The likelihood of finding mature sperm cells in men with AZFb or AZFb-c deletions: six new cases and a review of the literature ( )

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1 The likelihood of finding mature sperm cells in men with AZFb or AZFb-c deletions: six new cases and a review of the literature ( ) Sandra E. Kleiman, Ph.D., Leah Yogev, Ph.D., Ofer Lehavi, M.D., Ron Hauser, M.D., Amnon Botchan, M.D., Gedalia Paz, Ph.D., Haim Yavetz, M.D., and Ronni Gamzu, M.D., Ph.D. Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Objective: To reassess the predictive value of detecting sperm cells in men with AZFb or AZFb-c deletions. Design: Retrospective analysis of previously reported men with AZFb or AZFb-c deletions and the addition of six new cases. Setting: Fertility institution. Patient(s): Men with both sequence tagged site marker identification and testicular cytology/histology findings. Intervention(s): Systematic review of reported men with microdeletions that included eligibility, data extraction and analysis. Main Outcome Measure(s): Availability of sperm cells for intracytoplasmic sperm injection (ICSI) in men with AZFb/AZFb-c microdeletions. Result(s): The average prevalences reported for AZFb, AZFb-c, partial AZFb, and partial AZFb-c in azoospermic men were 0.9% 0.07%, 2.7% 0.93%, 1.23% 0.9%, and 1% 0.6%, respectively. Sperm cells were identified in 7% and 3% of the 28 and 71 men with complete AZFb and AZFb-c and in 57% and 43% of the 14 and 7 men with partial AZFb and AZFb-c deletions, respectively. The likelihood of finding sperm cells in men with complete versus partial AZFb and AZFb-c deletions was significantly lower. As yet, no clinical or chemical pregnancy after ICSI in cases with complete AZFb/b-c microdeletions has been reported. Conclusion(s): Determining the extent of AZFb or AZFb-c deletions is critical considering the frequency and the reasonable prospect of finding sperm cells in partial AZFb/AZFb-c deletions. Referring men with complete AZFb/ b-c microdeletions to testicular sperm extraction/icsi programs should be revaluated. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: Y-chromosome microdeletion, AZF microdeletion, male infertility, sperm cells, spermatogenesis Received November 2, 2010; revised January 24, 2011; accepted January 25, 2011; published online March 1, S.E.K. has nothing to disclose. L.Y. has nothing to disclose. O.L. has nothing to disclose. R.H. has nothing to disclose. A.B. has nothing to disclose. G.P. has nothing to disclose. H.Y. has nothing to disclose. R.G. has nothing to disclose. The research was carried out under the auspices of the Alan and Ada Selwyn Chair in Clinical Infertility Research and Molecular Medicine (Melbourne, Australia) granted to one of the authors (G.P.). Reprint requests: Sandra E. Kleiman, Ph.D., Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel ( ser@tasmc.health.gov.il). The involvement and prevalence of Yq-chromosome microdeletions in male factor infertility have been widely addressed during the past 15 years. These microdeletions are more common among men with nonobstructive azoospermia (7% 23%) compared with among men with severe oligozoospermia (1% 8%) (1 4). The presence of Yq microdeletions was historically detected in three regions referred to as azoospermia factor AZFa, AZFb (later demonstrated to include the proximal part of AZFc), and AZFc (5, 6). The most frequently deleted region (roughly 50%) is AZFc in its entirety, which is caused by recombination between two similar direct repeats (between amplicons b2 and b4) (7). The absence of the AZFb/b-c regions are the next most frequently observed deletions. Recombination between palindromes P5 and the proximal arm of palindrome P1 (P5/proximal P1) is the most common cause of complete AZFb deletion, while recombination between palindrome P5 or P4 and the distal arm of palindrome P1 (P5 or P4/distal P1) leads to complete AZFb-c deletion (6). The approximate Y-chromosome coordinates of these deletions are Mb (palindromes P5-P4) to 23.9 Mb or Mb (proximal or distal palindrome P1) for AZFb or AZFb-c, respectively. Absence of the complete AZFa region is rarely observed, and it is caused by recombination between two provirus sequences spaced by approximately 800 kb (8 10). The initial analyses of Y-chromosome microdeletions were performed with uncertified and heterogeneous markers (sequence tagged sites [STSs]) that were frequently uninformative or unreliable. The precise mapping of the Y-chromosome allowed the use of specific STSs of high quality (7, 11). Introducing guidelines for molecular diagnosis of Yq microdeletions (12, 13) enabled the Yq microdeletion test to become more homogeneous and reliable. The chances of finding mature sperm cells are different for each deleted region. Severe oligozoospermia is associated with 38% of men with AZFc deletion, and the chance of finding testicular sperm cells is up to 67% among azoospermic men with AZFc deletion (14). Men with complete AZFa or AZFb deletions or with larger deletions are usually azoospermic, with no likelihood of finding any mature sperm cells by testicular sperm extraction (TESE) (13, 15, 16). A number of studies have, however, reported the detection of mature sperm cells in men with AZFb/b-c deletions (17 27). Therefore, the degree of success in finding sperm cells in men with AZFb/b-c is still inconclusive /$36.00 Fertility and Sterility â Vol. 95, No. 6, May doi: /j.fertnstert Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 This paper aims to estimate the likelihood of finding mature sperm cells in a large group of men with AZFb/b-c deletions reported in the literature between 1994 and Only the men with these deletions who underwent TESE and their testicular histological or cytological assessment were available or sperm cells were detected in semen were enrolled. Several heretofore unreported men detected by our group were added to the cohort. METHODS Inclusion/Exclusion Criteria A search was done in PUBMED for all published studies on AZF microdeletions, and only original English publications on humans were retrieved for review (Supplementary Fig. 1). A meticulous analysis of the STS markers tested in each published case was performed in an attempt to estimate whether the AZFb deletion was partial or complete. Only papers that allowed linking between both the extent of the deletion and the testicular histology/ cytology findings or the presence of sperm cells in semen were included (Supplementary Fig. 1). The 19 men reported by Hopps et al. (28) and the single male reported by Stouffs et al. (22) were the exceptions: although the borders of the deletions were variable, they all included deletions of common STSs that clearly defined the existence of complete AZFb (from sy117 to sy143) and AZFb-c (from sy117 to sy254) microdeletions. Unreported cases detected in our institute were added. Cases of reported discontinuous or isolated single STS deletions were excluded to form more homogenous groups of cases with similar and frequent microdeletions. Sperm cell concentrations in semen and karyotypes were included when available. Only STS markers found in the MSY Breakpoint mapper ( breakpointmapper.wi.mit.edu/mapper.html) were taken into consideration to determine the extent of the deletion (29). This decision was based on the revision of the STS map after the Y-chromosome sequence was completely decoded. Single and multicopy STSs in the MSY mapper database were reliable markers that were accurately located, allowing precise determination of both the extent of the reported deletions (29) and the classification of the AZFb/b-c deletions into complete and partial deletions. Evaluation of AZF Microdeletions The new cases reported here for the first time were detected by multiplex polymerase chain reaction (PCR) as reported elsewhere (2), with the modification that each multiplex PCR mix also included ZFY/X (Supplementary Fig. 2). Whenever an AZFb/b-c deletion was identified, its extent was further assessed by multiplex PCR with the following additional STSs: sy1227, sy1228, sy1224, sy118, sy1192, sy1291, sy639, sy1257, sy1201, and sy160. RESULTS For simplification, we used the traditional terminology in which AZFb refers to P5/proximal P1 and AZFb-c refers to P5/distal P1 or P4/distal P1. Other breakpoints in the region were referred to as partial AZFb/b-c deletions. The 120-subject study cohort was made up of 28 men with complete AZFb microdeletion, 71 men with complete AZFb-c deletion, 14 men with partial AZFb microdeletion, and seven men with partial AZFb-c deletion (Tables 1, 2, and 3, respectively). Twenty-nine men in whom the assessed STSs could not determine whether or not the proximal area of the AZFb region was deleted were included in an separate table (Supplementary Table 1) (45 48). Frequency of Microdeletions That Include the AZFb Region AZFb-c is the most frequent microdeletion that includes the AZFb region among azoospermic men. The average prevalence (% SD) calculated for AZFb, AZFb-c, partial AZFb, and partial AZFb-c in azoospermic men was 0.9% 0.07%, 2.7% 0.9%, 1.2% 0.9%, and 1% 0.6%, respectively (Supplementary Table 2). Evaluation of Men with Complete AZFb/b-c Deletions The results of karyotyping were reported in 21 men with AZFb microdeletions and in 68 men with AZFb-c microdeletions. Mosaic karyotype 45,XO/46,XY was the most frequently detected defect, and it was observed with especially high frequency (seven of the 68 men with the AZFb-c deletion; Table 2). Only one of the 21 men from the study cohort with AZFb deletion was found to have the 45XO/46XY karyotype. Of the 99 reported men with complete AZFb/b-c deletion, 96 were found to be azoospermic. The remaining three men had sporadic sperm cells in sporadic semen analyses (Table 1 and 2) (22, 24, 26). Sperm cells were rarely observed in the testicular tissue of men with complete AZFb/b-c deletion (Table 4). Semen and testicular sperm cells were reported by three different groups in men with complete AZFb/b-c deletion (Tables 1 and 2) (21, 24, 26, 25, 39, 42). Another recent case report (not included in Table 2) described a man with an unusual AZFb-c deletion in which a concentration of one million sperm cells was observed in one semen sample followed by azoospermia in the subsequent tests: no mature sperm cells were detected in the TESE of this man for a further intracytoplasmic sperm injection (ICSI) procedure, but spermatids were identified (50). Maturation arrest was reported in 83% of the specimens with AZFb deletion and Sertoli cells only in 73% of the AZFb-c deleted specimens (Table 4). Data on assisted reproductive technology (ART) outcomes of men with AZFb microdeletions are rare. As yet, there are no published reports of an ongoing or even chemical pregnancy achieved via ICSI using sperm cells with complete AZFb/b-c deletion. Stouffs et al. (22) detected sperm cells in the first ejaculate of a man with AZFb microdeletion, and oocytes from his wife were retrieved, but ICSI was not accomplished because no sperm cells were found in the second ejaculate or in the subsequent TESE attempt. Other reports did not note ICSI attempts (24, 26). Evaluation of 21 Men with Partial AZFb/b-c Deletions Twenty of these 21 men had a normal karyotype. The one exception had partial AZFb-c deletion and a mosaic karyotype 45,X0/46,X,r (Y)/47,X,r(Y),r(Y) [10/89/1]. Severe oligozoospermia was frequent among men with partial AZFb or partial AZFb-c deletions (Tables 3 and 4). Sperm cells either in semen or in testes were found fairly frequently among men with partial AZFb/b-c deletions (Table 4). Among the 14 men with partial AZFb microdeletions, the proximal region (palindromes P5 and P4) was identified in five of them who had sperm cells (Table 3). The possibility of finding sperm cells among men with partial AZFb/b-c deletions was significantly higher than that for men with complete AZFb/b-c deletions in the current study (P<.001, Fisher s exact test). Hypospermatogenesis was often observed in the testis histology of partial deletions (Table 4). Choi et al. (30) reported a chemical pregnancy with sperm cells of a man with a partial AZFb deletion. A vertical natural transmission of a partial AZFb deletion through two generations was also reported (19), but this report was not included in Table 3 of the current review because it does not fulfill the inclusion criteria. The patient himself, his father, and his son had an unusual discontinuous deletion that included sy130 and sy143, but there were two STSs mapped between them (sy134 and sy142). Two twin pregnancies and the delivery of two female newborns was recently reported with the sperm of men with a partial AZFb deletion that did not remove the RBMY and PRY genes (51) Kleiman et al. Sperm cells and AZFb/AZFb-c microdeletions Vol. 95, No. 6, May 2011

3 TABLE 1 List of 28 men with complete AZFb microdeletion. Findings in histology/ cytology a Extent of the deletion (STS mapped by MSY) 5 0 Coordinate of deleted STS at the borders of the deletion, bp Proximal Distal Karyotype Reference no. Arrest spd ,XY 4 Arrest ,XY 2 NSSR 109- b ,XY 28 NSSR 109- b ,XY 28 NSSR 109- b ,XY 28 NSSR 109- b ,XY 28 NSSR 109- b NR 28 NSSR 109- b NR 28 NSSR 109- b NR 28 NSSR 109- b NR 28 NSSR 109- b NR 28 Arrest ,XY 22 Arrest ,XY 22 Arrest spg ,XY 22 c Hypo ,XY 21 Arrest NR 30 SCO ,XY 31 SCO, Arrest ,XY 31 Arrest ,XY 31 SCO ,X/46,X,idic(Y) [14/86] 25 HS ,XY 26 HS ,XY 26 HS ,XY 26 d Arrest ,XY 26 Arrest ,XY 32 NSSR NR Current report Arrest ,XY Current report Arrest ,XY Current report Note: Twenty-seven men had a P5/proximal P1deletion, and one man had a larger deletion that extended proximal from P5. Bold STS numbers denote the closest tested STS that was present before or after the extreme STS was deleted. STS ¼ sequence tagged site; NSSR ¼ no surgical sperm retrieval; Hypo ¼ hypospermatogenesis; HS ¼ quantitative reduction in the number of germ cells but no mature sperm cells detected; Arrest ¼ arrest at the spermatocyte stage unless it was otherwise specified; SCO ¼ Sertoli cell only; SPG ¼ spermatogonia; SPD ¼ spermatid; NR ¼ not reported. a Some papers reported different histology/cytology in each testis; all men were azoospermic except one (see footnote c). b Minimal proximal extent of the deletion (all deletions included sy117). c Reported to have 20 spermatozoa/ml in the first semen sample and none in the second sample. d A unique recombination after rearrangements between palindromes. Evaluation of Men with Inconclusive AZFb/b-c Deletions Twenty-nine men with inconclusive deletions that included the AZFb region are described in detail in a separate table (Supplementary Table 1). Since the STSs used in these men were not close enough to 19.2 Mb, it could not be accurately determined whether the deletion included the proximal AZFb region. The frequency of azoospermia as well as the frequency of sperm cell detection in semen or testis among these men was in between the complete and partial deletion frequencies, probably reflecting the fact that both kinds of deletions had been included (Table 4). Brandell et al. (17) identified round spermatids in two men, one with an AZFb microdeletion and one with an AZFb-c microdeletion, but the exact extent of the deletion was not given. No fertilization occurred when the round spermatids were injected into oocytes. A decade of experience in diagnostic, management, and clinical pregnancy outcomes of men with Y-chromosome microdeletions among American men was recently reported (52). Although the exact extent of the deletions for each case was not specified, no sperm cells were retrieved in 14 men with AZFb/b-c deletions. That report included one man with a partial AZFb-c microdeletion, in which the proximal portion of the AZFb region was not deleted: he had sperm in his ejaculate, but there was no mention of pregnancy achievement with these sperm cells. DISCUSSION The results of the present survey support the assessment of an AZF microdeletion as being an essential test for predicting the detection of sperm cells. Determining whether the AZFb region is partially or completely deleted seems to be critical for the prediction of the presence of sperm cells in the testis (Table 4), as previously proposed in a review (16). The extremely low chance of detecting sperm cells in men with complete deletion of AZFb is a common observation among all the reports. As yet, no oocyte fertilization nor clinical pregnancy or ongoing pregnancy with sperm cells with complete AZFb deletion have been reported. Understanding the rare cases of AZFb/b-c microdeletion with sperm cells that do have Fertility and Sterility â 2007

4 TABLE 2 List of the 71 men with complete AZFb-c (P5/distal P1 or P4/distal P1or P5-P4/terminal) microdeletion. Findings in histology/ cytology a Extent of the deletion (STS mapped by MSY) 5 0 Coordinate of deleted STS at the borders of the deletion, bp Proximal Distal Karyotype Reference no. SCO ,XY 4 Arrest t Xdel(Y) (q11.2) 4 SCO t X idicy 1 SCO ,XY 33 Arrest ,XY 34 SCO ,XY 35 SCO t ,XY 35 SCO ,XY 35 SCO t ,XY 35 SCO ,XY 35 SCO 88- c t ,XY 35 SCO t ,XY 35 SCO 88- c t ,XY 35 SCO 88- c t ,XY 35 SCO t ,XY 35 NSSR 88/109- d d -157/160/t X/46,XY 28 NSSR 88/109- d d -157/160/t X/46,XY 28 NSSR 88/109- d d -157/160/t XinvY (q11.2q12) 28 SCO þ few germ cells ,XY 36 SCO þ carcinoma in X/46,XY 37 situ, SCO SCO NR 30 Arrest NR 30 SCO ,XY 38 Arrest t X/46,XY [82/18] 39 Arrest ,XY 39 SCO (or NR ) SCO ,XY 22 SCO ,XY 22 Arrest ,XY 22 SCO ,XY 40 Hypo ,X/46,X,dic(Y)[12/38] 24 e SCO ,X/46,X,?dic(Y)(p10)/ 24 46,X,þmar/47,X?idic(Y) (p10)[33/9/5/3] SCO ,XY 31 Arrest ,XY 31 Arrest b ,XY 31 SCO b ,XY 31 SCO ,XY 32 SCO t ,XYdelY 32 SCO t ,XY 27 SCO t ,XY 27 SCO ,XY 27 SCO ,XY 27 SCO ,XY 41 SCO ,XY 41 Arrest ,XY 41 Arrest ,XY Kleiman et al. Sperm cells and AZFb/AZFb-c microdeletions Vol. 95, No. 6, May 2011

5 TABLE 2 Continued. Findings in histology/ cytology a Extent of the deletion (STS mapped by MSY) 5 0 Coordinate of deleted STS at the borders of the deletion, bp Proximal Distal Karyotype Reference no. SCO ,XY 41 SCO 88- c t ,X/46,Xdel(Y)/47, 42 Xdel(Y)del(Y) SCO ,XY 26 SCO ,XY 26 SCO ,XY 26 SCO ,XY 26 SCO ,XY 26 SCO ,XY 26 SCO ,XY 26 SCO ,XY 26 SCO ,XY 26 Arrest ,XY 26 Arrest ,XY 26 SCO t ,XY 26 Hypo t ,XY 26 f NSSR ,XY Current report NSSR t ,XY Current report Arrest 97- c t Xdel(Y) (q11.2) Current report Note: Bold STS numbers denote the closest tested STS that was present before or after the extreme STS was deleted. STS ¼ sequence tagged site; SCO ¼ Sertoli cell only; t ¼ terminal deletion; NSSR ¼ no surgical sperm retrieval; NR ¼ not reported. a Some papers reported different histology/cytology in each testis. All men were azoospermic except for two (see footnotes e and f). b The Alu polymorphism M1 localized upstream of sy127 was absent, inferring that the deletion was a complete AZFb-c deletion. c The deletion extent proximal to P5. d The minimal extent option of the deletions (all deletions included sy117 up to sy254, some extending up to the distal end of the Yq and including sy160). e Sporadic spermatozoa only in the first semen sample but azoospermia in successive samples, and Sertoli cells only were observed in testis histology. Some germ cells and three mature spermatids were detected in his cytology analysis. f This patient had %0.1 million/ml spermatozoa in semen and few spermatids in testis. a fertilization capability may contribute to the possibility of identifying the men with AZFb deletion in whom it would be worthwhile to search for spermatozoa. For the time being, referring couples in which the men have a complete AZFb/b-c microdeletion to a TESE/ICSI program should be revaluated. A clear correlation could be observed between the extent of the deletion and the histological findings (Table 4). Hypospermatogenesis in the testicular tissue is frequently observed among partial AZFb/ b-c deleted specimens, but more severe findings are observed in complete AZFb (83% with maturation arrest) and AZFb-c (73% Sertoli cells only) deleted specimens. Krausz et al. (16) expressed the need for precise knowledge of the extent and position of AZFb deletions to define whether the deletion was complete or partial and to determine the probability of finding mature spermatozoa. Accordingly, the STS marker sy127 (starting at location Mb) that was proposed by the European Academy of Andrology (EAA) as the first-choice STS for the proximal border of AZFb (12) must be complemented by a more proximal one located closer to location 19.2 Mb. The optional STS marker proposed by the EAA as being a second optional choice proximal marker (the multicopy sy114 located at 19.2 and 19.3 Mb) (13) or any other marker included in the MSY mapper and located close to 19.2 Mb (e.g., sy121, sy1224) should be seriously reconsidered for use as a definitive marker in the clinical setting. Partial AZFb/b-c deletions seem to be as frequent as complete AZFb deletions (Supplementary Table 2). The promising prospect of finding sperm cells by determining the extent of the deletion raises the level of importance of such an approach. Moreover, precise assessment should be considered crucial in light of the guidelines for Y-chromosomal microdeletions that emphasize that TESE should not be recommended in cases of complete AZFb/b-c deletion (13). Partial deletions of the AZFb interval should be valuable for analyzing the role of Y-chromosome genes in male factor infertility. These peculiar deletions should be corroborated by using additional markers and identification of the breakpoints. Validated STSs are valuable tools, but it should be borne in mind that the reaction methodology and the quality of the DNA may cause technical artifacts. The importance of an intact proximal region of AZFb for successfully achieved spermatogenesis was recently suggested (27). This region includes the testis-specific genes XKRY(X-linked Kell blood group precursor), CDY2 (chromodomain Y2), and HSFY (heat shock factor Y), whose copies remain intact in almost half of the partial AZFb deletions (Supplementary Fig. 1D). The findings collected in this review do not support that contention: all (5/5) men with a partial distal deletion of AZFb but also three of the nine men with a partial proximal AZFb deletion were reported to have sperm cells either in the testis or in the semen (Table 3). A severe oligoasthenotheratozoospermic man with a partial proximal AZFb deletion was recently reported (51), and his sperm cells have proven fertilization capacity. Men with karyotype abnormalities were included in the current review because linkage between AZFb-c deletions and chromosome Fertility and Sterility â 2009

6 2010 Kleiman et al. Sperm cells and AZFb/AZFb-c microdeletions Vol. 95, No. 6, May 2011 TABLE 3 List of men with partial AZFb (interval region between P4 and P3/P1 proximal; first 14 men) or partial AZFb-c (interval region between P4 and P3/P1distal or terminal; last seven men) microdeletion. Semen analysis (sperm/ml) Findings in histology/ cytology Extent of the deletion (STS mapped by MSY) 5 0 Coordinate of deleted STS at the borders of the deletion, bp Proximal Distal Karyotype Reference no. AZO Arrest NR 43 AZO Hypo ,XY 34 Oligoasthenozoospermia Complete spermatogenesis ,XY 21 < Hypo ,XY 44 AZO Hypo ,XY 44 AZO SCO ,XY 44 AZO SCO ,XY 44 AZO HS ,XY 26 AZO HS ,XY 26 AZO Arrest ,XY 26 % Hypo ,XY 26 < NR ,XY 27 AZO Hypo ,XY NR ,XY 45 AZO Arrest ,XY 36 AZO SCO ,XY 38 Few immotile NR ,X/46,X,r(Y)/47, 25 X,r(Y),r(Y) [10/89/1] AZO Hypo t ,XY 27 AZO Hypo ,XY 27 AZO SCO ,XY 31 AZO SCO ,XY 31 Note: Bold STS numbers denote the closest tested STS that was present before or after the extreme STS deleted. STS ¼ sequence tagged site; t ¼ terminal deletion; AZO ¼ azoospermia; Hypo ¼ hypospermatogenesis; HS ¼ quantitative reduction in the number of germ cells with no mature sperm cells; SCO ¼ Sertoli cell only; NR ¼ not reported.

7 TABLE 4 Frequency of azoospermia, sperm cells, and histological findings among all reported men with AZF microdeletions (n [ 149). AZF deletion Azoospermia, % (n) Sperm cells either in testis or in semen, % (n) Hypospermatogenesis Histological findings, % (n) Maturation arrest Sertoli cells only P5/proximal P1 96 (27/28) a 7 (2/28) 6 (1/18) 83 (15/18) b 11 (2/18) b(p) 64 (9/14) 57 (8/14) 42 (5/12) c 33 (4/12) b 17 (2/12) b(p?) 86 (12/14) 21 (3/14) 8 (1/13) 69 (9/13) 23 (3/13) P5-P4/distal P1-terminalYq 97 (69/71) d 3 (2/71) 3 (2/59) 24 (14/59) e 73 (43/59) b(p)c 86 (6/7) 43 (3/7) 33 (2/6) 17 (1/6) 50 (3/6) b(p?)c 87 (13/15) 13 (2/15) 0 (0/15) 33 (5/15) 67 (10/15) a One case who was reported to have 20 spermatozoa/ml in the first semen sample and none in the second sample was not counted as being azoospermic. b The cases with quantitative reduction in the number of germ cells (HS) but with undetected mature sperm cells were included in the maturation arrest group. c One case of oligoasthenozoospermia with apparently normal spermatogenesis was not included in the hypospermatogenesis category. d Two cases with AZFb-c deletion. One had sporadic spermatozoa in the first semen sample but was azoospermic in successive samples. Sertoli cells were observed in testis histology, but some germ cells and three mature spermatids were detected in the cytology analysis. The other one had %0.1 million/ml spermatozoa in semen and few spermatids in the testis. Both men were not considered as being azoospermic. e One case had few germ cells, but it was unclear up to which stage; n ¼ number; (p), that is, partial deletion; (p?), unconfirmed partial or complete deletion. instability had been proposed, although the primary cause of spermatogenic impairment in such men remains unresolved. Cells with AZFb-c deletions might have increased gonosome instability and thus a tendency to lose the whole Y-chromosome during embryonic development (26, 53). Large AZFb-c deletions that included the distal end of the Yq-chromosome, detected in 29% of the men in the current cohort, might contribute considerably to this chromosomal instability. Not surprising, all the men with an AZFb-c deletion who had an abnormal karyotype were among those without the distal end of the Yq-chromosome (sy160; Table 2). This may also explain the reported high frequency of Y-chromosome microdeletions in patients with sex chromosome mosaicism (frequently 45,X0/ 46,XY) (54, 55). Even though the cohort of the reviewed papers included 120 men with complete and 29 with incomplete AZF assessment, only a minor part of published papers on Y microdeletions included testicular histology or cytology, therefore the reported groups with AZFb deletions (except for the AZFb-c group with complete deletion) are still small. Future studies reporting additional men with deletions that include the extent of the deleted AZFb region and the relevant testicular findings will be helpful to validate the observations. Moreover, reporting pregnancy outcome of ART cycles of exceptional cases in which sperm cells were detected will help to prove the usability of these sperm cells in ART. It should be borne in mind that although a retrospective analysis was performed, flawed scientific and/or clinical methodology could not always be detected and may have contaminated the data. As suggested by the laboratory guidelines and reinforced by the results of the present large cohort, the extent of the AZFb deletions must be assessed carefully to improve the use of this measurement for application as a prognostic test. The results of the current report contribute to the improvement of Y-chromosome microdeletion testing, thus enabling clinicians to counsel their patients with greater confidence about the extremely low chance of detecting sperm in men with complete AZFb/b-c deletions. Acknowledgments: The authors thank Esther Eshkol for editorial assistance and Ilana Galernter (Statistical Department, Tel Aviv University) for expert statistical analysis. REFERENCES 1. Pryor JL, Kent-First M, Muallem A, Van Bergen A, Nolten WE, Meisner L, et al. Microdeletions in the Y-chromosome of infertile men. N Engl J Med 1997;336: Kleiman SE, Yogev L, Gamzu R, Hauser R, Botchan A, Lessing JB, et al. Genetic evaluation of infertile men. Hum Reprod 1999;14: PeterlinB, KunejT, Sinkovec J, Gligorievska N, ZornB. Screening for Y chromosome microdeletions in 226Slovenian subfertile men. Hum Reprod 2002;17: Girardi SK, Mielnik A, Schlegel PN. Submicroscopic deletions in the Y chromosome of infertile men. Hum Reprod 1997;12: Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F, et al. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 1996;5: Repping S, Skaletsky H, Lange J, Silber S, Van Der Veen F, Oates RD, et al. Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet 2002; 71: Kuroda-Kawaguchi T, Skaletsky H, Brown LG, Minx PJ, Cordum HS, Waterston RH, et al. The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet 2001;29: Blanco P, Shlumukova M, Sargent CA, Jobling MA, Affara N, Hurles ME. Divergent outcomes of intrachromosomal recombination on the human Y chromosome: male infertility and recurrent polymorphism. J Med Genet 2000;37: Kamp C, Hirschmann P, Voss H, Huellen K, Vogt PH. Two long homologous retroviral sequence blocks in proximal Yq11 cause AZFa microdeletions as a result of intrachromosomal recombination events. Hum Mol Genet 2000;9: Sun C, Skaletsky H, Rozen S, Gromoll J, Nieschlag E, Oates R, et al. Deletion of azoospermia factor a (AZFa) region of human Y chromosome caused by recombination between HERV15 proviruses. Hum Mol Genet 2000;9: Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier L, Brown LG, et al. The malespecific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 2003;423: Simoni M, Bakker E, Eurlings MC, Matthijs G, Moro E, M uller CR, et al. Laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. Int J Androl 1999;22: Fertility and Sterility â 2011

8 13. Simoni M, Bakker E, Krausz C. EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions. State of the art Int J Androl 2004;27: Oates RD, Silber S, Brown LG, Page DC. Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome, and of 18 children conceived via ICSI. Hum Reprod 2002;17: Kleiman SE, Bar-Shira Maymon B, Yogev L, Paz G, Yavetz H. The prognostic role of the extent of Y microdeletion on spermatogenesis and maturity of Sertoli cells. Hum Reprod 2001;16: Krausz C, Quintana-Murci L, McElreavey K. Prognostic value of Y deletion analysis: what is the clinical prognostic value of Y chromosome microdeletion analysis? Hum Reprod 2000;15: Brandell RA, Mielnik A, Liotta D, Ye Z, Veeck LL, Palermo GD, et al. AZFb deletions predict the absence of spermatozoa with testicular sperm extraction: preliminary report of a prognostic genetic test. Hum Reprod 1998;13: Maurer B, Gromoll J, Simoni M, Nieschlag E. Prevalence of Y chromosome microdeletions in infertile men who consulted a tertiary care medical centre: the M unster experience. Andrologia 2001;33: Rolf C, Gromoll J, Simoni M, Nieschlag E. Natural transmission of a partial AZFb deletion of the Y chromosome over three generations: case report. Hum Reprod 2002;17: Luetjens CM, Gromoll J, Engelhardt M, Von Eckardstein S, Bergmann M, Nieschlag E, et al. Manifestation of Y-chromosomal deletions in the human testis: a morphometrical and immunohistochemical evaluation. Hum Reprod 2002;17: Chiang HS, Yeh SD, Wu CC, Huang BC, Tsai HJ, Fang CL. Clinical and pathological correlation of the microdeletion of Y chromosome for the 30 patients with azoospermia and severe oligoasthenospermia. Asian J Androl 2004;6: Stouffs K, Lissens W, Tournaye H, Van Steirteghem A, Liebaers I. The choice and outcome of the fertility treatment of 38 couples in whom the male partner has a Yq microdeletion. Hum Reprod 2005;20: Song NH, Wu HF, Zhang W, Zhuo ZM, Qian LX, Hua LX, et al. Screening for Y chromosome microdeletions in idiopathic and nonidiopathic infertile men with varicocele and cryptorchidism. Chin Med J (Engl) 2005;118: Bettio D, Venci A, Rizzi N, Negri L, Setti PL. Clinical and molecular cytogenetic studies in three infertile patients with mosaic rearranged Y chromosomes. Hum Reprod 2006;21: Faure AK, Aknin-Seifer I, Satre V, Amblard F, Devillard F, Hennebicq S, et al. Fine mapping of rearranged Y chromosome in three infertile patients with non-obstructive azoospermia/cryptozoospermia. Hum Reprod 2007;22: Ferlin A, Arredi B, Speltra E, Cazzadore C, Selice R, Garolla A, et al. Molecular and clinical characterization of Y chromosome microdeletions in infertile men: a 10-year experience in Italy. J Clin Endocrinol Metab 2007;92: Costa P, Gonçalves R, Ferras C, Fernandes S, Fernandes AT, Sousa M, et al. Identification of new breakpoints in AZFb and AZFc. Mol Hum Reprod 2008;14: Hopps CV, Mielnik A, Goldstein M, Palermo GD, Rosenwaks Z, Schlegel PN. Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc regions. Hum Reprod 2003;18: Lange J, Skaletsky H, Bell GW, Page DC. MSY Breakpoint Mapper, a database of sequence-tagged sites useful in defining naturally occurring deletions in the human Y chromosome. Nucleic Acids Res 2008;36(Database issue):d Choi JM, Chung P, Veeck L, Mielnik A, Palermo GD, Schlegel PN. AZF microdeletions of the Y chromosome and in vitro fertilization outcome. Fertil Steril 2004;81: Yang Y, Ma MY, Xiao CY, Li L, Li SW, Zhang SZ. Massive deletion in AZFb/bþc and azoospermia with Sertoli cell only and/or maturation arrest. Int J Androl 2008;31: Mitra A, Dada R, Kumar R, Gupta NP, Kucheria K, Gupta SK. Screening for Y-chromosome microdeletions in infertile Indian males: utility of simplified multiplex PCR. Indian J Med Res 2008;127: Van Landuyt L, Lissens W, Stouffs K, Tournaye H, Liebaers I, Van Steirteghem A. Validation of a simple Yq deletion screening programme in an ICSI candidate population. Mol Hum Reprod 2000;6: Lin YM, Lin YH, Teng YN, Hsu CC, Shinn-Nan Lin J, Kuo PL. Gene-based screening for Y chromosome deletions in Taiwanese men presenting with spermatogenic failure. Fertil Steril 2002;77: Thangaraj K, Gupta NJ, Pavani K, Reddy AG, Subramainan S, Rani DS, et al. Y chromosome deletions in azoospermic men in India. J Androl 2003;24: Ambasudhan R, Singh K, Agarwal JK, Singh SK, Khanna A, Sah RK, et al. Idiopathic cases of male infertility from a region in India show low incidence of Y-chromosome microdeletion. J Biosci 2003;28: Giannouli C, Goulis DG, Lambropoulos A, Lissens W, Tarlatzis BC, Bontis JN, et al. Idiopathic nonobstructive azoospermia or severe oligozoosperia: a cross-sectional study in 61 Greek men. Int J Androl 2004;27: Tsujimura A, Matsumiya K, Takao T, Miyagawa Y, Koga M, Takeyama M, et al. Clinical analysis of patients with azoospermia factor deletions by microdissection testicular sperm extraction. Int J Androl 2004;27: Yogev L, Segal S, Zeharia E, Gamzu R, Maymon BB, Paz G, et al. Sex chromosome alignment at meiosis of azoospermic men with azoospermia factor microdeletion. J Androl 2004;25: M usl umanoglu MH, Turgut M, Cilingir O, Can C, Ozy urek Y, Artan S. Role of the AZFd locus in spermatogenesis. Fertil Steril 2005;84: Mohammed F, Al-Yatama F, Al-Bader M, Tayel SM, Gouda S, Naguib KK. Primary male infertility in Kuwait: a cytogenetic and molecular study of 289 infertile Kuwaiti patients. Andrologia 2007;39: Cui YX, Xia XY, Pan LJ, Wang YH, Hao LJ, Yao B, et al. Gonosomal mosaicism from deleted Y chromosomal nondisjunction. J Androl 2007;28: Krausz C, Quintana-Murci L, Barbaux S, Siffroi JP, Rouba H, Delafontaine D, et al. A high frequency of Y chromosome deletions in males with nonidiopathic infertility. J Clin Endocrinol Metab 1999;84: Ferlin A, Moro E, Rossi A, Dallapiccola B, Foresta C. The human Y chromosome s azoospermia factor b (AZFb) region: sequence, structure, and deletion analysis in infertile men. J Med Genet 2003;40: Plotton I, Ducros C, Pugeat M, Morel Y, Lejeune H. Transmissible microdeletion of the Y-chromosome encompassing two DAZ copies, four RBMY1 copies, and both PRY copies. Fertil Steril 2010;94: Martınez MC, Bernabe MJ, Gomez E, Ballesteros A, Landeras J, Glover G, et al. Screening for AZF deletion in a large series of severely impaired spermatogenesis patients. J Androl 2000;21: Patrat C, Bienvenu T, Janny L, Faure AK, Fauque P, Aknin-Seifer I, et al. Clinical data and parenthood of 63 infertile and Y-microdeleted men. Fertil Steril 2010;93: Kunej T, Zorn B, Peterlin B. Y chromosome microdeletions in infertile men with cryptorchidism. Fertil Steril 2003;79(Suppl 3): Imken L, El Houate B, Chafik A, Nahili H, Boulouiz R, Abidi O, et al. AZF microdeletions and partial deletions of AZFc region on the Y chromosome in Moroccan men. Asian J Androl 2007;9: Longepied G, Saut N, Aknin-Seifer I, Levy R, Frances AM, Metzler-Guillemain C, et al. Complete deletion of the AZFb interval from the Y chromosome in an oligozoospermic man. Hum Reprod 2010;25: Shi YC, Cui YX, Zhou YC, Wei L, Jiang HT, Xia XY, et al. A rare Y chromosome constitutional rearrangement: a partial AZFb deletion and duplication within chromosome Yp in an infertile man with severe oligoasthenoteratozoospermia. Int J Androl Stahl PJ, Masson P, Mielnik A, Marean MB, Schlegel PN, Paduch DA. A decade of experience emphasizes that testing for Y microdeletions is essential in American men with azoospermia and severe oligozoospermia. Fertil Steril 2010;94: Siffroi JP, Le Bourhis C, Krausz C, Barbaux S, Quintana-Murci L, Kanafani S, et al. Sex chromosome mosaicism in males carrying Y chromosome long arm deletions. Hum Reprod 2000;15: Alvarez-Nava F, Puerta H, Soto M, Pineda L, Temponi A. High incidence of Y-chromosome microdeletions in gonadal tissues from patients with 45, X/46, XY gonadal dysgenesis. Fertil Steril 2008; 89: Patsalis PC, Skordis N, Sismani C, Kousoulidou L, Koumbaris G, Eftychi C, et al. Identification of high frequency of Y chromosome deletions in patients with sex chromosome mosaicism and correlation with the clinical phenotype and Y-chromosome instability. Am J Med Genet 2005;35A: Kleiman et al. Sperm cells and AZFb/AZFb-c microdeletions Vol. 95, No. 6, May 2011

9 SUPPLEMENTARY TABLE 1 List of men with incomplete identification of the AZFb (n [ 14) or AZFb-c (n [ 15) microdeletion due to the absence of an STS at the proximal region of AZFb. Semen analysis, sperm/ml Findings in histology/cytology a Extent of the deletion (STS mapped by MSY) 5 0 coordinate of deleted STS at the borders of the deletion, bp Proximal Distal Karyotype Reference AZO Arrest ,XY 45 AZO Arrest, SCO NR 18 AZO SCO NR NR NR 18 AZO L- Arrest, R-Arrest NR 20 AZO L-Arrest, R-hypo NR 20 AZO Arrest (either SPG up NR 23 to late SPD) Olig Arrest (either SPG NR 23 up to late SPD) AZO Arrest ,XY 47 AZO Arrest SPD (no ,XY 47 sperm for ICSI) AZO SCO ,XY 47 AZO SCO ,XY 47 AZO Arrest ,XY 47 AZO Arrest ,XY 47 AZO SCO XdelYq 46 AZO SCO XdelYq 46 AZO SCO ,XY 46 AZO SCO ,X/46,XdicYp 46 AZO Arrest, SCO ,XY 48 AZO Arrest NR 20 AZO SCO, Arrest ,XY 3 AZO SCO Xdel(Y)(q11) 3 AZO SCO NR 48 Olig Arrest (either NR 23 SPG up to late SPD) Olig Arrest (either NR 23 SPG up to late SPD) AZO SCO NR 49 AZO SCO NR 49 AZO SCO ,X/46,XY [15/21] 47 AZO SCO ,X/46,X,idic(Y) (q11.2) [56/44] 47 Note: Bold STS numbers denote the closest tested STS that was present before or after the extreme STS was deleted. STS ¼ sequence tagged site; MSY ¼ MSY Breakpoint Mapper; AZO ¼ azoospermia; Olig ¼ oligozoospermia; Hypo ¼ hypospermatogenesis; Arrest ¼ arrest at the spermatocyte stage unless it was otherwise specified; SCO ¼ Sertoli cell only; SPG ¼ spermatogonia; SPD ¼ spermatid; NR ¼ not reported. a Some papers reported different histology/cytology in each testis. Fertility and Sterility â 2012.e1

10 SUPPLEMENTARY TABLE 2 Prevalence of AZFb/b-c deletions in azoospermia. Reference Prevalence reported in azoospermia, % (n) AZFb (P5/P1proximal) deletion Kleiman et al (2) 1 (1/105) Chiang et al (21) 0.9 (2/218) Yang et al (31) 0.8 (3/387) Ferlin et al (26) 1.1 (7/625) Mitra et al (32) 0.8 (1/119) Kleiman (present review) 0.8 (5/615) Average SD AZFb-c ((P5-P4/ P1distal-terminal Yq) deletion Girardi et al (4) 1.9 (2/108) Pryor et al (1) 3.8 (1/26) Van Landuyt et al (33) 0.9 (1/108) Lin et al (34) 0.8 (1/120) Giannouli et al (37) 2.3 (3/130) Tsujimura et al (38) 1.7 (1/60) M usl umanoglu 3.3 (1/30) et al (40) Yang et al (31) 1 (4/387) Mitra et al (32) 2.5 (3/119) Imken et al (49) 4.1 (2/48) Mohammed et al (41) 4.6 (5/108) Ferlin et al (26) 1.9 (12/625) Kleiman (present review) 2.3 (14/615) Average SD Partial AZFb deletion Krausz et al (43) 2.3 (3/131) a Lin et al (34) 0.8 (1/120) Ferlin et al (44) 0.6 (4/700) a Average SD Partial AZFb-c deletion Ambasudhan 0.7 (1/142) et al (36) Tsujimura et al (38) 1.7 (1/60) Yang et al (31) 0.5 (2/387) Average SD a The prevalence included azoospermic and oligozoospermic men e2 Kleiman et al. Sperm cells and AZFb/AZFb-c microdeletions Vol. 95, No. 6, May 2011

11 SUPPLEMENTARY FIGURE 1 Study selection chart. Fertility and Sterility â 2012.e3

12 SUPPLEMENTARY FIGURE 2 Six newly reported men evaluated in our institution with deletions in relation to the sequence of the Y-chromosome. (A) The palindromic structure encompassing the AZFb-c region. (B) The STSs used in the study: the tick marks show STS positions, ticks on the horizontal line denote single-copy STSs, and ticks down the horizontal line denote multicopy STSs. (C) Plus/minus STS results for the six infertile men with AZFb-c or AZFb deletions. Horizontal black bars indicate confirmed presence of the DNA, and minus signs indicate confirmed absence of the DNA region. Horizontal gray bars indicate multicopy STS positives that were disregarded because of cross-amplifying loci and negative results for flanking STSs. (D) Predicted and confirmed genes. (E) Apparently noncoding transcripts. Panels (A), (D), and (E) were according to Repping et al (6) e4 Kleiman et al. Sperm cells and AZFb/AZFb-c microdeletions Vol. 95, No. 6, May 2011