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The role of assisted conception techniques in male infertility
Current Obstetrics & Gynaecology (1996) 6, 18-23 © 1996 Pearson ProfessionaI Ltd
Mini-symposium: Male infertility
The role of assisted conception techniques in male infertility
S. Thornton, S. Fishel
In particular, the relative paucity of long-term follow-up data remains a concern and to this has to be added the very high cost of treatment. While some have suggested that ICSI should be the only treatment offered in assisted conception clinics, the principles of good medical practice would support a more cautious approach with ICSI reserved for the more severe cases at the extreme end of the sub-fertility spectrum.
It is estimated that in 30% of infertile couples, there is an identifiable defect either in functional competence or production of sperm. Although in the majority of sub-fertile men no discrete cause can be identified, modern molecular genetic techniques are beginning to shed light on the causes of sperm disorders in some men. Genes responsible for spermatogenesis have been located on the Y chromosome and microdeletions found in 15% of men with severe oligo- and azoospermia.1, 2 It is also of considerable concern that data are accumulating showing a decline in sperm counts over recent decades in westernised countries. No single aetiological factor has been identified to account for this decline though exposure of the male fetus in utero to environmental pollutants with oestrogen-like activity has been suggested. Ironically, as we begin to understand the molecular basis of male factor infertility, its management has been transformed by the arrival of a new empiric therapeutic technique (intracytoplasmic sperm injection-ICSI), where a single sperm is microinjected directly into the oocyte. The results with ICSI do not depend either on the original diagnosis or indeed on the severity of the sperm picture (provided sufficient sperm can actually be recovered to inject collected oocytes). Although the spread of ICSI through infertility clinics internationally has been phenomenal, there are still major concerns about treating all sperm problems with this method.
Patient assessment
There is a regrettable tendency in modern infertility practice to treat the man as a 'sperm count'. A careful history and physical examination is important to exclude the relatively few, but sometimes reversible, disorders that may effect semen and also to identify potentially serious associated conditions, such as testicular tumours. Having said that, the semen analysis remains the mainstay of male factor infertility diagnosis. Internationally recognised 'normal' criteria have been defined by the World Health Organization (Table 1). 3 It is extremely important to identify problems in production, collection or processing which can cause spurious diagnoses of male infertility. The two most common spurious diagnoses are incomplete collection or delays in processing which can lead to a very marked reduction in motility. With the developments in computer-aided sperm analysis (CASA) sperm count, motility and morphological characteristics can now all be assessed automatically and objectively. The equipment is costly but CASA has considerable advantages in a busy laboratory in terms of processing speed and also controlling for inter- and intra- observer variables. Recently, the morphological characteristics of
Dr Simon Thornton MA, MD, MRCOG, MHSM, Medical Director, Dr Simon Fishel PhD, Scientific Director, Nottingham University Research and Treatment Unit in Reproduction (NURTURE), Department of Obstetrics and Gynaecology, Floor B, East Block, Queen's Medical Centre, University Hospital, Nottingham NG7 2UH, UK Correspondence to: S. J. T 18
The role of assisted conception techniques in male infertility Table 1--WHO criteria for a 'normal' sperm count
2ml or more 7.2-8.0 20 x 106/mlor more Abnormal: azoospermia(no sperm) or oligozoospermia(reducednumbers) Motility: 50% or more with forwardprogressionor 25% or more with rapid progression (within 60 minutes of ejaculation) (Abnormal: asthenozoospermia) Morphology: 30% or more normal forms (Abnormal: teratozoospermia) (14%using Kruger strict criteria) MAR test: Fewer than 10% of sperm with adherent particles Immunobead test: Fewer than 20% of sperm with adherent particles
Volume: pH: Count:
sperm (particularly using the method of Kruger strict criteria) have had a dramatic impact on management of patients requiring assisted conception, especially those with severe male factor problems. The morphology can help to discriminate between couples who may benefit from in-vitro fertilisation (IVF) or those who require ICSI. A morphology of less than 5% has been shown to have a high risk of fertilisation failure with routine IVF. 4 In addition to the 'basic' semen analysis there are several more specialised sperm function which may more accurately define some aspects of the functional performance of sperm. These include the hamster egg penetration test, acrosome reaction ionophore challenge (ARIC) test, tests of sperm/mucus penetration and assays for reactive oxygen species. All of these tests have been developed because the absolute predictive value of the semen analysis is relatively poor in relation to fertility potential, either spontaneously or following assisted conception treatment. Unfortunately, this battery of tests, many of which can be extremely expensive, suffer from similar drawbacks to the basic semen analysis. As a result, many clinics have now largely abandoned the battery of sperm function tests and, after the standard basic investigations are completed, offer couples IVF both as a therapeutic but also as a diagnostic procedure. The ultimate test of a couples' fertility being IVF, in which the processes of sperm/egg interaction following gamete mixing may be observed closely. The role of specialised sperm function tests is further challenged by ICSI, the success of which is essentially independent of sperm function test results, which are designed to assess either the factors in transporting sperm through the female genital tract or the physiological events associated with fertilisation (Table 2). ICSI by its very nature, bypasses the transport aspect of sperm function totally and also most of the physiological processes preceeding fertilisation.
ing hormone (FSH), luteinizing hormone (LH), testosterone and prolactin may be useful in the elucidation of possible causes of hypogonadotrophic hypogonadism though the majority of these cases are clinically recognisable. Classically, FSH levels have been used to discriminate between the two common groups of azoospermic men: those with obstruction or those with testicular failure. Obstructive azoospermia is classically associated with a normal FSH, normal testicular volume and a normal or low ejaculatory volume. Testicular biopsy in such men would be expected to show normal spermatogenesis. In men with testicular failure, the picture is one with FSH levels greater than twice the upper limit of the normal range, the testes are small and testicular biopsy shows absent spermatogenesis. Unfortunately, the discrete separation of cases of azoospermia into these categories has been found to be fraught with problems with many men with maturation arrest on testicular biopsy having normal or only slightly reduced testicular volumes and normal FSH. In addition, many men with elevated FSH, azoospermia and small testes (even with histology suggesting Sertoli-only syndrome) have been shown to have small foci of spermatogenesis in the testes from which sperm can be recovered for use in the ICSI procedure. Selection of treatment for patients
In individual cases the optimal treatment for a couple will depend on a variety of factors (Table 3). Each of these has to be weighed up carefully with the couples and this is particularly important because the relative priority for different factors may differ between couples. For example, one couple who live near to the clinic may be prepared to come through for a cycle of IVF using a high insemination concentration, in spite of adverse sperm parameters, because they are concerned about the potential costs and unassessed long term hazards of ICSI, even though they recognise the risk of a possible failed fertilisation with IVE Another couple from overseas with the same sperm parameters might feel that, because of the logistic Table 2~Physiologicalevents associatedwith fertilisation
• Zona pellucida-spermbinding • Acrosomereaction • Spermhyperactivation • Focal digestion of the zona pellucida • Penetrationof the zona pellucida • Oocyte-spermmembranefusion • Membranehyperpolarisation
Other tests
Other tests have a relatively limited role to play in the assessment of male factor infertility. Follicle stimulat-
19
• Egg activation • Calciumdischarges • Corticalgranule release
20
Current Obstetrics & Gynaecology
Table3 Factorsto be takeninto considerationwhenconsidering treatmentoptions Duration of infertility Age of woman Previous spontaneousconception Outcomeof previousIVF attempts(eg fertilisationor failedfertilisation) Costs and availableresources Willingnessto acceptfailedfertilisation Ease of accessto furthertreatmentcycles Relativeconcernoverlongterm safetyof technique Religiousbeliefs Semenparameters on day of oocyterecovery (especiallymorphology) Oocytenumberson day of oocyterecovery
difficulties, they only have one chance at achieving a pregnancy so want ICSI which will virtually 'guarantee' them fertilisation and embryo transfer. It will be seen that the range of issues to consider in individual cases is wide and often extends beyond the realms of scientific evaluation. It is important for the couple to be provided with adequate information, ideally both verbal and written, from which to formulate their own views. Although much of this can be done at the time of consultation, evaluation of the sperm sample used for treatment and oocyte numbers on the day may require quite difficult re-evaluation and consideration after oocyte recovery. This important approach has led to > 97% of all couples achieving fertilisation at our centre. It is therefore very important that adequate 'scenario setting' is explored at the consultation so that couples can attempt to clarify their view, based on the likely range of scenarios that may arise on the day of oocyte recovery.
Treatment options Conventional IVF Conventional IVF was first reported in the successful management of oligozoospermia in 19825 and since then there have been many reports of successful fertilisation and delivery of normal, healthy babies resulting in IVF with semen samples that were either oligo, astheno or teratozoospermic or indeed with combinations of the three. In seeking to identify sub-groups of cases in which conventional IVF alone may be of value, Kruger et al4 made an important contribution by defining, using their strict morphological criteria, some of the prognostic limits for conventional IVF. In general terms, < 5% of normal forms will produce a fertilisation rate per oocyte of < 10%, compared with a fertilisation rate per oocyte of > 60% when the morphology is > 10% normal forms. Hence, conventional IVF is best restricted to cases where there are > 10% normal forms on repeated ejaculates, the duration of sub-fertility is short and there is no previous history of failed fertilisation. The use of conventional IVF
would be supported by previous Spontaneous conception or fertilisation in vitro.
High insemination concentration IVF (IVF-HIC) High insemination concentration IVF (IVF-HIC) may be useful for those patients where there is an acceptable sperm density and motility (mild to moderate oligoasthenozoospermia) with or without varying degrees of teratozoospermia. As many of these couples will in fact achieve fertilisation and embryo transfer, on the grounds of cost, many couples elect to use this approach in their first treatment cycle attempt. In this technique, the oocyte cumulus complex is added to a suspension of sperm with a concentration between 0.5-2x106 motile spermatozoa per ml, depending on the severity of the condition. IVF-HIC is useful in cases of teratozoospermia only where there was a previous failure of conventional IVF 6 and we have also recently demonstrated the value in cases of very severe teratozoospermia (morphology < 5% normal forms). 7 However, there has been some suggestion that in spite of acceptable fertilisation and cleavage rates, there is a risk of poorer embryo quality with an increased incidence of cytoplasmic fragmentation in embryos inseminated with high insemination concentration IVF in comparison with a sibling oocyte inseminated with ICSI. 8 We have proposed that the defects observed in embryo quality with IVF-HIC may be due to high concentrations of motile spermatozoa, immotile spermatozoa and other seminal debris present in the insemination medium. It is likely that such cellular products may raise the concentration of reactive oxygen species to supra-physiological levels, leading to damage to cell membranes. Thus poor quality embryos are probably associated with the aforementioned rather than fertilisation with morphologically abnormal sperms. Ord et al9 treated a group of patients with total motile Count in the pre-treatment sample of less than 5 million/ml and showed that 80% of patients and 38% of oocytes achieved fertilisation. The pregnancy rate per embryo transfer was 43%, demonstrating the relative efficacy. One therapeutic strategy that has proved invaluable in terms of addressing both the diagnostic uncertainties but also maximising the therapeutic potentials is to randomly assign the sibling oocytes at the time of egg recovery to IVF-HIC and ICSI. If fertilisation results from IVF-HIC this provides important diagnostic information for the couple as subsequent cycles, if necessary, should require IVF-HIC only (rather than the more expensive ICSI cycles). In addition, they have the benefit of having IVF embryos (about which a considerable amount of safety data and long term follow up exists) to transfer. However, in the event of a fertilisation failure from IVF, there should be fertilised embryos from ICSI. This approach inevitably provides the maximum amount of
The role of assisted conception techniques in male infertility 21 diagnostic information for couples whilst seeking to provide the optimum therapeutic balance in a first treatment cycle. Microassisted fertilisation
In recent years a variety of approaches have been developed to assist fertilisation in severe cases of male factor infertility. Major approaches have been: • Partial zona dissection (PZD). • Sub-zonal insemination (SUZI). • Intracytoplasmic sperm injection (ICSI). Clinical microassisted fertilisation was first recorded in 1985 by Metka et al. I° In 1987, fertilisation using the SUZI technique was demonstrated 11 and this was followed the following year by Lanzendorf's report of the direct injection of sperm into the cytoplasm of the oocyte.12This is the procedure now known internationally as ICSI. During this period there were other approaches to breaching the zona pellucida. In zona drilling a small hole is made in the zona pellucida either by enzyme or acid culture medium. The principle being that motile spermatozoa could then find their way through the breach if there was a dysfunction in sperm zona binding or penetration. In 1988, Ng et al ~3 reported the first human pregnancy after SUZI, followed in 1990 by Fishe114 reporting the birth of a set of normal healthy twins and a singleton after SUZI. However, publications by Palermo et a115,16 showed that ICSI represented a major step forward from the fertilisation and pregnancy rates achievable with earlier microinjection techniques. As a result, SUZI and PZD have largely been abandoned though there may be a role for the SUZI procedure, at least in certain cases. By depositing spermatozoa in the perivitelline space with subsequent sperm egg fusion, SUZI permits many of the normal physiological events of sperm selection and ultimately fertilisation (Table 2). In a recent study, using a novel approach developed at NURTURE, Computer Image Sperm Selection (CISS) technology has been linked to the SUZI technique. This procedure combines real-time image analysis of individual hyperactivated spermatozoa with the SUZ1 approach.17 The specific parameters associated with hyperactivation and the overall pattern of sperm motility of the selected sperms are considered to be a prerequisite for the acrosome reaction and sperm/egg fusion. Using this approach, we have been able to significantly increase the fertilisation rate using the SUZI technique, though there is still a gap between this and the fertilisation and pregnancy rates that we now see in our ICSI programme. Whether this technique will enable us to more objectively select 'good quality' sperm for 1CSI remains to be demonstrated.
Intracytoplasmic Sperm Injection (ICSI) 1992 saw a major transition point in clinical micro-
injection technology. The report by Palermo et a116 comparing SUZI and ICSI in couples with severely impaired semen parameters demonstrated fertilisation rates in 60% of injected oocytes and four pregnancies resulted from eight treatment cycles: two healthy boys were delivered from singleton pregnancies and a healthy boy and girl from a twin pregnancy. Further studies have demonstrated the value of ICSI in very severe cases of male factor infertility. ICSI has now gained worldwide acceptance as the ultimate microassisted approach to male factor infertility. Several groups now have considerable experience with ICSI and most expect a high incidence of fertilisation per oocyte (> 50%), per patient (> 90%) and a clinical pregnancy rate in excess of 25%. Provided that sperm can actually be recovered, the major limitation on success is technical rather than the condition of the sperm or from where it was extracted. Pregnancies have been reported in cases with 100% abnormal forms, spermatozoa obtained from the testes, spermatozoa obtained from men with extremely high follicle stimulating hormone and/or cryptozoospermia and globozoospermia. From a technical point of view two particular factors appear vital to the success of the technique. The first is successful penetration of the oolema and the second is the necessity to permanently immobilise the sperm prior to injection into the ooplasm. 18 Providing that permanent immobilisation of the sperm and full penetration of the oolema has occurred, fertilisation will occur in > 50% of mature oocytes. Failure of fertilisation may arise as a result of sperm residing in the furrow of the oolema, or lying free in the ooplasm but failing to activate the egg or undergo sperm/head decondensation.
Surgical sperm recovery and ICSI As the ICSI technique has evolved so the range of cases amenable to treatment has broadened. Surgical sperm recovery techniques have been used in cases of obstructive azoospermia to obtain mature spermatozoa from the epididymes by microepididymal sperm aspiration (MESA). This is an open approach to expose the epididymes and sperm are recovered microsurgically under direct vision. Simplification of this approach has been demonstrated by Craft et al, 19 with a percutaneous approach using simple apparatus with a butterfly needle and a syringe to generate negative pressure to aspirate sperm directly from the epididymes (PESA). The success of techniques of epididymal sperm recovery led inevitably to attempts to recover mature spermatozoa from the testes and in 1993 Schoysman et aF ° reported the first pregnancy from testicular spermatozoa. Although the majority o f initial cases of surgical sperm recovery were men suffering from obstructive azoospermia, sperm recovery from the epididymis or testis, by either open or percutaneous approaches, has been used for men with non-obstructive azoospermia
22
Current Obstetrics & Gynaecology
(including cases with cryptozoospermia but small foci of spermatozoa locatable in the testes). As between 5% and 10% of men having a vasectomy request a reversal, the field of surgical sperm recovery technique linked to ICSI is currently seeing phenomenal and dramatic development.
ICSI with immature sperm As much of the maturation and development of spermatazoa is dedicated to developing the processes necessary for sperm transport and oocyte penetration it was suggested that spermatids, containing a haploid chromosome compliment, might also be usable for fertility treatment in cases of maturation arrest. In 1995 we reported the first pregnancy (and livebirth in 1996) using elongated spermatids obtained from the testis, 21 Sofikitis et a122reported pregnancies from testicular round spermatid nuclear injunction (ROSNI) and Tesarik et aF 3 reported the first live births from ejaculated round spermatids injection (ROSI). Considerable work remains to be done to clarify the safety and efficacy of this approach to the most extreme form of male subfertility.
Risks of ICSI In spite of the rapid uptake of ICSI internationally, significant uncertainties remain about the longer term risks and hazards of the procedure. Thus far, initial paediatric follow up has been relatively reassuring. In a follow up study of 877 children, 24 major congenital abnormalities were found in 23 (2.6%) of the cases which is well within the expected population ranges (2-3.8%). A recent report from Holland 25 has, however, demonstrated a marked excess of sex chromosome abnormalities in offspring resulting from ICSI (five abnormal karyotypes in 15 fetuses). More reassuring data comes from the Brussels series of 585 fetal karyotypes which revealed only 5 (0.8%) de novo sex chromosome abnormalities. However, Moosani et a126 have shown, using individual sperm karyotyping, a significant increase in the frequency of XY disomy which may well account for some of the apparent increased incidence of sex chromosome anomalies seen in the ICSI cases from Holland. Finally, it is widely anticipated that, as many cases of male infertility may have a genetic component, the male offspring conceived as a result of ICSI will have a markedly increased risk of male factor infertility themselves. In addition to these issues, the oldest babies born as a result of ICSI treatment are only approaching their fourth birthday and longer term follow up studies, similar to those initiated for in vitro fertilisation in the early 1980s are obviously going to be important to establish the 'long term' safety and efficacy of the technique. U n t r e a t a b l e Infertility
In spite of the phenomenal developments in the field,
many couples remain untreatable. With advances in treatment this is now rarely due to pathology (e.g. aspermatogenesis). Many couples are limited by their financial resources to fund the complex and costly techniques which are rarely available through the National Health Service. Such couples require considerable and sensitive counselling. The availability of alternative options, such as adoption or the use of donor gametes should be explored with the couple. Conclusion
Assisted conception techniques, beginning with IVF and leading through to the microassisted fertilisation techniques, have resulted in the delivery of children to infertile men whose only other option previously had been adoption or the use of donor sperm. In our own practice we have seen that the increasing use of microinjection has dramatically reduced the number of cases requiring donor insemination. Progressive development however will be slow unless N H S funding for ICSI is provided universally and specialists begin to refer their patients to appropriate centres. In spite of the phenomenal success rates achievable with ICSI (now comparable to those achievable with standard IVF) concerns about the safety and costs remain. References
1. Ma K, Inglis JD, SharkeyA et al. A Y chromosomegene familywith RNA-binding protein homology;candidates for the azoospermiafactor (AZF) controllinghuman spermatogenesis.Cell 1993;75(7): 1287-1295 2. RiejoLR, Lee T-Y,Salo Pet al. Diversespermatogenicdefects in humans caused by Y chromosomedeletionsencompassing a novel RNA-binding protein gene. Nature Genetics 1995; 10: 383-393 3. WorldHealth Organization. WHO Laboratory manual for the examination of Human Semenand Sperm-CervicalMucus Interaction (3rd edn). Cambridge: CambridgeUniversity Press, 1992 4. KrugerTF, Acosta AA, Simmons KE Predictedvalue of abnormal sperm morphologyin IVF. Fertil Steril 1988;49: 11~117 5. Fishel SB and Edwards RG. Essentials of fertilisation.In: Edwards RG, Purdy JM (eds) Human Conceptionin vitro. London: AcademicPress 1982:157-159 6. OehningerS, Acosta AA, Morshedi Met al. Corrective measures and pregnancyoutcome in IVF in patients with sperm morphologyabnormalities. Fertil Steril 1988; 150: 283-287 7. Hall J, Fishel S, Green Set al. Intracytoplasmicsperm injection (ICSI) versus high inseminationconcentration(HIC) .IVF in cases of very severeteratozoospermia0/ST). Hum Reprod 1995; 10(3):493-496 8. Fishel S, Lisi F, Rinaldi L, Thornton Set al. ICSI versus high insemination concentration(HIC) for human conceptionin vitro. Reprod Fertil Dev 1995;7:169-175 9. Ord T, Patrizio P, BalmacedaJP et al. Can severemale-factor infertilitybe treated without micromanipulation?Fertil Steril 1993; 60:100 115 10. Metka M, Haromy T, Huper Jet al. Artificialinsemination using a micromanipulator.Fertilitat 1985; 1:41-47 11. Laws-KingA, TrounsonA, Sathananthan H et al. Fertilisation of human oocyte by micro-injectionof a single spermatozoonunder the zona pellucida. Fertil Steril 1987;48: 637-642 12. Lanzendorf E, Maloney MK, Veek LL. A preclinical evaluation of human spermatozoainto human oocytes.Fertil Steril 1988;49:835-842
The role of assisted conception techniques in male infertility 23 13. Ng S-C, Bongso TA, Ratnam SS et al. Pregnancy after transfer of multiple sperm under the zona. Lancet 1988; 2:790 14. Fishel SB, Antinori S, Jackson Pet al. Twin birth after sub-zonal insemination. Lancet 1990; 2:722 15. Palermo G, Joris H, Derde M-P et al. Sperm characteristics and outcome of human-assisted fertilisation by sub-zonal insemination and intracytoplasmic sperm injection. Fertil Steril 1993; 59:826-835 16. Palermo G, Joris H, Devroey Pet al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340:1%18 17. Green S, Fishel S, Hobson Get al. Computer image sperm selection (CISS) as a novel approach to sub-zonal insemination. Hum Reprod 1995; 10(6): 1430-1434 18. Fishel S, Lisi F, Rinaldi Let al. Systematic examination of immobilising spermatozoa before intracytoplasmic sperm injection in the human. Hum Reprod 1995; 10(3): 49%500 19. Craft I, Tsirigotis M, Bennett Vet aL Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia. Fertil Steril 1995; 63:1038-1042
20. Schoysman R, Vanderzwalmen P, Nij Met al. Pregnancy after fertilisation with testicular spermatozoa. Lancet 1993; 342:1237 21. Fishel S, Green S, Bishop Met al. Pregnancy after intracytoplasmic injection of spermatid. Lancet 1995; 345: 1641-1642 22. Sofikitis N, Mayagawa I, Sharlip Iet al. Human pregnancies achieved by intra-ooplasmic injections of round spermatid nuclei isolated from testicular tissue of azoospermic men (Abs). J Urol 1995; 153(4): 302A 23. Tesarik J, Mendoza C, Testart J. Viable embryos from injection of round spermatids into oocytes. N Engl J Med 1995; 333:525 24. Van Steirteghem A. Follow up of 877 children born following intracytoplasmic sperm injection. Keynote Address, American Society for Reproductive Medicine meeting, Seattle, 7-12 October 1995 25. In't Veld P, Brandenburg H, Verhoeff Aet al. Sex chromosome abnormalities in pregnancies estabished by intracytoplasmic sperm injection and in vitro fertilisation (IVF-ICSI). Lancet 1995; 346:773 26. Moosani N, Cox D, Pattinson H et al. Chromosomal analysis of sperm from men with idopathic infertility using sperm karyotyping and fluorescence in situ hybridisation. Fertil Steril 1995; 64(4): 811-817
Mini-symposium: Male infertility
The role of assisted conception techniques in male infertility
S. Thornton, S. Fishel
In particular, the relative paucity of long-term follow-up data remains a concern and to this has to be added the very high cost of treatment. While some have suggested that ICSI should be the only treatment offered in assisted conception clinics, the principles of good medical practice would support a more cautious approach with ICSI reserved for the more severe cases at the extreme end of the sub-fertility spectrum.
It is estimated that in 30% of infertile couples, there is an identifiable defect either in functional competence or production of sperm. Although in the majority of sub-fertile men no discrete cause can be identified, modern molecular genetic techniques are beginning to shed light on the causes of sperm disorders in some men. Genes responsible for spermatogenesis have been located on the Y chromosome and microdeletions found in 15% of men with severe oligo- and azoospermia.1, 2 It is also of considerable concern that data are accumulating showing a decline in sperm counts over recent decades in westernised countries. No single aetiological factor has been identified to account for this decline though exposure of the male fetus in utero to environmental pollutants with oestrogen-like activity has been suggested. Ironically, as we begin to understand the molecular basis of male factor infertility, its management has been transformed by the arrival of a new empiric therapeutic technique (intracytoplasmic sperm injection-ICSI), where a single sperm is microinjected directly into the oocyte. The results with ICSI do not depend either on the original diagnosis or indeed on the severity of the sperm picture (provided sufficient sperm can actually be recovered to inject collected oocytes). Although the spread of ICSI through infertility clinics internationally has been phenomenal, there are still major concerns about treating all sperm problems with this method.
Patient assessment
There is a regrettable tendency in modern infertility practice to treat the man as a 'sperm count'. A careful history and physical examination is important to exclude the relatively few, but sometimes reversible, disorders that may effect semen and also to identify potentially serious associated conditions, such as testicular tumours. Having said that, the semen analysis remains the mainstay of male factor infertility diagnosis. Internationally recognised 'normal' criteria have been defined by the World Health Organization (Table 1). 3 It is extremely important to identify problems in production, collection or processing which can cause spurious diagnoses of male infertility. The two most common spurious diagnoses are incomplete collection or delays in processing which can lead to a very marked reduction in motility. With the developments in computer-aided sperm analysis (CASA) sperm count, motility and morphological characteristics can now all be assessed automatically and objectively. The equipment is costly but CASA has considerable advantages in a busy laboratory in terms of processing speed and also controlling for inter- and intra- observer variables. Recently, the morphological characteristics of
Dr Simon Thornton MA, MD, MRCOG, MHSM, Medical Director, Dr Simon Fishel PhD, Scientific Director, Nottingham University Research and Treatment Unit in Reproduction (NURTURE), Department of Obstetrics and Gynaecology, Floor B, East Block, Queen's Medical Centre, University Hospital, Nottingham NG7 2UH, UK Correspondence to: S. J. T 18
The role of assisted conception techniques in male infertility Table 1--WHO criteria for a 'normal' sperm count
2ml or more 7.2-8.0 20 x 106/mlor more Abnormal: azoospermia(no sperm) or oligozoospermia(reducednumbers) Motility: 50% or more with forwardprogressionor 25% or more with rapid progression (within 60 minutes of ejaculation) (Abnormal: asthenozoospermia) Morphology: 30% or more normal forms (Abnormal: teratozoospermia) (14%using Kruger strict criteria) MAR test: Fewer than 10% of sperm with adherent particles Immunobead test: Fewer than 20% of sperm with adherent particles
Volume: pH: Count:
sperm (particularly using the method of Kruger strict criteria) have had a dramatic impact on management of patients requiring assisted conception, especially those with severe male factor problems. The morphology can help to discriminate between couples who may benefit from in-vitro fertilisation (IVF) or those who require ICSI. A morphology of less than 5% has been shown to have a high risk of fertilisation failure with routine IVF. 4 In addition to the 'basic' semen analysis there are several more specialised sperm function which may more accurately define some aspects of the functional performance of sperm. These include the hamster egg penetration test, acrosome reaction ionophore challenge (ARIC) test, tests of sperm/mucus penetration and assays for reactive oxygen species. All of these tests have been developed because the absolute predictive value of the semen analysis is relatively poor in relation to fertility potential, either spontaneously or following assisted conception treatment. Unfortunately, this battery of tests, many of which can be extremely expensive, suffer from similar drawbacks to the basic semen analysis. As a result, many clinics have now largely abandoned the battery of sperm function tests and, after the standard basic investigations are completed, offer couples IVF both as a therapeutic but also as a diagnostic procedure. The ultimate test of a couples' fertility being IVF, in which the processes of sperm/egg interaction following gamete mixing may be observed closely. The role of specialised sperm function tests is further challenged by ICSI, the success of which is essentially independent of sperm function test results, which are designed to assess either the factors in transporting sperm through the female genital tract or the physiological events associated with fertilisation (Table 2). ICSI by its very nature, bypasses the transport aspect of sperm function totally and also most of the physiological processes preceeding fertilisation.
ing hormone (FSH), luteinizing hormone (LH), testosterone and prolactin may be useful in the elucidation of possible causes of hypogonadotrophic hypogonadism though the majority of these cases are clinically recognisable. Classically, FSH levels have been used to discriminate between the two common groups of azoospermic men: those with obstruction or those with testicular failure. Obstructive azoospermia is classically associated with a normal FSH, normal testicular volume and a normal or low ejaculatory volume. Testicular biopsy in such men would be expected to show normal spermatogenesis. In men with testicular failure, the picture is one with FSH levels greater than twice the upper limit of the normal range, the testes are small and testicular biopsy shows absent spermatogenesis. Unfortunately, the discrete separation of cases of azoospermia into these categories has been found to be fraught with problems with many men with maturation arrest on testicular biopsy having normal or only slightly reduced testicular volumes and normal FSH. In addition, many men with elevated FSH, azoospermia and small testes (even with histology suggesting Sertoli-only syndrome) have been shown to have small foci of spermatogenesis in the testes from which sperm can be recovered for use in the ICSI procedure. Selection of treatment for patients
In individual cases the optimal treatment for a couple will depend on a variety of factors (Table 3). Each of these has to be weighed up carefully with the couples and this is particularly important because the relative priority for different factors may differ between couples. For example, one couple who live near to the clinic may be prepared to come through for a cycle of IVF using a high insemination concentration, in spite of adverse sperm parameters, because they are concerned about the potential costs and unassessed long term hazards of ICSI, even though they recognise the risk of a possible failed fertilisation with IVE Another couple from overseas with the same sperm parameters might feel that, because of the logistic Table 2~Physiologicalevents associatedwith fertilisation
• Zona pellucida-spermbinding • Acrosomereaction • Spermhyperactivation • Focal digestion of the zona pellucida • Penetrationof the zona pellucida • Oocyte-spermmembranefusion • Membranehyperpolarisation
Other tests
Other tests have a relatively limited role to play in the assessment of male factor infertility. Follicle stimulat-
19
• Egg activation • Calciumdischarges • Corticalgranule release
20
Current Obstetrics & Gynaecology
Table3 Factorsto be takeninto considerationwhenconsidering treatmentoptions Duration of infertility Age of woman Previous spontaneousconception Outcomeof previousIVF attempts(eg fertilisationor failedfertilisation) Costs and availableresources Willingnessto acceptfailedfertilisation Ease of accessto furthertreatmentcycles Relativeconcernoverlongterm safetyof technique Religiousbeliefs Semenparameters on day of oocyterecovery (especiallymorphology) Oocytenumberson day of oocyterecovery
difficulties, they only have one chance at achieving a pregnancy so want ICSI which will virtually 'guarantee' them fertilisation and embryo transfer. It will be seen that the range of issues to consider in individual cases is wide and often extends beyond the realms of scientific evaluation. It is important for the couple to be provided with adequate information, ideally both verbal and written, from which to formulate their own views. Although much of this can be done at the time of consultation, evaluation of the sperm sample used for treatment and oocyte numbers on the day may require quite difficult re-evaluation and consideration after oocyte recovery. This important approach has led to > 97% of all couples achieving fertilisation at our centre. It is therefore very important that adequate 'scenario setting' is explored at the consultation so that couples can attempt to clarify their view, based on the likely range of scenarios that may arise on the day of oocyte recovery.
Treatment options Conventional IVF Conventional IVF was first reported in the successful management of oligozoospermia in 19825 and since then there have been many reports of successful fertilisation and delivery of normal, healthy babies resulting in IVF with semen samples that were either oligo, astheno or teratozoospermic or indeed with combinations of the three. In seeking to identify sub-groups of cases in which conventional IVF alone may be of value, Kruger et al4 made an important contribution by defining, using their strict morphological criteria, some of the prognostic limits for conventional IVF. In general terms, < 5% of normal forms will produce a fertilisation rate per oocyte of < 10%, compared with a fertilisation rate per oocyte of > 60% when the morphology is > 10% normal forms. Hence, conventional IVF is best restricted to cases where there are > 10% normal forms on repeated ejaculates, the duration of sub-fertility is short and there is no previous history of failed fertilisation. The use of conventional IVF
would be supported by previous Spontaneous conception or fertilisation in vitro.
High insemination concentration IVF (IVF-HIC) High insemination concentration IVF (IVF-HIC) may be useful for those patients where there is an acceptable sperm density and motility (mild to moderate oligoasthenozoospermia) with or without varying degrees of teratozoospermia. As many of these couples will in fact achieve fertilisation and embryo transfer, on the grounds of cost, many couples elect to use this approach in their first treatment cycle attempt. In this technique, the oocyte cumulus complex is added to a suspension of sperm with a concentration between 0.5-2x106 motile spermatozoa per ml, depending on the severity of the condition. IVF-HIC is useful in cases of teratozoospermia only where there was a previous failure of conventional IVF 6 and we have also recently demonstrated the value in cases of very severe teratozoospermia (morphology < 5% normal forms). 7 However, there has been some suggestion that in spite of acceptable fertilisation and cleavage rates, there is a risk of poorer embryo quality with an increased incidence of cytoplasmic fragmentation in embryos inseminated with high insemination concentration IVF in comparison with a sibling oocyte inseminated with ICSI. 8 We have proposed that the defects observed in embryo quality with IVF-HIC may be due to high concentrations of motile spermatozoa, immotile spermatozoa and other seminal debris present in the insemination medium. It is likely that such cellular products may raise the concentration of reactive oxygen species to supra-physiological levels, leading to damage to cell membranes. Thus poor quality embryos are probably associated with the aforementioned rather than fertilisation with morphologically abnormal sperms. Ord et al9 treated a group of patients with total motile Count in the pre-treatment sample of less than 5 million/ml and showed that 80% of patients and 38% of oocytes achieved fertilisation. The pregnancy rate per embryo transfer was 43%, demonstrating the relative efficacy. One therapeutic strategy that has proved invaluable in terms of addressing both the diagnostic uncertainties but also maximising the therapeutic potentials is to randomly assign the sibling oocytes at the time of egg recovery to IVF-HIC and ICSI. If fertilisation results from IVF-HIC this provides important diagnostic information for the couple as subsequent cycles, if necessary, should require IVF-HIC only (rather than the more expensive ICSI cycles). In addition, they have the benefit of having IVF embryos (about which a considerable amount of safety data and long term follow up exists) to transfer. However, in the event of a fertilisation failure from IVF, there should be fertilised embryos from ICSI. This approach inevitably provides the maximum amount of
The role of assisted conception techniques in male infertility 21 diagnostic information for couples whilst seeking to provide the optimum therapeutic balance in a first treatment cycle. Microassisted fertilisation
In recent years a variety of approaches have been developed to assist fertilisation in severe cases of male factor infertility. Major approaches have been: • Partial zona dissection (PZD). • Sub-zonal insemination (SUZI). • Intracytoplasmic sperm injection (ICSI). Clinical microassisted fertilisation was first recorded in 1985 by Metka et al. I° In 1987, fertilisation using the SUZI technique was demonstrated 11 and this was followed the following year by Lanzendorf's report of the direct injection of sperm into the cytoplasm of the oocyte.12This is the procedure now known internationally as ICSI. During this period there were other approaches to breaching the zona pellucida. In zona drilling a small hole is made in the zona pellucida either by enzyme or acid culture medium. The principle being that motile spermatozoa could then find their way through the breach if there was a dysfunction in sperm zona binding or penetration. In 1988, Ng et al ~3 reported the first human pregnancy after SUZI, followed in 1990 by Fishe114 reporting the birth of a set of normal healthy twins and a singleton after SUZI. However, publications by Palermo et a115,16 showed that ICSI represented a major step forward from the fertilisation and pregnancy rates achievable with earlier microinjection techniques. As a result, SUZI and PZD have largely been abandoned though there may be a role for the SUZI procedure, at least in certain cases. By depositing spermatozoa in the perivitelline space with subsequent sperm egg fusion, SUZI permits many of the normal physiological events of sperm selection and ultimately fertilisation (Table 2). In a recent study, using a novel approach developed at NURTURE, Computer Image Sperm Selection (CISS) technology has been linked to the SUZI technique. This procedure combines real-time image analysis of individual hyperactivated spermatozoa with the SUZ1 approach.17 The specific parameters associated with hyperactivation and the overall pattern of sperm motility of the selected sperms are considered to be a prerequisite for the acrosome reaction and sperm/egg fusion. Using this approach, we have been able to significantly increase the fertilisation rate using the SUZI technique, though there is still a gap between this and the fertilisation and pregnancy rates that we now see in our ICSI programme. Whether this technique will enable us to more objectively select 'good quality' sperm for 1CSI remains to be demonstrated.
Intracytoplasmic Sperm Injection (ICSI) 1992 saw a major transition point in clinical micro-
injection technology. The report by Palermo et a116 comparing SUZI and ICSI in couples with severely impaired semen parameters demonstrated fertilisation rates in 60% of injected oocytes and four pregnancies resulted from eight treatment cycles: two healthy boys were delivered from singleton pregnancies and a healthy boy and girl from a twin pregnancy. Further studies have demonstrated the value of ICSI in very severe cases of male factor infertility. ICSI has now gained worldwide acceptance as the ultimate microassisted approach to male factor infertility. Several groups now have considerable experience with ICSI and most expect a high incidence of fertilisation per oocyte (> 50%), per patient (> 90%) and a clinical pregnancy rate in excess of 25%. Provided that sperm can actually be recovered, the major limitation on success is technical rather than the condition of the sperm or from where it was extracted. Pregnancies have been reported in cases with 100% abnormal forms, spermatozoa obtained from the testes, spermatozoa obtained from men with extremely high follicle stimulating hormone and/or cryptozoospermia and globozoospermia. From a technical point of view two particular factors appear vital to the success of the technique. The first is successful penetration of the oolema and the second is the necessity to permanently immobilise the sperm prior to injection into the ooplasm. 18 Providing that permanent immobilisation of the sperm and full penetration of the oolema has occurred, fertilisation will occur in > 50% of mature oocytes. Failure of fertilisation may arise as a result of sperm residing in the furrow of the oolema, or lying free in the ooplasm but failing to activate the egg or undergo sperm/head decondensation.
Surgical sperm recovery and ICSI As the ICSI technique has evolved so the range of cases amenable to treatment has broadened. Surgical sperm recovery techniques have been used in cases of obstructive azoospermia to obtain mature spermatozoa from the epididymes by microepididymal sperm aspiration (MESA). This is an open approach to expose the epididymes and sperm are recovered microsurgically under direct vision. Simplification of this approach has been demonstrated by Craft et al, 19 with a percutaneous approach using simple apparatus with a butterfly needle and a syringe to generate negative pressure to aspirate sperm directly from the epididymes (PESA). The success of techniques of epididymal sperm recovery led inevitably to attempts to recover mature spermatozoa from the testes and in 1993 Schoysman et aF ° reported the first pregnancy from testicular spermatozoa. Although the majority o f initial cases of surgical sperm recovery were men suffering from obstructive azoospermia, sperm recovery from the epididymis or testis, by either open or percutaneous approaches, has been used for men with non-obstructive azoospermia
22
Current Obstetrics & Gynaecology
(including cases with cryptozoospermia but small foci of spermatozoa locatable in the testes). As between 5% and 10% of men having a vasectomy request a reversal, the field of surgical sperm recovery technique linked to ICSI is currently seeing phenomenal and dramatic development.
ICSI with immature sperm As much of the maturation and development of spermatazoa is dedicated to developing the processes necessary for sperm transport and oocyte penetration it was suggested that spermatids, containing a haploid chromosome compliment, might also be usable for fertility treatment in cases of maturation arrest. In 1995 we reported the first pregnancy (and livebirth in 1996) using elongated spermatids obtained from the testis, 21 Sofikitis et a122reported pregnancies from testicular round spermatid nuclear injunction (ROSNI) and Tesarik et aF 3 reported the first live births from ejaculated round spermatids injection (ROSI). Considerable work remains to be done to clarify the safety and efficacy of this approach to the most extreme form of male subfertility.
Risks of ICSI In spite of the rapid uptake of ICSI internationally, significant uncertainties remain about the longer term risks and hazards of the procedure. Thus far, initial paediatric follow up has been relatively reassuring. In a follow up study of 877 children, 24 major congenital abnormalities were found in 23 (2.6%) of the cases which is well within the expected population ranges (2-3.8%). A recent report from Holland 25 has, however, demonstrated a marked excess of sex chromosome abnormalities in offspring resulting from ICSI (five abnormal karyotypes in 15 fetuses). More reassuring data comes from the Brussels series of 585 fetal karyotypes which revealed only 5 (0.8%) de novo sex chromosome abnormalities. However, Moosani et a126 have shown, using individual sperm karyotyping, a significant increase in the frequency of XY disomy which may well account for some of the apparent increased incidence of sex chromosome anomalies seen in the ICSI cases from Holland. Finally, it is widely anticipated that, as many cases of male infertility may have a genetic component, the male offspring conceived as a result of ICSI will have a markedly increased risk of male factor infertility themselves. In addition to these issues, the oldest babies born as a result of ICSI treatment are only approaching their fourth birthday and longer term follow up studies, similar to those initiated for in vitro fertilisation in the early 1980s are obviously going to be important to establish the 'long term' safety and efficacy of the technique. U n t r e a t a b l e Infertility
In spite of the phenomenal developments in the field,
many couples remain untreatable. With advances in treatment this is now rarely due to pathology (e.g. aspermatogenesis). Many couples are limited by their financial resources to fund the complex and costly techniques which are rarely available through the National Health Service. Such couples require considerable and sensitive counselling. The availability of alternative options, such as adoption or the use of donor gametes should be explored with the couple. Conclusion
Assisted conception techniques, beginning with IVF and leading through to the microassisted fertilisation techniques, have resulted in the delivery of children to infertile men whose only other option previously had been adoption or the use of donor sperm. In our own practice we have seen that the increasing use of microinjection has dramatically reduced the number of cases requiring donor insemination. Progressive development however will be slow unless N H S funding for ICSI is provided universally and specialists begin to refer their patients to appropriate centres. In spite of the phenomenal success rates achievable with ICSI (now comparable to those achievable with standard IVF) concerns about the safety and costs remain. References
1. Ma K, Inglis JD, SharkeyA et al. A Y chromosomegene familywith RNA-binding protein homology;candidates for the azoospermiafactor (AZF) controllinghuman spermatogenesis.Cell 1993;75(7): 1287-1295 2. RiejoLR, Lee T-Y,Salo Pet al. Diversespermatogenicdefects in humans caused by Y chromosomedeletionsencompassing a novel RNA-binding protein gene. Nature Genetics 1995; 10: 383-393 3. WorldHealth Organization. WHO Laboratory manual for the examination of Human Semenand Sperm-CervicalMucus Interaction (3rd edn). Cambridge: CambridgeUniversity Press, 1992 4. KrugerTF, Acosta AA, Simmons KE Predictedvalue of abnormal sperm morphologyin IVF. Fertil Steril 1988;49: 11~117 5. Fishel SB and Edwards RG. Essentials of fertilisation.In: Edwards RG, Purdy JM (eds) Human Conceptionin vitro. London: AcademicPress 1982:157-159 6. OehningerS, Acosta AA, Morshedi Met al. Corrective measures and pregnancyoutcome in IVF in patients with sperm morphologyabnormalities. Fertil Steril 1988; 150: 283-287 7. Hall J, Fishel S, Green Set al. Intracytoplasmicsperm injection (ICSI) versus high inseminationconcentration(HIC) .IVF in cases of very severeteratozoospermia0/ST). Hum Reprod 1995; 10(3):493-496 8. Fishel S, Lisi F, Rinaldi L, Thornton Set al. ICSI versus high insemination concentration(HIC) for human conceptionin vitro. Reprod Fertil Dev 1995;7:169-175 9. Ord T, Patrizio P, BalmacedaJP et al. Can severemale-factor infertilitybe treated without micromanipulation?Fertil Steril 1993; 60:100 115 10. Metka M, Haromy T, Huper Jet al. Artificialinsemination using a micromanipulator.Fertilitat 1985; 1:41-47 11. Laws-KingA, TrounsonA, Sathananthan H et al. Fertilisation of human oocyte by micro-injectionof a single spermatozoonunder the zona pellucida. Fertil Steril 1987;48: 637-642 12. Lanzendorf E, Maloney MK, Veek LL. A preclinical evaluation of human spermatozoainto human oocytes.Fertil Steril 1988;49:835-842
The role of assisted conception techniques in male infertility 23 13. Ng S-C, Bongso TA, Ratnam SS et al. Pregnancy after transfer of multiple sperm under the zona. Lancet 1988; 2:790 14. Fishel SB, Antinori S, Jackson Pet al. Twin birth after sub-zonal insemination. Lancet 1990; 2:722 15. Palermo G, Joris H, Derde M-P et al. Sperm characteristics and outcome of human-assisted fertilisation by sub-zonal insemination and intracytoplasmic sperm injection. Fertil Steril 1993; 59:826-835 16. Palermo G, Joris H, Devroey Pet al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340:1%18 17. Green S, Fishel S, Hobson Get al. Computer image sperm selection (CISS) as a novel approach to sub-zonal insemination. Hum Reprod 1995; 10(6): 1430-1434 18. Fishel S, Lisi F, Rinaldi Let al. Systematic examination of immobilising spermatozoa before intracytoplasmic sperm injection in the human. Hum Reprod 1995; 10(3): 49%500 19. Craft I, Tsirigotis M, Bennett Vet aL Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia. Fertil Steril 1995; 63:1038-1042
20. Schoysman R, Vanderzwalmen P, Nij Met al. Pregnancy after fertilisation with testicular spermatozoa. Lancet 1993; 342:1237 21. Fishel S, Green S, Bishop Met al. Pregnancy after intracytoplasmic injection of spermatid. Lancet 1995; 345: 1641-1642 22. Sofikitis N, Mayagawa I, Sharlip Iet al. Human pregnancies achieved by intra-ooplasmic injections of round spermatid nuclei isolated from testicular tissue of azoospermic men (Abs). J Urol 1995; 153(4): 302A 23. Tesarik J, Mendoza C, Testart J. Viable embryos from injection of round spermatids into oocytes. N Engl J Med 1995; 333:525 24. Van Steirteghem A. Follow up of 877 children born following intracytoplasmic sperm injection. Keynote Address, American Society for Reproductive Medicine meeting, Seattle, 7-12 October 1995 25. In't Veld P, Brandenburg H, Verhoeff Aet al. Sex chromosome abnormalities in pregnancies estabished by intracytoplasmic sperm injection and in vitro fertilisation (IVF-ICSI). Lancet 1995; 346:773 26. Moosani N, Cox D, Pattinson H et al. Chromosomal analysis of sperm from men with idopathic infertility using sperm karyotyping and fluorescence in situ hybridisation. Fertil Steril 1995; 64(4): 811-817