genetic basis of male fertility

Genetic basis of male fertility

T B HargreaveFertility Problem Clinic, Department of Urology, Western General Hospital, Edinburgh, UK

We are in the age of genetic discovery. Now the human genome has beencompletely sequencedi, there will be increasing understanding and ability tomanipulate biochemical pathways downstream of genes. At the same time, furtherdevelopment of in vitro fertilization (IVF) and intracytoplasmic sperm injection(ICSI) will enable procreation in situations that were formerly impossible and whenthere may be an increased possibility of genetic abnormality. Furthermore, pre-implantation diagnosis will enable defects to be diagnosed and will give theopportunity for the couple to decide whether to continue with treatment towardsa pregnancy or not. Thus, there is a need for clinicians to have a good knowledgeof the genetic and hereditary aspects of male (and indeed female) infertility andfor couples to have access to correct information and expert counselling. Also,there are ethical implications of these scientific and clinical advances for the futurechild, the individual, the couple and society. There is increasing public uneaseabout this new science of reproduction and, in the UK, there is regulation by law;thus, there is a need for clinicians and scientists to give accurate information ineveryday language to the public.

Correspondence toMr 7 B Hargreave,

Fertility Problem Clinic.Department of Urology,

Western GeneralHospital,

Edinburgh EH4 2XU. UK

The normal human cell has 46 chromosomes arranged as 22 pairs ofautosomes and either two X chromosomes in the female or an X and Y inthe male. On these chromosomes are arranged approximately 50,000-100,000 genes that encode proteins2. The chromosomes package contains6-7 billion base pairs of deoxyribonucleic acid (DNA). The DNA isarranged as sequences of the four DNA bases (adenosine, cytosine, thiamineand guanine; ACTG) and it is this sequence that spells out the sequence ofamino acids in proteins. Not all of the genetic code sequence translates intoprotein production. The sequences of the DNA bases are arranged intoprotein describing areas called exons, non-protein describing areas calledintrons and also special sequences to indicate the beginning and end ofgenes. Usually the sequence of amino acids m a protein is derived frominformation from several exons and processing is under the control ofsplicing enzymes. Splicing allows the formation of different proteins withshared common sequences to use the same exon and thus maximizes theefficiency of the genetic information.

Genes can be classified into functional classes; for example; oncogenes,check point genes, genes regulating apoptosis, etc. Genes may be arranged in

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functional clusters and with respect to the male gender the sex-determin-ing gene SRY is located on the short arm of the Y chromosome and thereare several genes involved with spermatogenesis on the long arm of the Ychromosome.

Humans share common genes with all living creatures. We differ from ourclosest relatives the chimpanzees by approximately 1.6% of our DNA anddiverged from a common ancestor about 7 million years ago. Gorillas differin approximately 2.3% of their DNA from chimpanzees and man anddiverged from our (humans and chimpanzees) common ancestor about 10million years ago. All the above species diverged from monkeys about 32 mill-ion years ago and there is a DNA difference of only 7.5%3. Our kinship withprimates and mammals allows appropriate animal models to be used to studyhuman genetic mechanisms both generally and with respect to male fertility.

Spermatogenesis is unique because the DNA content of sperm is halfthat of the spermatogonia. In the initial stages, mitotic divisions give riseto spermatocytes. These undergo meiotic division to form haploid roundspermatids. The round spermatids elongate in a process called spermio-genesis and, during this process, the DNA becomes compacted in thesperm head. These changes are under genetic control, but we are only nowbeginning to understand the mechanisms. It is estimated that there are2000 genes that regulate spermatogenesis, most of these being present onthe autosomes, but there are approximately 30 genes on the Y chromo-some. Y genes are present in only half of humanity. In general, autosomalgenes that regulate spermatogenesis are concerned with regulation ofmetabolic processes in other cells in the body as well as in the cells ofspermatogenesis, whereas Y genes are not essential for vital functionsoutwith reproduction.

Y chromosome genes are subject to different evolutionary pressure com-pared with all other genes. Spermatogenesis is the result of rapid cell divisionand there is much greater opportunity for mutation than in oogenesis whichoccurs early in life and is followed by a long inert period. The Y chromosomehas been present for all of evolutionary time in the mutagenic environment ofspermatogenesis, whereas the X chromosome and all the autosomes havespent half of evolutionary time within the relatively inert environment of theovum4. Normally, autosomal genes can undergo DNA repair when they pairwith their opposite number during mitotic division, but for Y genes thispairing cannot happen. These differences in the environment of Y genes mayexplain the multicopy nature of some of the Y gene families and the manyinert copies (pseudogenes) that are found. Unfortunately, this tendency formany copies makes if more difficult to map the Y chromosome accuratelyand more difficult to locate active genes. All Y genes are haploid and defectsin a single gene are likely to have effects.

Our new understanding of the genetics of spermatogenesis holds thepromise for the development of novel non-hormonal methods of male

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contraception and this, in terms of overall scientific priorities for thehuman race, must rate very highly and is justification for funding thisarea of genetic research.

Chromosomal abnormalities

Disorders of the chromosomes can be classified as numerical or structural.Numerical disorders are polyploidy or aneuploidy. Polyploidy cells (e.g.diploid [46 chromosomes], triploid [69 chromosomes], tetraploid [92chromosomes]) are seen in spontaneous abortions and certain cancer cellsand are essentially incompatible with life. Aneuploidy is where there is gainor loss of one or more chromosomes. In the context of male infertility, oneof the most common aneuploid forms is 46XXY or Klinefelter's syndrome.

Structural chromosome disorders may be the deletion of material fromone or more chromosomes, duplication of material, re-arrangement forthe normal order within one chromosome or swapping of materialbetween chromosomes; where there is no net loss of material this isknown as a balanced translocation. A single chromosome may fuse withitself to form a ring. These structural abnormalities may lead to pheno-typic disorder or may predispose to severe congenital abnormality whengametes are formed.

Chromosomal abnormalities are more common in infertile men and insubfertile male partners of ICSI couples. In a survey of pooled data from11 publications reporting 9766 infertile men there was an incidence ofchromosomal abnormalities of 5.8%5. Of these, sex chromosome abnor-malities accounted for 4.2% and autosomal abnormalities for 1.5%. Forcomparison, the incidence of abnormalities in pooled data from 3 seriestotalling 94,465 newborn male infants was 0.38% and of these 131(0.14%) were sex chromosome abnormalities and 232 (0.25%) auto-somal abnormalities6. In a population of 781 male partners of couplesundergoing ICSI, 30 men (3.8%) had chromosomal abnormalities; ofthese, there were 10 (1.2%) sex chromosome aberrations and 20 (2.6%)autosomal aberrations7. There is less information about the incidence ofchromosome abnormalities in spermatozoa. It is now possible to studylarge numbers of sperm using multicolour fluorescent in situ hybridizationanalysis (FISH), whereas previously sperm karyotyping was by hamsterpenetration assay - a method that was both laborious and insensitive.Two studies using FISH reveal increased frequency of aneuploidy inspermatozoa involving the autosomes and particularly the sexchromosomes8'9. There is a need for further FISH sperm studies usingsperm from men in well-defined clinical categories (e.g. Table 3). Also,when considering such studies, it may be relevant to study the populationof sperm that would normally be used in an ICSI procedure. The ICSI

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biologist will normally select by motility and secondarily by morphologyand it is probably appropriate to select in the same way sperm forchromosome studies.

Sex chromosomal abnormalities

Klinefelter's syndrome and variants (47XXY, 46XY;47XXY mosaicism)

Klinefelter's syndrome is the most frequent sex chromosome abnormality,which occurred in 66 (0.07%) of phenotypically newborn males in pooleddata from cytogenetic analysis of 94,465 newborn infants6. Adult maleswith Klinefelter's syndrome have small firm testicles that are devoid of germcells; this is to be distinguished from men with other causes of damage tospermatogenesis who have small soft testicles. The phenotype can vary froma normally virile man to one with the stigmata of androgen deficiencyincluding female hair distribution, scanty body hair, and long arms and legs,because of late epiphyseal closure.

Leydig cell function is commonly impaired in men with Klinefelter'ssyndrome10. Testosterone levels may be normal or low, oestradiol levelsnormal or increased and FSH levels increased. Surprisingly, libido is oftennormal despite low testosterone levels, but with ageing there is often a needfor androgen replacement; thus men with Klinefelter's syndrome should beoffered longer-term supervision in addition to the management of theirfertility problems.

s Men with Klinefelter's mosaicism 46XY;47XXY have variable germ cellpresence and variable sperm production. Until the advent of IVF and ICSIthis was academic, but it is now important to diagnose mosaicism, as somesperm retrieved may be expected to be normal and can be used to fertilize.Pre-implantation FISH analysis of cells from embryos can be used toconfirm normality11. The production of 24XY sperm has been reported in0.9-2.1% of men with Klinefelter's mosaicism12'13 and in 1.36-25% ofmen with somatic karyotype 47XXY14"18. All this indicates that some47XXY cells are able to achieve meiosis and produce mature spermatozoa.Conversely, it is not known whether haploid sperm in Klinefelter'ssyndrome are always the result of a clone of normal cells in a mosaicpopulation or whether in certain circumstances some 47XXY male germcells are viable and capable of producing haploid sperm.

Increased frequency of sex chromosome abnormalities in ICSI babies

There are a number of reports that indicate higher than normal frequencyof sex chromosome abnormalities in children born of ICSI compared with


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the normal population. The underlymg mechanism is not yet known, butthere are several possibilities discussed in the literature: these includeKlinefelter's mosaicism with an aneuploid cell line confined to the germcells and thus not detectable by peripheral blood karyotyping19, or theproduction of s47XY sperm chromosome diploid sperm and this hasreported in sperm from severely oligozoospermic men with a normalkaryotype20. Another possibility is that low levels of mosaicism may bemore common than is thought and may be missed during routinekaryotype analysis.

47XYY a rarer abnormality and often associated with an apparently normalsperm analysis

Males with 47XYY are seen more frequently in the infertile populationand FISH analysis of spermatozoa indicates an increase in disomy21.

Y chromosome haplotype and male fertility

In 1992, there was a report of decreasing human sperm concentrationover a number of years22. This observation has been confirmed byothers, although there is conflicting information from differentcountries. The most accepted hypothesis is that this is a deleterious effectof environmental contamination with oestrogenic chemicals. However,much less is known about the genetic contribution to male fertilitypotential. A Japanese group23 used PCR to look at DNA differences inthe Y chromosome The results were used to classify the Y chromosomeinto four haplotypes - I, II, HI, IV. Men with haplotype II had a lowersperm concentration compared with men with haplotypes HI and IV,and the frequency of haplotype II is more common in azoospermic mencompared with normal men. This work indicates a significant geneticcontribution to male fertility potential and could provide the basis foralternative hypotheses to explain changes in sperm concentrations indifferent populations with time.

Autosomal abnormalities

It is well known that apart from those men with Klinefelter's syndromethere is an excess of autosomal abnormalities in populations of menwith non-obstructive azoospermia or severe oligozoospermia24*25.Usually, autosomal disorders do not cause infertility in isolation, butreduced spermatogenesis is the consequence of a more general


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disturbance in phenotype; patients with these problems are often knownto doctors because of other developmental abnormalities and do notpresent de novo with infertility. However, in view of the known excessof these disorders in men with a low sperm count, it is appropriate toperform karyotyping for all men with low sperm counts where it isproposed to use ICSI to enable fertility and to offer genetic counsellingwhen abnormalities are discovered. Karyotype analysis may be normal,but this does not exclude genetic defects; patients and infertilityclinicians need to understand the limitations of karyotype analysis.From time to time, men may ask for ICSI where there is already a knownautosomal defect. In these cases, the genetic counselling is the samenormal and independent of the need for ICSI.

Genetic defects

Disorders of the sequence of the DNA bases are called mutations and,while mutations may occasionally be beneficial and become preserved inevolution, often they result in defective genetic function. It is also likelythat many mutations occur in the testis during spermatogenesis and thatthe external position of the testes is because a cooler temperature dampsdown the mutation rate4. Mutations may involve relatively largeportions of the gene, but there are also many examples of where thealteration of a single base pair on the original DNA (point mutation) canhave a profound effect on phenotype. Genetic defects may be single ormultiple and may be inherited or the result of new mutation. They maybe inherited in a dominant pattern, as in Huntington's chorea, or arecessive pattern, as in cystic fibrosis, and may have variable penetrance.

When mutations are in exons, there is an alteration in the proteinproduced and, in many cases, a marked resultant alteration in phenotype.Mutations of non-coding areas (introns) are less well understood, but canaffect the amount of protein produced making the synthesis of proteinfrom the original DNA less efficient; for the andrologist, one of the bestknown examples of this is the 5T intron abnormality that occurs in thecystic fibrosis transmembrane gene complex (CFTR) and which is foundeither alone or with exon mutations in men with congenital absence of thevas deferens (see below).

Most, if not all, genes on the Y chromosome are to do with theregulation of male differentiation and defects will be manifest as pheno-typic abnormality except where there are autosomal homologues of thegene, e.g. DAZ. X-linked genes may be dominant {e.g. hypo-phosphatemic rickets) or recessive (Duchenes muscular dystrophy). X-linked dominant genes will alter the phenotype in the female, but in themale the recessive defect will also be manifest.


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Human reproduction: pharmaceutical and technical advances

X linked genetic disorders and male fertility

Each man has only one X chromosome and, therefore, any X-hnkedrecessive disorders will be manifest in males and the defect will betransmitted through his daughters to his grandsons.

Kallman's syndrome

The commonest X-linked disorder in infertility practice is Kallman'ssyndrome and the most common form of this is X-linked recessive causedby a mutation in the KALIG-1 gene on Xp22.326. This gene is involvedwith the regulation of cell adhesion and axonal path finding. Patients withKallman's syndrome have hypogonadotrophic hypogonadism and mayhave other clinical features including anosmia, facial asymmetry, cleftpalate, colour blindness, deafness, maldescended testes and renal abnor-malities. Other rare forms of Kallman's syndrome have been describedincluded an autosomal dominant form27 and autosomal recessive. It isimportant to note some men with Kallman's syndrome have an isolatedgonadotrophin deficiency without any other phenotypic abnormalitiesand that these men may sometimes present de novo with infertility, whichcan be treated successfully by hormone replacement therapy.

Androgen insensitivity - Reifensteine syndrome

The rare disorder of androgen insensitivity may sometimes first presentwith infertility. The condition has X-linked recessive inheritance due to adefect in the androgen receptor gene located on Xqll-12. The phenotypemay range from complete testicular feminisation with an immature femalephenotype to an apparently normal male with infertility, although thelatter is rare. In our clinic, we conducted a structured genetic search forandrogen deficiency amongst those men with high testosterone and lowsperm counts, but failed to find any cases using base pair mismatchanalysis technology28. In the course of the study, we reported several denovo mutations of the androgen receptor, but in all cases these wereassociated with obvious genital abnormalities, such as hypospadias. Weconcluded that androgen insensitivity in the infertile male in the absenceof any genital abnormality is rare.

Other X disorders

There is a case report of an azoospermic man with biopsy provenspermatogenetic arrest who has been found to have a submicroscopic


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interstitial deletion on the Xp pseudoautosomal region in peripheralblood and skin fibroblast samples. Other genetic and chromosomestudies were entirely normal including probing of the Yq region29. It isalso worth noting the report of two men with azoospermia and Xpseudoautosomal deletions30. So far, there are no other examples of X-linked disorders affecting male fertility and, if such disorders exist andare not associated with azoospermia, these would skip generations ofmales and will be very difficult to define.

X-linked disorders that are not associated with male infertility

There are many rare X-linked disorders not associated with infertility.When recessive these appear in male babies but skip several generationsand, therefore, family history is important. For example, Menkes diseaseis an X-linked recessive disturbance of copper metabolism associatedwith progressive neurological symptoms31. The main problem is therecognition of such disorders especially if there has been severalgenerations of female births where a recessive X-linked has been carriedthrough these generations without any clinical stigmata. Management isin the normal way by giving the couple choices after appropriate geneticcounselling, including consideration about the severity of any disorderthat may result. It may be appropriate to offer the couple pre-implantation sex determination and replacement of female embryos onlyor, in some situations, not to embark on fertility treatment at all.

Y genes and male infertility

In 1992, we reported three men with severe damage to spermatogenesisand an apparently normal chromosome analysis, but where molecularprobes revealed microdeletions on the long arm of the Y chromosome32^3.We were stimulated to probe the long arm of the Y chromosome becauseof a report by Tiepolo and Zuffardi34 who described men with azoo-spermia and deletions of the long arm of the Y chromosome transectinginterval 6 with the loss of all distal genetic material and by our ownfinding of an infertile man with a short arm dicentric Y35. Following our1992 report, there have been a large number of publications of case seriesand it is clear that, while microdeletions may occur in the fertilepopulation36, they are more prevalent in the infertile populations. To date,the microdeletions detected have been rather large, but there arepreliminary reports of much smaller deletions within genes37.Microdeletions have been found in three non-overlapping regions of theY chromosome AZF a-b-c38. Several genes have been described and these


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The Human Y Chromosome

Genetic Functions / PhenotypesStature

PAR1Tesns Determination (TDF)

Turner Syndrome Yp/Xq213(Lymphoedema) Homology

Yp/Xq22.3|-HomologyL

GBY (Qonadoblastoma)

GCY(Growth)

Turnersyndrome(Skeletal

anomalies]

AZFa(Sertoli Cel

OnlySyndrome)

t

AZFbDiverse

Spermatogenic ]Phenotypes

Mosaic tun?

AZFbDiverse

SpermatogenicPbenotypes

Yq/Xpll.2|Homology |

Yq Hetrochromatin

PAR 2

1A1A1A1B1A2IB1CIDIE2A2B2C3A3B

3C

3D3E3F3G

5B

5C

SD5E5F5G5H515J5K

5L

5M5N5O5PSQ6A6B

6C

SRYRPS4YZFY

GenesSHOX/PHOQ—•Mouse Chromosome 3CSF2RA •Mouse Chromosome 19IL3RA •Mouse Chromosome 14ANT3 •Possibly deleted in mouse?ASMTXE7MIC2RMIC2XG

RBM

TTYITSPYA

5A

PRKYAMELYPRYITY1

4A

3G3F3E3D

3C

TTYIPRYAMELYPRKY

TSPYATTYI (Teats transcript Y)

TTY2 (Testls transcript Y)TSPYB

~|Yq/Xp22.3ARS-D I HomologyXGYP —DFFRY (Drosophlia Fat Facets related) "I Yq/X 11.3-pl 1.4DBY (Dead box Y - posstblt helicase) jHomoIogy

~ Autosomal copies includingchromosome 21(Thymosin p4Y)

TB4\

KALpBPY1

|STSp

(Basic protein Y)RBM I, RBM II

3Homology

MIA

CIA (ckromodomatn protein Y)XKRY (XK related Y- putative membrane

transport protein)

1SMCY (HYA)IE1F1AY » Autosomal copy on lp1RBM1 (Translation Initiation factor 1 A)

PRYTTY2RBM I, RBM IIDAZ ~| RBM UBPY2 IPRY \(PTP-BLretiatedY- tyrotSnephosphatase)

cnv J

iSYBLl (synaptobrevin-lUce protein)JFLR9

Fig. 1 The Y chromosome and fertility genes. Figure prepared by Dr N Affara (Cambridge University) and presentedat the VGene Conference, Royal College of Physicians, Edinburgh, September 1998.

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include RBM39, DAZ, DFFRY40, DBY and CDY (Fig. 1). The abnormalitymost commonly reported in the literature is a microdeletion in the AZFcregion and encompassing the DAZ gene. However, there is no exactcorrelation between DAZ deletion and the presence of absence ofspermatogenesis, but this may be because for the DAZ gene there is alsoan autosomal copy (see below).

DAZ and RBM genes

The first candidate gene for spermatogenesis was identified from our unitand is now called RBM (RNA binding motif). There is a family of up to 50RBM genes41 and, while most copies are probably inactive, deletions of theAZFb region causes functional inactivation of RBM. The DAZ gene(formerly known as SPGY) was the second candidate gene to be described;initially it was thought to be a single copy gene42, but it is now known to bea member of a family of 6-10 genes4445. Both DAZ and the RBM genefamilies encode proteins with a similar structure, which are probablyinvolved in the metabolism of RNA. RBM is a nuclear protein and itsexpression is restricted to the male germ line in humans and in mice,whereas DAZ protein is cytoplasmic. RBM and DAZ proteins are relatedto the protein hnRNPG and this family of proteins are involved in RNAmetabolism including packaging of RNA, transport to the cytoplasm andsplicing. However, hnRNPG is autosomally encoded and ubiquitouslyexpressed indicating a function that is required for all cell types46"48. RBMis probably regulating splicing events essential for spermatogenesis49, where-as DAZ/SPGY with its autosomal contribution is important for gameto-genesis in both sexes, perhaps in the male regulating translational repressionduring spermatogenesis; knockout female mice without the DAZL genehave a failure of proper development of the female genital tract.

RBM is a highly conserved gene and has been found on the Y chromo-somes of all mammalian species so far tested50, including marsupials51.DAZ/SPGY is found only in humans and old world primates, but anautosomal homologue is present in mammals and in mice is found onchromosome 3. This autosomal homologue is also present in humans onchromosome 17 and may account for the varied spermatogenesis in menwith AZFc deletions. DAZL protein is expressed only in male and femalegerm cells in mice and men and these observations have led to thesuggestion that RBM and DAZLA might have been acquired from theirrespective autosomal homologue during the course of evolution and thatgenes associated with spermatogenesis will tend to accumulate on the Ychromosome. There is considerable sequence identity between the X andthe Y and it has also been postulated that there has been inactivation of Xgenes leaving the active copy on the Y52*53.

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Other Y genes

Several other genes on the long arm of the Y have been described (Fig. 1),but less is known about the clinical importance of these as, in general,microdeletions in regions other than AZFc are less frequent. Also, theinteraction between genes may be complex; for example, in the case ofDFFRY and DBY genes, there is interaction between the two genes so thatcomplete azoospermia only occurs when both genes are deleted together.

Clinical implications of Y microdeletions

There are no reports that men with microdeletions have any phenotypicabnormalities other than abnormal spermatogenesis and men withmicrodeletions appear to be in perfect health m every other respect38'42'54.As there is only one Y chromosome, we may predict that Y microdeletionswill be transmitted to their sons although this is hkely to be rare in thenormal population because without treatment with ICSI men with verylow sperm counts are less likely to be father of children. However, foursuch cases have been reported in the literature (Table 1). It may beimportant that in two of the cases the microdeletion appears to be larger

Table 1 Transmission of Y chromosome deletion from father to son

Authors

Kobayashi etal 199472

Vogt etal 19963"

Kent-First et al 199673

Pryor etal 1997*

Stuppia etal 1997"

Mulhall etal 1997"

Silber etal 1998"

Kamischke etal 1999"

Y deletion son

AZFc

AZFc

Small near AZFcSmall near AZFcLargeAZFc-AZFb

SY153-5Y267SY207-SY272

AZFc

Ongoing twin pregnancy

Two ongoing pregnanciesTwo ongoing, 1 twin AZFc

AZFc

Y deletion father

AZFc

AZFc

Small near AZFcNot detected

Not detected

SY153-5Y267

AZFc (smaller)

AZFc

AZFc AzoospermicAZFc oligospermic

AZFc

Phenotype son

7

< 0 1 x 10*

ICSINormal

0 3 x 1 0 *

< 2 x 106

ICSI

TESA-ICSIICSI

ICSINormal

Adapted from a table presented by Dr K McElreavey, Institute Pasteur, Pans at the Y Gene Conference,Royal College of Physicians Edinburgh, 1998


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in the son than the father. More information is needed from father/son pairswhere the son has a very low sperm count and also about the outcome ofICSI attempts where sperm have been used from men with microdeletions.There is a need for long-term follow-up of any male children. However,although it may be desirable to obtain information about the genetic statusof ICSI babies, there are ethical questions about whether young babiesshould be tested and, if so, whether the test results should be identifiable.

Testing for Y microdeletions

Testing for microdeletions is now widespread in IVF/ICSI units, but thereis no standardised methodology and, therefore, it is difficult to makedirect comparison between reported results. Several centres havedeveloped screening methodologies38-54"56. As there is no correlationbetween histopathology and deletion of DAZ, it is premature to rely onspecific gene probes as these will fail to detert a significant proportion ofmen with microdeletions. Also testing using peripheral blood may not bereliable. Lack of DAZ mRNA in testicular cells has been reported in aman with apparently normal DAZ gene constitution on DNA extractionfrom leukocytes57. These findings may be explained by unrecognized verysmall deletions encompassing active copies of DAZ, mosaicism, orabnormalities of DAZ transcription.

Advice to couples where the man has a Y microdeletion

What advice can we give our patients? There is no point in testing men formicrodeletions where ICSI is being used to overcome obstructiveazoospermia, as in these men spermatogenesis should be normal. For othermen with severely damaged spermatogenesis, testing for microdeletionsbefore ICSI is desirable; but, as these men and their male children areunlikely to have any phenotypic abnormality other than damaged sperm-atogenesis, it is reasonable to take into account the cost and limitations ofpresent methods of testing and to discuss this with the couple. If a manwith microdeletions and his partner wish to proceed with ICSI, they can beadvised that microdeletions will be passed to sons but not to daughters;however, it is unknown to what extent a son who inherits a microdeletionwill, in turn, have a fertility problem. The couple may be told that there isno evidence of any other health consequences of microdeletions. In a studyof the actual decisions taken by couples in this situation in TheNetherlands and Belgium, it was found that most chose to proceed withICSI but that 2 1 % refrained from treatment or chose DI; the decision wasstrongly influenced by the opinion of the counsellor58.


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Autosomal defects with severe phenotypic abnormalities as well as infertility

There are a number of inherited disorders with severe or considerablegeneralized abnormalities as well as infertility (Table 2). Such patients willbe well-known to doctors often from childhood and any fertility problemhas to be managed in the context of the care of the man as a whole andwith consideration of his and his partner's ability to care for a child shouldtreatment be successful.

Autosomal defects causing male infertility but in the absence of overt phenotypicabnormality

Lillford et alS9 published an epidemiological study indicating that reducedmale fertility may run in some families and there is some molecularevidence that there may be autosomal defects that could account for this.It is difficult to know how many autosomal genes are specific for fertility,but there are Likely to be relatively few as with evolution these tend toaccumulate on the Y chromosome.

Roest et al reported a mutation affecting the ubiquitin conjugatingenzyme hHR6A and hHR6B in mice60. This enzyme is implicated in post-replication repair. Heterozygous male mice and knockout female mice arecompletely normal and able to transmit the defect which, however, causedderanged spermatogenesis in homozygous mice. It is possible that similarhHR6B mutations may cause male infertility in man. If this is the case, thenthere may be an enhanced chance of passing such defect by ICSI as themale partner will be more likely than normal to carry such a defect and will

Table 2 Less common inherited disorders associated with infertility and other alterations to phenotype

Disorder Phenotype Genetic basis

Prader-Willi

Bardet-Biedle

Cerebellar ataxia and

hypogonadotrophichypogonadism

Noonan's syndrome

Myotonic dystrophy

Dominant polycystickidney disease

5-a-reductase deficiency

Obesity, mental retardation

Obesity, mental retardation,retinitis pigmentosa, polydactyly

Deletion of 15q12 on paternally inheritedchromosome

Autosomal recessive 16q21

Eunuchoidism and disturbances of gait and speech Autosomal recessive

Short stature, webbed neck, cardiac and pulmonary Autosomal dominantabnormality, cryptorchidism

Muscle wasting, cataract testicular atrophy

Renal cysts, obstruction from epididymal cysts

Perineal or scrota I hypospadias, vaginal pouchimmature female phenotype

Autosomal dominant 19q13.3

Autosomal dominant 16p13 3 and 4q

Autosomal recessive

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thus pass the defect to half of his offspring. The chance of the offspringbeing affected will depend on the prevalence rate of these mutations in thegeneral community. This may be quite low as there will have been atendency to select against such mutations because of their antifertility effect.

Cystic fibrosis mutations and male infertility

Cystic fibrosis, a fatal autosomal recessive disorder is the most commongenetic disease of Caucasians; 1 in 25 are carriers of gene mutationsinvolving the cystic fibrosis transmembrane conductance regulator gene(CFTR). This gene, located on the short arm of chromosome 7, encodesfor a membrane protein that functions as an ion channel and alsoinfluences the formation of the ejaculatory duct, seminal vesicle, vasdeferens and distal two-thirds of the epididymis. Congenital bilateralabsence of the vas deferens is associated with CFTR mutations and isfound in approximately 2% of men with obstructive azoospermiaattending our clinic in Edinburgh61. However, the incidence in men withobstructive azoospermia will vary in different countries depending onthe prevalence of cystic fibrosis mutations in the population and theprevalence of other causes of obstruction; in those countries with a highprevalence of sexually transmitted infection, CBAVD as a cause ofazoospermia will be relatively infrequent compared with azoospermiaassociated with post gonococchal epididymitis. However, the clinicalfinding of absent vasa is easy to miss and all men with azoospermiashould be very carefully examined to exclude CBAVD and particularlyif semen analysis reveals azoospermia in association with a semenvolume of less that 1.0 ml and the pH is acidic (<7.0)

In recent years, increasing numbers of mutations of the CFTR genehave been characterized and more than 400 have been described62.There have been at least 17 published series of men with CBAVD whohave been tested for varying numbers of mutations. In general, the moremutations tested for, the higher the percentage of men found to havemutations, so that in more recent publications61'63"65 detection rates havebeen higher (75%, 70%, 81%, 76%, respectively) whereas in olderpublications detection rates have been around 40%. In a review ofpublished series of 449 men with CBAVD, the Delta F508 mutation wasdetected in 244 men, the R117H mutation in 54 men and the W1282Xmutation in 37; additionally, 63 other mutations were detected inbetween 1-9 men, but not all mutations were tested for in all caseseries66. It seems likely that, as more and more mutations are defined andtested for, approaching 100% of men with CBAVD will be found to havemutations. At present it is not practical to test for all known mutationsas many have a very low prevalence in a particular populations and in


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most places testing is restricted to the 20-30 mutations that occur mostcommonly in that community.

Mutations may be found in both copies of the CFTR gene, but in mostmen with CBAVD they are found in only one copy. In some of these sup-posedly heterozygous cases there may be an unknown second mutation, butthere is also another interesting mechanism. In up to 63 % of these, a DNAvariant the 5T allele can be detected in a non-coding region of CFTR67 andwe have confirmed these observations in our own patients. Further work isneeded to understand the genetics of CBAVD fully. It is noteworthy that theheterozygous men with CBAVD whom we see in our clinic often have mildclinical stigmata of cystic fibrosis, e.g. history of chest infections. It will,therefore, be important to follow-up children born after ICSI and where thefather has CBAVD and is either hetero or homozygous. It is also worthadvising those men with any mild clinical stigmata to avoid smoking as theirrespiratory reserve will be reduced compared with normal.

There have been reports of CFTR mutations in men with severe oligozoo-spermia, but without absence of the vas deferens, and it has been postulatedthat the CFTR complex may also relate to spermatogenesis68. While therelationship between absence of the vas deferens and CFTR mutations isbecoming well established, the role of these mutations in spermatogeneticdefects is as yet unclear.

Advice for couples where the man has CBAVD

CFTR mutations have implications for clinical infertility practice. Whenthe male partner has CBAVD, it is important to test the female partnerfor CF mutations as well. If she is also found to be a carrier, then theremust be very careful consideration about whether the couple wishes toproceed with ICSI using the husband's sperm as the chance of a babywith cystic fibrosis will be 25% if he is heterozygous or 50% if he ishomozygous. If the female partner is negative for known mutations, herchance of being a carrier of unknown mutations is about 0.4% and, inthese circumstances, the chance of her heterozygous partner siring achild with cystic fibrosis is approximately 1:410. These figures areestimates calculated in the light of known mutation frequency inCaucasian populations, but will vary depending on the frequency of thedifferent mutations in different populations.

Unilateral (and bilateral absence/abnormality of the vas) and renal abnormalities -a different genetic disorder

Unilateral absence of the vas deferens is usually associated with ipsilateralabsence of the kidney69. This probably has a different genetic cause and is


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most commonly encountered as an incidental finding in the vasectomyclinic. CF tests should be performed when a man is found to have aunilateral absence of the vas and the kidneys are normal; conversely,when a man has bilateral vas abnormality and CF tests are normal, thenultrasound is indicated to define renal anatomy.

Mitochondrial abnormalitiesVery few, if any, mitochondria are a parental contribution to the embryoand, therefore, these are unlikely to be a significant factor in inheritedmale infertility, although clearly defective mitochondrial function insperm may, by way of poor sperm motility, be a cause of infertility.

Cytoplasmic disorders that may be inheritedThere is evidence that in humans the cytoskeleton is a paternal contri-bution and thus, in theory, there is the possibility of non-DNA transmittedabnormalities when very defective sperm is used70. Whether this actuallyoccurs is not known.

Unknown genetic disordersIntracytoplasmic sperm injection (ICSI) is now used to enable men withsevere damage to spermatogenesis to father children in situationsformerly considered hopeless and where only a very few spermatozoacan be obtained. This has led to worries that children may be born withfetal abnormality because, by bypassing the selective processes of thefemale genital tract and coverings of the egg, the process could enabledefective sperm to fertilize or, alternatively, eggs may be fertilized thatwould otherwise not do so. It is, therefore, very re-assuring that thecollected statistics of fetal abnormality from ICSI centres do not indicateany increase in congenital malformations compared with the generalpopulation. However, the indications for ICSI are constantly beingextended to include fertilisation with immature sperm forms and it willbe particularly important to continue to monitor fetal abnormality rateswith detailed subgroup analysis according to the clinical and moleculardiagnosis of the father (see Table 3).

Ethical considerations, genetic counselling and ICSI

The main difficulties will occur where there is a conflict of interestbetween the wishes of the couple and the interests of a future child.

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Table 3 Suggested male risk categories for the follow up of children born by ICSI

1 Men with sex chromosome abnormality on peripheral blood karyotyping

2 Men with autosomal chromosome abnormality on peripheral blood karyotyping

3 Men with obstructive azoospermia secondary to CBAVD This category has been discussed above

4 Men with damaged spermatogenesis and Y microdeletions, again discussed above

5 Men with other defined genetic disorders Some possibilities have been discussed above

6 Men with normal spermatogenesis and obstructive azoospermia, for example after failedvasectomy reversal but where karyotyping and genetic tests have not been undertaken or if theyhave been performed are normal In this category there is no reason to predict any geneticabnormality different from the general population

7 Men with normal karyotype and normal genetic tests but where spermatids have been used Incases where karyotype or genetic tests are abnormal then the followed up category shouldprobably be considered under one of the above groups

8 Men with testicular maldescent

9 Men with damaged spermatogenesis secondary to cancer chemotherapy

10 Men with damaged spermatogenesis secondary to known mrtogen, e g occupation hazard

11 Men with damaged spermatogenesis of unknown aetiology

The male diagnostic categories descend in order of genetic and diagnostic precision The actual risk tothe potential child of fathers in these categories is unknown, but use of pooled category data will helpdefine risks which will not be apparent from even large data sets

The best initial management is to give the couple full informationabout the risks to the child and then for the couple to decide whether toproceed or not. However, in the situation where both partners areknown to carry defects (for example, cystic fibrosis mutations) there canbe up to a 50% chance of the birth of a child who will develop clinicalcystic fibrosis and die young after a number of years of morbidity. In thissituation, many clinicians and infertility clinic personnel may feel thattheir duty of care to the future child and the interests of society as awhole outweigh the wishes of the mdividual couple and that it is notethical to proceed and that ICSI should not be offered to the couple. Insome countries, law may govern these matters and then there is nochoice, but, in the absence of law, this type of conflict makes the doctorsrole very difficult. Each case has to be judged on its merits and in thecontext of what is available and affordable in the local health caresystem. When there is a conflict that cannot be resolved by agreement,the interests of a future child probably take precedence over the interestsof a couple. If the decision is taken to proceed, it is important for thecouple to fully appreciate what may be in store for a future child and itis often appropriate for arrangements to be made for the couple to visitanother family where there is a teenager or older person suffering from

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Table 4 Risk of germline therapy78

1 Uncertainty and risk

2 The slippery slope particularly in regard to genetic enhancement

3 The lack of consent by future generations

4 Inappropriate allocation of healthcare resources

5 Intrinsic immorality

the condition. Also, the couple will need to give consideration to pre-implantation diagnosis and replacement only of normal embryos or, if thisis not available, amniocentesis and genetic diagnosis and the possibility oftermination.

ICSI is new technology, but perhaps the greatest potential application isthe opportunity for germ line therapy. At present in most countries this isconsidered unethical and sometimes illegal. Nevertheless, the followinghypothetical situation may help focus debate. Suppose a couple decide togo ahead with ICSI in a situation where both partners are known to havecystic fibrosis mutations, but after successful pregnancy would notcontemplate amniocentesis and abortion. If, in this situation, it waspossible to correct the mutation before embryo replacement, which wouldbe less harmful - to repair the defect and enable the birth of a childwithout cystic fibrosis, or not to repair the defect and for a child to beborn with cystic fibrosis? These arguments can be extended to correctionof oncogenes and other defects. The risks of germ line therapy (Table 4)have been summarized by Fiddler and Pergament71 in a review article,which gives powerful argument in favour of germ line therapy.

Summary

Our new knowledge of the genetic basis of infertility and the advent of ICSInecessitates good understanding of genetics by clinicians and the public atlarge. In this chapter, the emphasis is on the description of those geneticdisorders more likely to be encountered by the infertility clinician. Ad-vances in genetic diagnosis will cause discovery of the genetic basis of moredisorders and easier and cheaper diagnosis of known disorders; for someof these genes, therapy may become practicable. We live in the age ofgenetic discovery and infertility clinicians will need to keep up-to-date withfast moving scientific advances. It will also be important that infertilityclinicians keep in mind the fundamental ethical principles of beneficence,autonomy and justice and to be able to manage situation where theseprinciples may be in conflict.

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Acknowledgements

References

I would like to acknowledge the 19 year friendship and co-operationwith Dr Ann C Chandley (former Senior Scientist, MRC HumanGenetics Unit, Edinburgh) and in the last 6 years for work on the Ygenes with Prof. Howard Cooke and colleagues (MRC Human GeneticsUnit). I also acknowledge co-operation on work on androgen receptorswith Dr Phdlipa Saunders and colleagues (MRC Reproductive BiologyUnit, Edinburgh) and for work on CF mutations with Prof. David Brockand colleagues (MRC Human Genetics Unit, Edinburgh). I would alsolike to thank Dr Chanda Ghosh (Urology Department, Western GeneralHospital, Edinburgh) for help with references and sub-editing.

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