S.J.Qureshi 1, A.R.Ross 1, K.Ma 1, H.J.Cooke 1, M.A.M c lntyre 2, A.C.Chandley 1 and T.B.Hargreave Introduction

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1 Molecular Human Reproduction vol. no. pp , 1996 Polymerase chain reaction screening for Y chromosome microdeletions: a first step towards the diagnosis of geneticallydetermined spermatogenic failure in men S.J.Qureshi 1, A.R.Ross 1, K.Ma 1, H.J.Cooke 1, M.A.M c lntyre, A.C.Chandley 1 and T.B.Hargreave MRC Human Genetics Unit, department of Pathology, and 3 Department of Urology, Western General Hospital NHS Trust, Edinburgh EH4 XU, Scotland, UK 4 To whom correspondence should be addressed Overall, 11% of men attending infertility clinics suffer unexplained oligo or azoospermia. Cytogenetic observations of loss of the distal portion of the Y chromosome long arm (Yq) were found to be associated with disrupted spermatogenesis. The existence of a gene locus involved in the regulation of spermatogenesis, the azoospermia factor (AZF), was thus postulated. It is suggested that microdeletions, or mutations, at the AZF locus could result in impaired spermatogenesis in chromosomally normal men. In order to test this hypothesis we have carried out Y chromosome genetic screening of 0 oligo or azoospermic 46XY patients. We have also assessed phenotype/genotype relationships in those patients whose infertility has an underlying genetic aetiology. Patients were screened by polymerase chain reaction (PCR) with a set of Y chromosomespecific sequence tagged sites (STS) for submicroscopic deletions of their Y chromosome. Our results show that as many as 8% of cases of unexplained male infertility may have an underlying genetic aetiology related to microdeletions in two specific regions of the Y chromosome. Positive results from such a screen will be important when deciding the suitability of a patient for assisted conception schemes such as intracytoplasmic sperm injection. Key words: azoospermia/male infertility/oligozoospermia/spermatogenesis/y chromosome Introduction In the UK, ~8% of marriages are childless and amongst male partners who attend fertility clinics, worldwide surveys show that the largest diagnostic category is those with nonobstructive oligo or azoospermia, accounting for 11.% of all cases (Hargreave, 1994). The Y chromosome is an important carrier of genetic information in the control of spermatogenesis, with an early insight being gained from studies indicating a clear correlation between deletion of the long arm (Yq) distal region and a testicular histology of absent or severely impaired spermatogenesis (Tiepolo and Zuffardi, 1976). It was postulated that a locus important in the control of human spermatogenesis (the azoospermia factor, AZF) had been lost. The AZF locus has subsequently been mapped, using collections of deleted Y chromosomes, to interval 6 (Vergnaud et al, 1986), lying within cytological band Yqll.3 (Andersson et al, 1988). However, the phenotypic abnormalities of absent or severely impaired spermatogenesis found in men with Yq cytological deletions are also seen amongst chromosomally normal (46XY) individuals. This raised the possibility that, at least in a proportion of cases, oligo or azoospermia might have, as its underlying aetiology, a disruption by microdeletion or mutation of the AZF locus. Confirmation of this came in 199 when a panel of 19 chromosomally normal azoospermic men was screened by Southern analysis with a series of Yspecific D probes and two individuals with microdeletions were detected (Ma et al., 199). One individual, codename Jolar, showed deletion of two probes in proximal subinterval I of interval 6; the second, Klard, showed deletion of 11 probes mapping across distal subintervals XIIXIV. These proximal and distal microdeletions did not overlap, indicating either that the AZF locus is very large, or that more than one gene, or different members of one gene family, might be involved in the process of spermatogenesis. Other similar screening programmes have confirmed the common occurrence of microdeletions in Yq amongst patients with unexplained oligo or azoospermia (Kobayashi et al, 1994; Reijo et al, 1995, 1996). In 1993 a gene, RBM (Rbinding motif; formerly YRRM) was isolated from the distal Klard deletion interval (Ma et al, 1993). RBM belongs to a gene family containing up to 30 members, distributed across the length of the Y chromosome (Schemmp et al, 1995). Due to their Yspecific location, testisspecific expression, crossspecies conservation and partial deletion in some oligo and azoospermic patients, they have been proposed as candidates for AZF (Ma et al, 1993; Chandley and Cooke, 1994; Schemmp et al, 1995). More recently, a second Y chromosome gene, DAZ (deleted in azoospermia), which also expresses specifically in the testis and bears an R recognition motif, has been isolated and proposed as another candidate for AZF (Reijo et al, 1995). However, neither RBM nor DAZ have so far yielded information concerning specific point mutations in nondeleted oligoor azoospermic men, and the identity of AZF is still unknown. In the absence of a test for a specific AZF gene, it is nevertheless still possible to test for the presence or absence of Y chromosomespecific sequences known to be associated with oligo and azoospermia. European Society for Human Reproduction and Embryology 775 Downloaded from on 1 December 017

2 S.J.Qureshi et al. In the present paper, we describe a screening exercise carried out amongst 0 chromosomally normal men all of whom were severely oligozoospermic or azoospermic, together with information from a series of fertile controls (n = 80), 1 of whom were fertile brothers or fathers of the cases. Materials and methods Patients Men were selected from a large infertility practice, including those with unexplained severe oligozoospermia and those with nonobstructive azoospermia. Excluded were those with history of testicular maldescent, history of testicular malignancy or cancer chemotherapy or radiotherapy and those men with history of testicular injury including torsion. All men selected for polymerase chain reaction (PCR) analysis appeared chromosomally normal when karyotyped. Their seminal analyses showed azoospermia (n = 51) or oligozoospermia (0.10X 6 /ml; n = 47, two remaining individuals having mean sperm counts of 36.8 and 60.3X lovml). Clinical features in many included high concentrations of follicle stimulating hormone (FSH) and reduced testis volumes, each indicative of spermatogenic impairment. Approximately half of all cases had undergone testicular biopsy making phenotype/genotype correlations possible. Grandfathers and fathers from the Centre Etudes Polymorphism Humaines, Paris (CEPH) reference families were used as fertile controls. Where possible the fathers and/or fertile brothers of patients with microdeletions were also analysed. A total of 80 normal controls was tested. Sequence tagged site analysis Human genomic Ds were prepared from blood or lymphoblastoid cell lines using standard techniques. A series of Y chromosomespecific STSs have been characterized previously and the primer sequences described (Foote et al., 199). A total of 3 loci, spanning the Jolar and Klard deletion intervals was examined (Ma et al., 199). PCR was performed in 50 ul volumes in 1.5 mm MgCI^, mm Tris (ph 8.3), 50 mm KC1, 0.01% gelatin, 00 um dntps, with 1 IU of Taq polymerase, 000 ng of human genomic D and each primer at 1 \im. Thermocycling consisted of 3 min at 94 C for one cycle, 1 min at 94 C, 1 min at 58 C (Reijo et al., 1995), 1 min at 7 C for 35 cycles. Reactions were stored at 4 C until loading onto 4% gels for analysis. To prevent scoring of false negatives, each PCR reaction included internal control primers, usually a second STS. An STS was considered to be absent after three amplification failures in the presence of positive amplification of the control primers. Results The phenotypes of the patients included in this study are summarized in Table I. The average age at ascertainment was 31 years (range 1947 years) with no difference between the five subdivisions based on mean sperm counts (azoospermic, >05, >5, > 0, >0xl0 6 /ml). Of the 38 patients with mean sperm counts >5X lofyml, 1 had counts <1 X 6 / ml. Testis volumes showed a small decline as the sperm counts declined but even for the azoospermic group, the average testis volume was within the normal range. Of 16 azoospermics who had been biopsied, four were found to have Sertoli cellonly testes. Concentrations of luteinizing hormone (LH) and testosterone also remained within the normal ranges as sperm counts declined, although there was a tendency for concentrations of these hormones to increase as the sperm count decreased. STS screening identified eight patients with microdeletions in Yq. In order to show that these deletions are related to phenotype rather than being polymorphisms, we asked for blood from fertile male relatives. We found that neither the fathers nor brothers of Klard, Eltor or Jolar were deleted. Permission was sought but not granted from the relatives of Kupau, Nikei, Amgal, Sayer or Hamil. We also screened a further 74 men of proven fertility and found that none carried microdeletions in either of the two regions tested. A representative PCR is shown in Figure 1. Four individuals had a proximal microdeletion of Yq, in the Jolar interval, while a further four individuals had a more distal microdeletion, in the Klard interval. The extent of these deletions is summarized in Figure. In the Jolar interval, three patients, Jolar, Amgal and Eltor, were deleted for three consecutive STS, sy84, sy85 and sy86. A fourth patient, Sayer, was also missing an adjacent STS, sy87. The size of this deletion interval is estimated at 500 kb. In the Klard interval, the largest deletion was found in Kupau. The smallest deletion interval was found in patient Hamil, who was deleted for only one of the STS tested, sy15. The phenotypic features of these eight patients are presented in Table II. All four patients deleted in the Jolar region, and two, Nikei and Hamil, deleted in the Klard region, underwent testicular biopsy. A variable picture was found histologically. Jolar and Eltor, deleted proximally, were azoospermic with Sertoli cellonly syndrome (SCO); Amgal, also azoospermic, was mainly SCO, with a few spermatogonia present in some tubules; Sayer showed diminished spermatogenesis with occasional tubules containing spermatogonia, spermatocytes or spermatids. His mean sperm count was 4.3X 6 /ml. Among the patients deleted distally, Nikei showed depression of spermatogenesis with partial arrest and degeneration at the spermatocyte and spermatid stages but a very few spermatozoa were seen both in the testis section and in the ejaculate. Hamil showed severely diminished spermatogenesis, only a minority of tubules containing spermatozoa. His mean sperm count was 0.6X1 Otyml. Klard and Kupau were not biopsied; seminal analysis gave azoospermia in the former and a mean count of 0.4X lofyml in the latter. For Sayer, Nikei, Kupau and Hamil, the seminal analysis also indicated poor sperm motility, and, where analysed, poor morphology. Discussion It is worth noting that our series of patients includes a significant number with very low sperm counts (<lx 6 /ml). At the time we began this work, microdeletions had not been discovered; included in the series reported here are the original patients in which we first described the presence of microdeletions (Ma et al., 199). Our rationale for choosing patients with severe oligozoospermia was that if there was a genetic abnormality, it is in this group it would be likely to be found and subsequent events have shown this to be a correct assumption. Using a simple PCRbased strategy we have used a set of 776 Downloaded from on 1 December 017

3 Y chromosome microdeletions and male infertility Table I. Phenotypic features of all patients. Figures in parentheses are ranges Mean sperm count (X 6) All patients (Azoospermic69.3) Azoospermic >05 >5 >0 >0 nc No. cases Mean SD age at Mean testis vol. (ml) Hormone concentrations*" R L LH FSH Testosterone 17 6 (630) ( ) 15 dt 6 15 it 8 19 it 5 0 Jt 7 3 dt ( ) ( ) (1947) I Normal range for Caucasians = 1545 ml; R = right, L = left. b Normal range: luteinizing hormone (LH) = 16.5 IU/1; follicle stimulating hormone (FSH) = 16 IU/1; testosterone = 30 ng/ml. TData not available for every patient O LU * ^^34194 Figure 1. A representative polymerase chain reaction. The control male sample and one patient, codename Kekir, are positive for all four sequence tagged sites tested (sy83, sy84, sy85, sy86). Only sy83 is amplified in Eltor. Y chromosomespanning STS to screen a subset of 0 oligoand azoospermic patients for Yq microdeletions. We have demonstrated that 8% of these patients carry a microdeletion on Yq. The microdeletions were not found at random but were restricted to two specific intervals, proximal subinterval I of interval 6 (the Jolar interval), and distal subintervals XIIXIV (the KJard interval, see Figure ). A total of 80 fertile men, including 1 who were relatives of patients, was also screened. None carried a microdeletion. We conclude that Y chromosome microdeletions may be associated with spenmatogenic disturbance, suggesting that a significant genetic aetiology underlies some male infertility. In the Jolar region the minimum deletion interval is defined by three of the patients, Jolar, Amgal and Eltor: Sayer has a larger deletion. The phenotypes of these patients are described in Table II. The three patients with the smallest deletion (Figure ) have stronger phenotypes; SCO or partial SCO. In the Klard interval, three patients, Klard, Kupau and Nikei have similar deletion intervals (Figure ). However, the fourth patient, Hamil, is deleted for just one STS, sy15. This seems to place the critical breakpoint region to between sy15 and sy3. Neither Jolar, Amgal, Eltor, Sayer nor Hamil are deleted for DAZ, questioning the role of DAZ in spermatogenesis. However, as RBM genes are located at several positions along Yq, it is not clear at this point whether any or all of the patients have lost critical RBM genes. The existence of two distinct deletion intervals suggests that more than one AZF gene is important, or that AZF may belong to a gene family, with loci at several positions along the Y chromosome. In our opinion it is premature at this time for Vogt (Vogt, 1995; Vogt et ai, 1996) to suggest that three AZF regions, AZFa (Jolar; STS 8486), AZFb (middle; STS 07143) and AZFc (Klard; STS 3158), exist on the long arm of the human Y chromosome, each with a distinct phenotype. Our patient, Sayer, showed microdeletion in 'AZFa', had spermatogenesis (albeit diminished), and a sperm count of 4.3XlO6/ml, a phenotype very similar to those of Nikei and Hamil who were deleted in 'AZFc'. Also, among the azoospermic patients reported by Reijo et al. (1995) who were deleted in 'AZFc', two had SCO, which would be expected for deletion in 'AZFa' on the Vogt hypothesis. In conclusion, we have demonstrated how PCR screening can be used to detect a genetic basis for male infertility. The technique is very easy to apply and results can be obtained quickly. Many of the men in this study might well be considered as candidates for intracytoplasmic sperm injection (ICSI) at assisted reproduction clinics. We would predict that the use of ICSI for such men could result in the passing of a microdeletion (and presumably impaired function) to a son. This is presently the subject of a multicentre cooperative study (study of ICSI male infertility and microdeletions in the Y chromosome; SIMMY), based in our Unit. We would recommend that no patient with non obstructive azoospermia or severe oligozoospermia should undergo ICSI without first being tested for Y chromosome microdeletions. If a deletion were detected, the 777 Downloaded from on 1 December 017

4 S.J.Qureshi et al. a. b. (Q3;!!'~ c. a > CD ^ u CO CO CO CO ^y ^^ c o m c o t o c o c o c o t t CO GO CO CO CO CO CO CD d. Fertile Males JOLAR AMGAL ELTOR SAYER I Figure. Diagram of the Y chromosome microdeletions. (a) Y chromosome; (b) Y chromosome subintervals; (c) sequence tagged sites (STS); (d) deletion pattern of patients. The solid box indicates presence of an STS. Blank spaces indicate absence of an STS. Table II. Phenotypic features of men with Yq microdeletion Patient Age (years) al codename am:eruunmeni Testis vol (ml)' R L Hormone concenlrations b LH FSH Testosterone Testicular histology findings Johnsen d score R L Mean count (X IO*/ml) Seminal analysis Motility^ Morphology 0 (% normal) (% normal) JOLAR AMGAL ELTOR SAYER KLARD NIKE1 KUPAU HAMIL Sertoli cellonly Mainly Sertoli cellonly Sertoli cellonly Dimished spermatogenesis NB Dimished spermatogenesis with partial arrest and degeneration at the spermatocyte and spermatid stages NB Severely diminished spermatogenesis Three nonmotile spermatozoa seen on a wet preparation _ _ Abnormal heads seen Normal range for Caucasians = 1545 ml; R = right L = left. b Normal range: luteinizing hormone (LH) = 16.5 IU/1; follicle stimulating hormone (FSH) = 16 IU/1: testosterone = 30 ng/ml. c See text for further details. d Johnsen (1970) = not applicable; = not ascertainable: NB = not biopsied. family should be given appropriate genetic counselling to warn them of the risks (Chandley and Hargreave, 1996; Reijo et al., 1996; Persson et al, 1996; Martin, 1996). At this stage it is not possible to draw conclusions about the position or extent of a microdeletion and its effect on fertility. Although there is a tendency for a more severe phenotype of spermatogenic failure when a deletion occurs proximally on Yq, this is by no means absolute and the need still is for many more cases to be analysed before a meaningful understanding of genotype/phenotype correlations can be obtained. Clearly there is still much to be learned about the distribution and activity of Ylinked genes which control the 778 Downloaded from on 1 December 017

5 Y chromosome microdeletions and male infertility spermatogenic process in man, and only by the continued screening of oligo and azoospermic individuals will our understanding develop. Acknowledgements Dr P.Ellis and the cytogeneticists of the Department of Genetic Pathology, Royal Hospital for Sick Children, Edinburgh, are thanked enormously for their assistance in providing information on the somatic karyotypes of all of the men included in this study. This work was funded by the Medical Research Council. References Andersson, M., Page, D.C., Pettay, D. et al. (1988) Y autosome translocations and mosaicism in the aetiology of 45,X maleness: assignment of fertility factor to distal Yqll. Hum. Genet., 79, 7. Chandley, A.C. and Cooke, HJ. (1994) Human male fertilityylinked genes and spermatogenesis. Hum. Mol. Genet., 3, Chandley, A.C. and Hargreave, T.B. (1996) Genetic anomaly and ICSI. Hum. Reprod., 11, Foote, S., Vollrath, D., Hilton, A. et al. (199) The human Y chromosome: Overlapping D clones spanning the euchromatic region. Science, 58, Hargreave, T.B. (1994) Human infertility. In Hargreave, T.B. (ed.), Male Infertility. nd edn. Springer Verlag, London, pp Johnsen. S.G. (1970) Testicular biopsy score counta method for registration of spermatogenesis in human testes. Normal values and results of 335 hypogonadal males. Hormones, 1, 14. Kobayashi, K., Mizuno, K, Hida, A. et al. (1994) PCR analysis of the Y chromosome long arm in azoospermic patients: evidence for a second locus required for spermatogenesis. Hum. Mol. Genet., 3, Ma, K., Sharkey, A., Kirsch, S. et al. (199) Towards the molecular localisation of the AZF locus: mapping of microdeletions in azoospermic men within 14 subintervals of interval 6 of the human Y chromosome. Hum. Mol. Genet., 1, 933 Ma, K., Inglis, J., Sharkey, A. et al. (1993) A Y chromosome gene family with Rbinding protein homology: candidates for the azoospermia factor controlling human spermatogenesis. Cell, 75, Martin, R.H. (1996) The risk of chromosomal abnormalities following ICSI. Hum. Reprod., 11, Persson, J.W., Peters, G.B. and Saunders, D.M. (19%) Is ICSI associated with risks of genetic disease? Implications for counselling, practice and research. Hum. Reprod., 11, Reijo, R., TienYi, L., Salo, P. et al. (1995) Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel Rbinding protein. Nature Genet.,, Reijo, R., Alagappan, R.K., Patrizio, P. et al. (1996) Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet, 347, Schemmp, W., Binkele, A., Amemann, J. et al. (1995) Comparative mapping of YRRM and TSPYrelated cosmids in man and hominoid apes. Chromosome Res., 3, 734. Tiepolo, L. and Zuffardi, O. (1976) Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum. Genet., 34, Vergnaud, G., Page, D.C., Simmler, M.C. et al. (1986) A deletion map of the human Y chromosome based on D hybridisation. Am. J. Hum. Genet., 38, 914. Vbgt, P. (1995) Genetic aspects of artificial fertilization. In Ombelet, W. and Vereecken, A. (eds), Modem Andrology. Hum. Reprod., (Suppl. 1), Vbgt, PH., Edelmann, A., Kirsch, S. et al. (1996) Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yqll. Hum. Mol. Genet., 5, Received on June 17, 1996; accepted on August 3, Downloaded from on 1 December 017