Male infertility and intracytoplasmic sperminjection (ICSI)

Alastair J Campbell and D Stewart IrvineMRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, Edinburgh, UK

Micro-assisted fertilization in the form of intracytoplasmic sperm injection (ICSI)has truly revolutionised the treatment options for couples with impaired semenquality, and those with both obstructive and non-obstructive azoospermia. Ingeneral, the major issues which relate to the success of ICSI are those related tothe success of conventional IVF, and the high multiple pregnancy rate remains amajor cause for concern. There is growing evidence that the short-term healthof ICSI offspring is relatively unremarkable, but our growing understanding ofthe genetic basis of much of male subfertility and of the impaired genomicintegrity which characterises the oligozoospermic phenotype indicate a cautiousapproach to the longer term health of ICSI offspring.

Correspondence to.Dr D Stewart Irvine,

MRC Human ReproductiveSciences Unit Centre for

Reproductive Biology,37 Chalmers St.

Edinburgh EH3 9ET, UK

Infertility remains a common problem causing significant psychologicaldistress to those couples affected, who are increasingly seeking medicaladvice. The substantial contribution of reproductive dysfunction in themale partner to this infertility has been highlighted, and the developmentof micro-assisted fertilization techniques, principally intracytoplasmicsperm injection (ICSI), in the context of assisted conception has revol-utionised the management of couples with so-called male factor infertility.This article focuses on the diagnosis and causes of male infertility and thedevelopment of ICSI to help such couples.

Infertility is commonly defined as the failure of conception after at least12 months of unprotected intercourse1; however, accurate assessment ofthe prevalence of infertility has always been difficult because of thescarcity of large-scale population based studies2'3. Estimates suggest thatsome 14-17% of couples may be affected at some time in their reprod-uctive lives4"5, with recent European data suggesting that as many as onein four couples may experience difficulties in conceiving6.

Contribution of the male partner to infertility

The accurate diagnosis of male infertility is fraught with problems and,therefore, establishing the extent of the male partner's contribution to

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infertility is difficult. The majority of studies of the epidemiology andaetiology of male infertility have used the criteria of semen qualitydevised by the World Health Organization7"9. However, a significantproportion of men with a normal semen analysis according to thesecriteria will be infertile because of defective sperm function10'11, whilst aproportion of men with abnormal 'WHO' semen parameters will havenormal sperm function12-13. Hence, most studies use descriptive asopposed to functional diagnostic criteria, and it is becoming clear fromrecent studies on semen quality in relation to the achievement ofspontaneous pregnancy that these descriptive criteria may be in urgentneed of revision14. Despite these shortcomings, numerous workers haveconcluded that the single most common factor contributing to infertilityis abnormal semen quality5'6'15"19.

Diagnosis and classification of 'male infertility'

From the above, it can be seen that a major obstacle to the understandingand, therefore, treatment of male infertility is its accurate detection. It isimportant to emphasise the value of a proper clinical history andexamination, as few positive diagnoses in clinical andrology can be madeon the examination of semen samples alone20. Whilst the WHO hasdevised a diagnostic classification of male infertility to introducestandardisation and facilitate multi-centre research, recent advances inunderstanding the genetic causes of male infertility argue for a review ofthe current classification21"25. Diagnosis is traditionally based on criteria ofsemen quality promulgated by internationally recognised guidelines9-26,which incorporate information on ejaculate volume, sperm concentration,graded sperm motility and morphological appearance. This complexassessment is subject to a range of shortcomings which might be expectedto limit its diagnostic value. Many attributes of semen quality examinedmay be subjective in interpretation, although the recent introduction ofquality control into routine diagnostic laboratory andrology practiceshould serve to limit inconsistencies in values between differentlaboratories27"29. Additionally, marked interejaculate variation in semenquality within one mdividual is well recognised, and requires theassessment of multiple samples for diagnostic purposes8*30"31. Lastly, it hasbecome apparent that there is substantial geographical variation in humansemen quality32, so while the most recent WHO guidelines providereference ranges for 'normal' values, these are not well evidence-based andhighlight the importance of individual laboratories establishing normalvalues for their own local populations.

Given the difficulties which beset the meaningful diagnosis of 'maleinfertility' it is perhaps unsurprising that progress in understanding its

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aetiology has been slow7 and that effective treatments are few33. Theclear exception to this is the application of techniques of micro-assistedfertilization, principally intracytoplasmic sperm injection (ICSI), in thecontext of assisted conception, which have revolutionised themanagement of couples with male factor problems.

IVF and male infertility

Early developments in in vitro fertilization (IVF) focused on couples withfemale factor infertility and particularly women suffering from bilateraltubal occlusion34. Conventional IVF rapidly became established as aneffective treatment option for couples with tubal disease and withunexplained infertility35; however, it soon became apparent that it yieldedgenerally poor pregnancy rates for couples with male factor infertility3*"38.Tournaye et aP6, for example, compared in vitro fertilization and embryotransfer (IVF-ET) in a group of couples with male infertility and a similargroup with tubal infertility. In cases of male infertility, more oocytes wererecovered but fewer oocytes were fertilized, fewer embryo transfers wereperformed, the average number of embryos per transfer was lower and thetotal pregnancy rate per cycle was also lower at 12.8% versus 22.9%.They concluded that male infertility could be treated by IVF-ET, but thatthe results were disappointing when compared to a control group withnormal spermatozoa. Although there was much discussion in the literatureon the fine-tuning of the IVF procedure for couples with problems in themale partner39, management options for couples with poor semen qualityremained very limited until the breakthrough of effective micro-assistedfertilization in 199240.

Micro-manipulation techniques

Although the successful clinical application of techniques of micro-assisted fertilization took place some 14 years after the first successfulhuman in vitro fertilization34, animal experimentation with micro-assisted fertilization had in fact pre-dated human IVF41'42. In earlyhuman studies, a range of approaches was initially explored.

Partial zona dissection

Partial zona dissection (PZD) was the first micromanipulation techniquestudied in animal models with clinical intent43, and early reports inhuman practice of clinical pregnancies were encouraging44-45, suggesting

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that monospermic fertilization and cleavage rates could be doubled bythese approaches. However, concerns existed over the risk of poly-spermy, along with doubts about appropriate case selection4*.

Sub zonal insemination

Sub zonal insemination (SUZI) involves the injection of spermatozoa into theperivitelline space and, again, initial reports of its use were encouraging47'48,although other groups found the technique to be less successful49'50.

Intracytoplasmic sperm injection

The developments in human micro-assisted fertilization culminated inICSI, with the first human pregnancies resulting from this technique beingdescribed by the Brussels group in 199240. This approach involvesinjection of a single spermatozoon directly into the cytoplasm of theoocyte through the intact zona pellucida, and it very soon becameapparent that this technique produced superior results to PZD or SUZI,with pregnancy rates of 22% per started cycle being reported51"52. Indeed,such has been the success of ICSI that commentators have been moved tosuggest that it might be considered the treatment of choice for all caseswhere in vitro conception is indicated53. Verheyen et alSA reported on acontrolled comparison of conventional IVF and ICSI in patients withasthenozoospermia (which they defined as <5% rapidly progressive sperm-atozoa) and noted a very much reduced fertilization rate and substantialrisk of failure of fertilization associated with the use of conventional IVF.The same group55 reported on a study comparing IVF with ICSI in siblingoocytes from couples with tubal infertility and normal semen quality. Theyobserved that the use of ICSI in this group was not detrimental to embryoquality or implantation potential. The results of a large multi-centrerandomized controlled trial comparmg IVF with ICSI in normozoospermiccouples are eagerly awaited. Most recently, a meta-analysis has concludedthat, for couples with normal semen, there is no evidence of any benefiteither in fertilization rates per retrieved oocyte, or in pregnancy rates,between ICSI and conventional IVF. In contrast, for couples withborderline semen, ICSI results in higher fertilization rates than IVF, andcouples with very poor semen will have better fertilization outcomes withICSI than with SUZI or additional IVF56.

ICSI with epididymal spermatozoa

Initially, clinical ICSI was used in the treatment of couples in whom themale partner had substantially abnormal semen quality, but it was not long

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before the technology was applied to the significant numbers of men whopresent with no sperm in their ejaculates. Amongst men with obstructiveazoospermia, attention focused on spermatozoa derived from theepididymis. The first pregnancies achieved with epididymal sperm weredescribed using conventional IVF57-58. However, fertilisation rates werelow, so whilst it was established that sperm from the epididymis had adegree of functional competence, this was limited59-60, and animal experi-mentation suggested that micro-assisted fertilization was likely to bebeneficial42-61. Initially, the use of SUZI was described62-63, but this wasrapidly replaced in clinical practice by ICSI64'65, which achieved verysatisfactory success rates.

As a consequence of the successful use of epididymal spermatozoa mICSI, techniques have been described to facilitate the surgical retrieval ofspermatozoa66'67. The major approaches include microsurgical epididymalsperm aspiration (MESA), and percutaneous epididymal sperm aspiration(PESA). MESA mvolves a formal scrotal exploration, is commonlyperformed under general anaesthetic, and hence is a significant surgicalintervention68. A major advantage of this technique is that it permits fullscrotal examination and, therefore, has diagnostic power. The number ofspermatozoa retrieved is high, which facilitates cryopreservation69 and, ifindicated, it can be combined with definitive reconstructive surgery, suchas vaso-vasostomy or epididymo-vasostomy70. PESA is a widely used tech-nique which is less invasive, can be performed under local anaesthesia71"73,and can be performed repeatedly74. PESA provides less diagnosticinformation, and the yield of spermatozoa may be lower, with one recentreview suggesting that at least 20% of attempts at PESA are unsuccessfuland require resort to MESA66. It has also been argued that, in patientswith presumed obstructive azoospermia, direct testicular fine needleaspiration (TFNA) may be the procedure of choice67.

ICSI with testicular spermatozoa

In contrast to the position in men with obstructive azoospermia, amongstmen with non-obstructive azoospermia attention naturally focuses on thetestis as a site for sperm recovery. With the availability of ICSI, it hasbecome clear that non-obstructive azoospermia is a very heterogeneouscondition, and that testicular histology is similarly heterogeneous, with fociof apparently normal spermatogenesis adjacent to seminiferous tubulesdevoid of germ cells75. These observations led to attempts at the surgicalrecovery of sperm from men with non-obstructive azoospermia, and thesuccessful achievement of pregnancies76. Since these exciting initialobservations, surgical sperm recovery from men with non-obstructiveazoospermia has become a routine part of clinical infertility practice77"81

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and, as with the epididymis, cryopreservation of testis derived spermatozoahas also become routine82. A recent review concluded that surgical spermrecovery would be successful in some 48% of men with non-obstructiveazoospermia66.

Various approaches to the surgical recovery of sperm from the testis havebeen described, both in the context of obstructive and non-obstructiveazoospermia. Testicular sperm aspiration (TESA) is a simple percutaneousaspiration technique, performed under local anaesthetic, and which has ahigh success rate of sperm retrieval in those men with normalspermatogenesis. The advantages of TESA are its simplicity, quickness andnon-invasiveness. Testicular sperm extraction (TESE) on the other handmvolves an open excisional biopsy, equivalent to a diagnostic biopsy.Sperm are extracted using either mincing techniques or enzymatic digestionto disrupt the tissue and allow seminiferous tubules to releasespermatozoa. TESE also allows cryopreservation of tissue. Girardi andSchlegel66 and Tournaye67 in recent reviews of the subject have argued thatin men with non-obstructive azoospermia, surgical access to the testis isrequired, since pockets of spermatogenesis are isolated, and larger ormultiple biopsies are more often needed. This view has been supported byrecent comparative studies in which TESE was required in almost 80% ofmen with non-obstructive azoospermia83.

Undoubtedly, one of the major problems confronting the process ofsurgical sperm recovery from men with non-obstructive azoospermia is thefact that there are currently no good predictors of which patients will havesperm recovered successfully, and which will not84. Against this back-ground, a number of groups have argued that surgical recovery of spermfrom the testis coupled with cryopreservation should precede ovarianstimulation in the female partner85'86.

For those men m whom mature spermatozoa cannot be recovered, thereis currently interest in the possibility of using less mature cells, commonlyelongating or round spermatids, to achieve fertilization87. Work in animalmodels has suggested that this may be a viable approach88-89, and there area number of clinical case reports m the literature90-91. At the present time,however, uncertainties over the safety92 and efficacy of this approachshould confine its use to properly designed clinical trials.

Success rates of ICSI

ICSI has become well established as an effective form of treatment forcouples with male factor infertility. In the last year for which data areavailable (1997/98) the UK's Human Fertilization and EmbryologyAuthority reported 9295 cycles of ICSI, alongside 24,889 cycles of IVF- in other words, some 27% of assisted conception treatment in the UK

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is for severe male factor infertility. In the same year, the reported livebirth rate for ICSI was 20.7% per cycle compared to 14.9% for IVF. Inkeeping with the major issues in assisted conception in general, themultiple birth rate was high at over 25%, and the age of the femalepartner was a major determinant of success, with a woman in her earlythirties achieving a 20-25% per cycle success rate, dropping to 5% orless for a woman over the age of 40 years93.

Tarlatzis recently reviewed the world-wide experience of ICSP4. Henoted that over the 3 year period (1993-1995), the number of centresundertaking ICSI in Europe increased from 35 to 101, and the totalnumber of ICSI cycles per year rose from 3157 to 23,932. The incidenceof oocytes damaged by the procedure was low (< 10%), and thefertilization rates obtained with ejaculated, epididymal, and testicularspermatozoa for 1995 were 64%, 62%, and 52%, respectively. Ultimately,between 86-90% of couples achieved embryo transfer, and the viablepregnancy rate was 2 1 % for ejaculated, 22% for epididymal, and 19% fortesticular sperm, with an incidence of multiple gestations of 29%, 30%,and 38%, respectively. It was noteworthy that no difference was found inICSI results concerning the aetiology of azoospermia; for example,obstructive (congenital or acquired) or non-obstructive. The Brussels grouphave recently reviewed their own data on ICSI outcomes95 in a group ofBelgian couples, aged less than 37 years, who had their first ICSI cyclebetween 1992/93. The per cycle delivery rate was high at 31%, but the realcumulative pregnancy rate was 60% after 6 cycles of treatment. Again,female age had a powerful effect on success rates.

Outcomes of ICSI

Given that ICSI is effective, is it also safe? The rapid development of micro-assisted conception techniques and the wide-spread use of in ICSI inalleviating male infertility have raised concerns about the health of theoffspring. ICSI, by directly injecting individual spermatozoa into a matureoocyte bypasses the natural physiological processes of normal spermselection, raising concerns over the potential risk of congenitalmalformations and genetic defects in children born after ICSI. It has alsostimulated debate regarding associations between obstetric and perinataloutcome and type of conception. Recent work showing that poor qualitysemen tends to contain gametes with compromised DNA integrity96, butthat this does not compromise its fertilizing ability at ICSP7 furtherhighlights the importance of interest in the area of ICSI outcome.

Without doubt, the most thorough and detailed follow-up studies of ICSIoffspring have been those orchestrated by the Brussels group98 who haveundertaken a prospective follow-up study of 1987 children born after ICSI,

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aiming to compile data on karyotypes, congenital malformations, growthparameters and developmental milestones. They were able to collect dataon 1699, 91 and 118 children born after ICSI with ejaculated, epididymaland testicular spermatozoa, respectively, as well as on 79 children bornfrom cryopreserved ICSI embryos. Of 1082 karyotypes determined byprenatal diagnosis, 1.66% (18) were abnormal de novo (nine each ofautosomal and sex chromosomal aberrations), and 0.92% (10) wereinherited structural aberrations. Of these, nine (eight balanced structuralaberrations and one unbalanced trisomy 21) were transmitted from thefather. Forty-six major malformations (2.3%) were observed at birth.Seven malformations, observed by prenatal ultrasound, were terminated.Twenty-one (1.1 %) stillbirths, including four with major malformations,occurred later than 20 weeks of pregnancy.

In Denmark, a national cohort study of 730 infants born after ICSP9

included all clinical pregnancies obtained after ICSI registered in Denmarkbetween January 1994 and July 1997 at five public and eight privatefertility clinics. Only 183 women (28.5%) underwent prenatal diagnosis,resulting in 209 karyotypes with seven (3.3%) chromosome aberrations.Six major chromosomal abnormalities (2.9%) and one inherited structuralchromosome aberration (0.5%) were found, but no sex chromosomeaberrations. The frequency of multiple birth, Caesarean section rate,gestational age, preterm birth, and birth weight were comparable withprevious studies. The perinatal mortality rate was 13.7 per 1000 childrenborn with a gestational age of 24 weeks or more. In 2.2% of the live-borninfants, and in 2.7% of all infants, the parents reported major birth defects.Minor birth defects were found in nine live-born infants (1.2%).

Most recently, Wennerholm et alm studied the incidence of congenitalmalformations in a complete cohort of 1139 infants, 736 singletons, 200sets of twins and one set of triplets born after ICSI. The number of infantswith an identified anomaly was 87 (7.6%), 40 of which were minor. Theincidence of malformations in children born after ICSI was compared withall births in Sweden using data from the Swedish Medical Birth Registryand the Registry of Congenital Malformations. For ICSI children, the oddsratio (OR) for having any major or minor malformation was 1.75 (95%confidence interval (CI) 1.19-2.58) after stratification for delivery hospital,year of birth and maternal age. If stratification for singletons/twins wasalso done, the OR was reduced to 1.19 (95% CI, 0.79-1.81). Theincreased rate of congenital malformations is thus mainly a result of a highrate of multiple births. Of interest, the only specific malformation whichwas found to occur in excess in children born after ICSI was hypospadias(relative risk 3.0, exact 95% CI, 1.09-6.50). The same group examined theobstetric outcome of pregnancies after ICSI101. Deliveries occurred in75.9% and early spontaneous abortion, late spontaneous abortion andectopic pregnancy in 21.4%, 1.0% and 1.2% of pregnancies, respectively.

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Multiple birth occurred in 21.3% (almost all twins) of deliveries, andpreterm birth occurred in 15.7% of all deliveries. Preterm birth was notrelated to sperm origin or quality but was related to multiple birth. Theprematurity rate was 8.4%, 42.3% and 100% for singletons, twins andtriplets, respectively. The perinatal mortality rate was 11.7 per 1000 borninfants; 7.3% of infants had a malformation, 40 of which were minor.They concluded that the obstetric outcome of ICSI pregnancies was similarto that of conventional IVF and was not influenced by sperm origin orquality. The high incidence of multiple births remains the major concern.Other groups have reached similar conclusions102.

In other words, as the database of ICSI offspring grows larger, theavailable evidence on the short-term health of these offspring is generallyreassuring. It is important, however, to appreciate the important role thata genetic aetiology plays in the origins of much male subfertility, and theability of ICSI to promote the transgenerational transmission of geneticdefects causing gametogenic failure. It seems clear that male, but notfemale, fertility problems show a distinct pattern of familial aggregation,and that couples with male infertility have fewer siblings than fertilecontrols103. The significantly increased risk of chromosomal abnormalitiesin men with impaired semen quality21 is easily managed by the appropriateinvestigation and counselling which are required prior to treatment104.Some groups have even advocated chromosomal studies on bothpartners105. It is less easy to be certain how to respond to the availableevidence on microdeletions of the Y chromosome in men with severelyimpaired semen quality22'24'25'106. The strength of the association between Ychromosome deletions and severely impaired semen quality is impressive,and it is increasingly suggested that these lesions may result in progressionfrom oligozoospermia to azoospermia over time107. In a recent study, Pryoret a/106 found deletions in 7% of infertile men, and in only 2% of normalmen, but observed no clear relationships between the size and location ofthe deletions and the severity of the spermatogenic failure. Moreover, it isclear that these genetic deletions, if present, can be transmitted to offspringvia ICSI107'108. On the basis of this evidence, some authorities now advocatescreening of men for Y chromosome microdeletions prior to ICSI, andadvocate testing of offspring and reproductive monitoring for those foundto have inherited deletions.

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