJ, Warnes GM, Kirby C et al (1993a) The outcome of GIFT following the elective or non-elective replacement of 2, 3 or 4 oocytes. Human Reproduction 8(8): 1231-1234. Wang XJ, Ledger W, Payne D et al (1994) The contribution of embryo cryopreservation to in vitro fertilization/gamete intra-fallopian transfer: 8 years experience. Human Reproduction 9(1): (in press). Wilton L, Shaw JM & Trounson AO (1989) Successful single cell biopsy and cryopreservation preimplantation mouse embryos. Fertility and Sterility 51: 513-517. World Health Organization (1987) WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Cambridge: Cambridge University Press. Young D (1970) Surgical treatment of male infertility. Journal of Reproduction and Fertility 23: 541-542.