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Histological and genetic analysis and risk assessment for chromosomal aberration after ICSI for patients presenting with CBAVD

Histological and genetic analysis and risk assessment for chromosomal aberration after ICSI for... Abstract Intracytoplasmic sperm injection (ICSI) has opened a new field in the treatment of male infertility, leading to a debate concerning its genetic safety. In this study we present an analysis of 11 patients presenting congenital bilateral absence of the vas deferens (CBAVD). In all 11 cases, genetic counselling, histological analysis of testicular biopsies, cystic fibrosis transmembrane conductance regulator (CFTR) mutation screenings of both partners and spermatozoa three-colour fluorescent in-situ hybridization (FISH) analysis were performed. A total of 31 CFTR mutations were screened and mutations were found in eight out of 11 cases, with ΔF508 being the most common mutation found. Histological analyses showed that seven out of 11 patients had normal tubule/membrane/interstitium (TMI) and Johnsen scores, while the remaining four patients had mild impairment of testicular parenchyma. The average aneuploidy rate was 6.8 ± 3.9% compared with two control subjects with 4.4 and 5.4% aneuploidy rates respectively, using FISH analysis. After ICSI, the fertilization and pregnancy rates were 66.2 and 22.7% respectively. Thus, in our case of CBAVD, the risk of chromosomal aberration following ICSI, in the absence of a CFTR mutation in the male patient and/or in his partner, was not higher than in normal fertile men. Furthermore, the pregnancy success rate following ICSI of these CBAVD patients was comparable to the general ICSI population, even when histological analysis showed limited spermatogenesis. congenital absence of the vas deferens, fluorescent in-situ hybridization, histological analysis, ICSI, testicular sperm extraction Introduction The successful development of intracytoplasmic sperm injection (ICSI) for assisted reproduction (Palermo et al., 1992) represents a milestone in the treatment of male infertility. ICSI is now offered to couples either suffering from an andrological subfertility or having experienced repeated fertility failures following conventional IVF procedures. ICSI pregnancies have thus been achieved with ejaculated, epididymal and testicular spermatozoa. Since its introduction, however, there have been major safety concerns regarding the violation, by the microinjection pipette, of the natural barriers to fertilization (Lamb, 1999; Schlegel, 1999). Further, although ICSI does not appear to result in an increase in congenital malformations, some controversy concerning the genetic risks of such an invasive technique remains (Bonduelle et al., 1996a,b; Engel et al., 1996; Govaerts et al., 1996). In a short series of prenatal diagnostic tests, a high incidence of sex-chromosome aberrations was found after ICSI (In't Veld et al., 1995). In a more recent and larger study comprising 1082 prenatal tests performed upon pregnancies obtained after ICSI, a six-fold increase in sex chromosomal aberrations and a two-fold increase in autosomal aberrations was reported (Bonduelle et al., 1998). This difference in incidence between sex chromosomal and autosomal aberrations may be explained by the fact that autosomal aberrations often lead to early termination which is not the case with sex chromosomal aberrations, the latter being more compatible with full term pregnancies. Although the reason for the increase in chromosomal aberrations following ICSI remains unclear, many hypotheses have been proposed. Bonduelle et al. (1998) suggested that it may result from the selection of spermatozoa which would otherwise be unable to naturally fertilize an oocyte. Two recent reports, however, equally offer alternative explanations (Hewitson et al., 1999; Luetjens et al., 1999). The latter authors explained that the increase in sex chromosome aberrations may be due to non-random chromosome positioning and differential nuclear decondensation following the injection of non-acrosome-reacted spermatozoa. In agreement with this, another study (Hewitson et al., 1999) showed that, at least in the rhesus monkey, ICSI resulted in abnormal sperm decondensation, uncharacteristic vesicle-associated membrane protein and perinuclear theca retention and nuclear mitotic apparatus exclusion from the decondensing nuclear apex. Furthermore, they showed a time delay in the replication of both parental genomes and variable positioning of the first polar body relative to the meiotic spindle. Microinjection targeting, therefore, using the first polar body as reference may result in direct contact between the micropipette and the meiotic spindle thus resulting in its destruction and a subsequent embryonic lethality. There is general agreement for prerequisite genetic counselling, karyotyping and, in cases of a congenital bilateral absence of the vas deferens (CBAVD), screening for cystic fibrosis transmembrane conductance regulator (CFTR) mutations before ICSI can be performed (Meschede and Horst, 1997; Pauer et al., 1997). A high incidence of CFTR mutations has been associated with CBAVD (Chillon et al., 1995; De Braekeleer and Ferec, 1996; Lissens et al., 1996; Boucher et al., 1999). Recently, studies of ICSI patients have been performed to assess spermatozoa chromosomal content and thus evaluate any potential genetic risk (Yurov et al., 1996; In't Veld et al., 1997). An overview of structural and numerical abnormalities in spermatozoa of normal men and carriers of chromosome aberrations has been recently published (Guttenbach et al., 1997). In the context of our ICSI programme, and when possible, we systematically perform fluorescent in-situ hybridization (FISH) analysis in all cases of testicular sperm extraction (TESE) in order to establish the genetic risks, according to the specific type of azoospermia. Our goal is to both evaluate the chances of a successful pregnancy and further be in a position to inform couples of potential embryonic genetic risks linked to the procedure. Here we present an analysis of 11 cases of azoospermia resulting from CBAVD. In all 11 cases, genetic counselling, histological analyses of testicular biopsies, CFTR mutation screenings of both partners and spermatozoa three-colour FISH analyses were performed. Materials and methods Patients Included in this study were 11 couples in which the male partner presented with obstructive azoospermia due to CBAVD. No renal malformations were observed in these patients. ICSI with spermatozoa recovered by open testicular biopsy was carried out as previously described (Silber et al., 1995). Testicular tissues Testicular tissues were obtained on the same day as oocytes were retrieved from the female partner. Biopsy samples were placed in G1.2 medium (IVF Science Scandinavia; Gothenburg, Sweden) and immediately squashed between two microscopic slides. Spermatozoa were recovered from all biopsies. Surplus spermatozoa were frozen to avoid a second biopsy for any future ICSI. Remaining spermatozoa used during the injection procedure and thus not suitable for the freezing procedure were analysed by FISH. These samples were washed once in phosphate-buffered saline (PBS) and placed in an area previously delimited with a diamond pen on superfrosted slides (CML, France). Slides were air-dried at room temperature, washed once in PBS, dehydrated in an ethanol series (70, 90, 100%), air-dried and stored at –20°C. Histological analysis of testicular biopsies Diagnostic testicular biopsies were performed in parallel at the urological surgery ward. Recovered tissue was fixed for 1 h in a 0.1 mol/l cacodylate buffered 2% glutaraldehyde solution at +4°C. Fragments were rinsed for 30 min in the same buffer and post-fixed for 1 h in 1% OsO4 (osmic acid) After dehydratation, the samples were embedded in epoxy resin. Semi-thin sections (1 μm) were cut and stained with Toluidine Blue. DNA probes Probes specific for chromosomes X (pBamX5), Y (pCY98) and 1 (pUC1.77) were directly labelled, as described previously (Viville et al., 1997), by nick translation (Boehringer, Mannheim, Germany) with (i) rhodamine-4-dUTP (Amersham, Les Ullis, France); (ii) fluorescent isothiocyanate (FITC)-12-dUTP (Boehringer); and (iii) a mixture of (1:1) FITC-12dUTP and rhodamine-4-dUTP respectively. Probes were dissolved in 60% formamide/2×sodium chloride/sodium citrate (SSC) for use at a final concentration of 1–2 ng/μl. The use of two sex chromosomal probes and an autosomal specific probe allowed the determination of ploidy status of each spermatozoa analysed. FISH Sperm nuclei decondensation was performed by a 5 min incubation in 1 mol/l Tris buffer at pH 9.5 containing 25 mmol/l dithiothreitol (DTT) (Martin et al., 1995). Slides were then washed for 5 min in 2×SSC and 5 min in PBS, dehydrated through an ethanol series and air-dried. Slides were then treated with pepsin (100 mg/ml) in 0.01 N HCl for 20 min at 37°C, rinsed twice in bi-distilled water, fixed in methanol/acid acetic (3:1) for 10 min at 4°C, rinsed twice in bi-distilled water and dehydrated through an ethanol series. The hybridization solution (60% formamide, 2×SSC, 10% dextran) containing the three probes was applied to the slides under a coverslip. The probes and the nuclei were denatured at 78°C for 3 min. Hybridization was performed in a dark, moist chamber at 37°C for 1–2 h. Post hybridization washes included 5 min in 60% formamide/2×SSC and 5 min in 2×SSC at 42°C, followed by two 5 min washes in 4×SSC/0.05% Tween 20 at room temperature. After dehydratation through an ethanol series, slides were counterstained with 4′,6 diaminidino-2-phenylindol (DAPI) in antifading solution (Vector, Burlingame, USA) Signal analysis was performed on a Zeiss microscope according to scoring criteria described previously (Hopman et al., 1988). Results Patients In seven of the 11 cases, azoospermia represented the only apparent reason for infertility whereas in the remaining four a female factor (endocrinological or tubal) was also implicated. In total, 31 different CFTR mutations, including ΔF508, R117H and the variant IVS5T, which represent the more common mutations found in CBVAD patients (Chillon et al., 1995), responsible for 86% of all cystic fibrosis cases in the Caucasian population were investigated. Eight patients (73%) presented at least one mutation, three patients (27%) presented only the ΔF508 mutation and five (45%) presented compound heterozygosity for ΔF508/R347H, ΔF508/R117C, ΔF508/5T, ΔF508/D443Y and R117H–7T/5T (see Table I) As expected the ΔF508 mutation was the most frequently found (64%). For three patients no mutations, according to our criteria, were detectable, although one patient demonstrated a positive sweat test. For four patients (patients 2, 3, 4 and 5), in addition to CFTR mutation screenings, karyotypic analyses were also performed. All four were normal. For three of the patients (patients 4, 6 and 10) presenting a CFTR mutation, the families accepted genetic counselling and genetic screening revealed a familial origin of the defect. In two of these families, siblings had also consulted the fertility clinic regarding a CBAVD reproductive problem. All patients reported in this study presented normal serum concentrations of FSH. Histopathological analyses In all 11 cases, histopathological analysis of the testis demonstrated spermatogenesis characterized by elongated spermatids and spermatozoa in the majority of tubule sections. Seven of the patients had normal tubule/membrane/interstitium (TMI) (Valette et al., 1976) and Johnsen scores (Johnsen, 1970; Yoshida et al., 1997) see Table I. For four patients, histological analysis revealed a mild impairment of the testicular parenchyma, with irregularities in tubule diameter, decreases in germ cell density, exfoliation and necrobiosis of the seminiferous epithelia, hernia, tubule breaks and moderate thickening of the tunica propria (Figure 1). Table I shows that semi-quantitative evaluation using the TMI score is more discriminative than the Johnsen score, in agreement with a previous study (Meyer et al., 1988). TESE results Sufficient motile spermatozoa were recovered from the testicular biopsies of all 11 patients. Table II presents an overview of the TESE results. A total of 109 oocytes were collected (mean 9.9 per woman, range 0–18) following ovarian stimulation. After removal of cumulus and corona cells 74 oocytes exhibited the first polar body and were thus chosen for sperm injection. ICSI was carried out only in 10 couples, as no oocytes could be recovered from one patient. Fertilization occurred in 49 cases, thus giving a fertilization rate of 66.2% (range 36–100% for each couple). Embryo transfer took place in all 10 couples. Five clinical pregnancies, including one twin pregnancy resulted, from which five healthy babies were born. One case of spontaneous abortion occurred (patient 11). The pregnancy rate per embryo transferred was 22.7% (5/22). FISH results Considering the relative scarcity of samples which results from the biopsy procedure when compared to conventional IVF procedures which use ejaculated spermatozoa, priority was given to the freezing protocol. From the 11 biopsies tested, one gave no FISH signal (patient 11). Of the remaining 10, the number of spermatozoa analysed varied between 64 and 699. The results are shown in Table III. We present the results with aneuploidy rate as the cumulative frequency of disomy, diploidy and nullisomy signals. Statistical analysis was not possible due to the limited number of spermatozoa available. In each of the 10 cases, 72.5–98.2% of spermatozoa presented a normal X,1 or Y,1 signal. In patient number 5, 25% spermatozoa presented a nullisomy for the sex chromosomes (0, 1 signal). Furthermore, only 64 spermatozoa from patient 5 could be analysed of which 40 gave an interpretable FISH signal and a distorted sex chromosome ratio in favour of the Y chromosome (42.5% of Y1 signals versus 30.0% of X1 signals). Therefore, not considering patient 5, the average aneuploidy rate of our patients was 6.8 ± 3.9% (Table III) and was thus largely comparable with our fertile control subjects (two patients: 4.4 and 5.4%) and to the average general population rate, which was 1–4% (Guttenbach et al., 1997). Discussion We present here the results of a study employing a three-colour FISH analysis of testicular spermatozoa chromosomal content from patients presenting CBAVD. Our purpose was to establish a correlation between (i) the quality of testicular function, and (ii) the genetic safety of the TESE technique, as judged by histological criteria and FISH analysis, and success of the TESE/ICSI procedure. Because of the high prevalence of CFTR mutations in CBVAD patients (Chillon et al., 1995; De Braekeleer and Ferec, 1996; Lissens et al., 1996; Boucher et al., 1999) a screening for 31 different CFTR mutations covering 86% of all known cases was performed for all patients. For eight patients, at least one mutation (73%) was found, five patients were detected as compound heterozygotes (45%) and one patient for which no mutation found had a positive sweat test. These results are in accordance with previously published data (Lissens et al., 1996). With the exception of patient 5, and bearing in mind the limited number of spermatozoa available for analysis, patients presenting with obstructive subfertility due to CBAVD did not show an elevated spermatic aneuploidy rate overall, in comparison with normal fertile controls. This is in agreement with previously published results (Martin et al., 1993; Bischoff et al., 1994; for review see, Guttenbach et al., 1997). We presented the aneuploidy rate as the cumulative frequency of disomy, diploidy and nullisomy which may represent an over-estimation of the aneuploidy rate since some nullisomy certainly results from probe non-hybridization. Therefore, the rate of aneuploidy for these patients is within an acceptable range. Thus, although only a limited number of spermatozoa could be analysed, these results suggest that the genetic risks for children conceived by TESE from patients presenting with CBAVD are not increased. The higher level of chromosomal abnormalities found in patient 5 is difficult to explain. Indeed, of 64 spermatozoa obtained only 40 gave sufficient FISH signals for analysis. It must be mentioned that sperm FISH signals obtained by testicular biopsy are of a poorer quality than signals obtained from the ejaculated spermatozoa of fertile males (C.Falquet and S.Viville personal observations) For this patient, therefore, it is difficult to conclude whether the high level of aneuploidy is real or due to a technical artefact limited to this specific case. Nevertheless 72.5% of this patient's spermatozoa did present a normal chromosomal composition, at least for the chromosomes analysed. We believe, therefore, that we can still safely offer a TESE to this couple. It is interesting to note that the incidence of aneuploidy did not correlate with the presence of a CFTR mutation and furthermore, that the success rate of IVF following TESE/ICSI was independent of the quality of spermatogenesis, as judged by TMI and Johnsen scores. This latter result is in agreement with previous reports (Tournaye et al., 1996; Jezek et al., 1998; Nagy et al., 1998). For these couples where the male partner presents with CBAVD, a pregnancy can thus be expected as soon as spermatozoa are recovered. In conclusion, considering the three chromosomes analysed during this study and according to the pregnancy rate (22.7% per embryo transferred) obtained, we believe it is appropriate to propose this TESE/ICSI technique to patients with CBAVD. This implies genetic counselling and a search for CFTR mutations in order to establish the risk of having a child suffering from cystic fibrosis. If mutations are found in both partners then we offer either prenatal or preimplantation genetic diagnosis. Furthermore, according to this present study spermatozoa recovered from patients with CBAVD do not present an elevated aneuploidy rate compared with normal fertile males and lastly the TESE/ICSI pregnancy rate is as high as that observed for classical IVF or ICSI techniques. This study is currently being extended in order to aid geneticists to establish counselling programmes for infertile males presenting CBAVD. Table I. Patients' history Patient no.  TMI score  Johnsen score  CF mutation screening  Sweat test  Karyotype  Family history  TMI = tubule/membrane/interstitium; CF = cystic fibrosis; ND = not determined; Neg = negative; Pos = positive; NF = not found in 31 screened mutations, including ΔF508, R117H and the variant IVS5T.  1  1  12  NF  ND  ND  No  2  1  12  ΔF508/NF  ND  Normal  No  3  3  11  R117H-7T/5T  ND  Normal  No  4  3  11  NF  ND  Normal  Yes  5  1  11  ΔF508/NF  ND  Normal  No  6  2  11  ΔF508/R347H  ND  ND  Yes  7  4  10  ΔF508/5T  ND  ND  No  8  3  10  ΔF508/NF  Neg  ND  No  9  1  11  NF  Pos  ND  No  10  2  11  ΔF508/R117C  Pos  ND  Yes  11  2  11  ΔF508/D443Y  Pos  ND  No  Patient no.  TMI score  Johnsen score  CF mutation screening  Sweat test  Karyotype  Family history  TMI = tubule/membrane/interstitium; CF = cystic fibrosis; ND = not determined; Neg = negative; Pos = positive; NF = not found in 31 screened mutations, including ΔF508, R117H and the variant IVS5T.  1  1  12  NF  ND  ND  No  2  1  12  ΔF508/NF  ND  Normal  No  3  3  11  R117H-7T/5T  ND  Normal  No  4  3  11  NF  ND  Normal  Yes  5  1  11  ΔF508/NF  ND  Normal  No  6  2  11  ΔF508/R347H  ND  ND  Yes  7  4  10  ΔF508/5T  ND  ND  No  8  3  10  ΔF508/NF  Neg  ND  No  9  1  11  NF  Pos  ND  No  10  2  11  ΔF508/R117C  Pos  ND  Yes  11  2  11  ΔF508/D443Y  Pos  ND  No  View Large Table II. testicular sperm extraction Patient no.  Female partner  Fertilization per oocyte injected (%)  No. of day 2 embryos  No. of day 3 embryos  No. of embryos transferred  Pregnancy  Birth  1  Tubal factor  7/7 (100)  6  2  2  0  0  D2 and D3 = days 2 and 3 post-fertilization respectively; N = none.  aSpontaneous abortion.  2  N  4/10 (40)  4  3  3  0  0  3  Endocrinological  3/4 (75)  3  2  2  0  0  4  Endocrinological  6/6 (100)  6  6  2  0  0  5  N  0  0  0  0  0  0  6  N  5/7 (71)  4  4  2  1 (twin)  2  7  N  2/2 (100)  2  2  2  1  1  8  Endocrinological  3/6 (50)  3  2  2  0  0  9  N  4/11 (36)  4  2  2  1  1  10  N  7/13 (54)  6  5  2  1  1  11  N  8/10 (80)  7  6  3  1  0a  Total    54/83 (65.1)  50  39  22  5  5  Patient no.  Female partner  Fertilization per oocyte injected (%)  No. of day 2 embryos  No. of day 3 embryos  No. of embryos transferred  Pregnancy  Birth  1  Tubal factor  7/7 (100)  6  2  2  0  0  D2 and D3 = days 2 and 3 post-fertilization respectively; N = none.  aSpontaneous abortion.  2  N  4/10 (40)  4  3  3  0  0  3  Endocrinological  3/4 (75)  3  2  2  0  0  4  Endocrinological  6/6 (100)  6  6  2  0  0  5  N  0  0  0  0  0  0  6  N  5/7 (71)  4  4  2  1 (twin)  2  7  N  2/2 (100)  2  2  2  1  1  8  Endocrinological  3/6 (50)  3  2  2  0  0  9  N  4/11 (36)  4  2  2  1  1  10  N  7/13 (54)  6  5  2  1  1  11  N  8/10 (80)  7  6  3  1  0a  Total    54/83 (65.1)  50  39  22  5  5  View Large Table III. Fluorescent in-situ hybridization (FISH) results in spermatozoa Patient No.  No. analysed  No. with interpretable FISH signals (%)  FISH results  Total % of Aneuploidy        Normal haploid  Diploid  Aneuploid          X,1(%)  Y,1(%)  X,Y,1,1(%)  X,Y,1(%)  1(%)  X,1,1(%)  Y,1,1(%)  Y,Y,1(%)  Y(%)    1  699  677 (96.8)  257 (38.0)  396 (58.5)  0  4 (0.6)  19 (2.8)  0  0  0  1 (0.1)  3.5  2  444  433 (97.5)  194 (44.8)  226 (52.2)  3 (0.7)  3 (0.7)  6 (1.4)  0  0  0  1 (0.2)  3.0  3  112  104 (92.8)  49 (47.1)  48 (46.2)  0  0  6 (5.8)  1 (1.0)  0  0  0  6.7  4  297  281 (94.6)  150 (53.4)  116 (41.3)  0  5 (1.8)  9 (3.2)  0  0  1 (0.4)  0  5.3  5  64  40 (62.5)  12 (30.0)  17 (42.5)  0  2 (5.0)  9 (22.5)  0  0  0  0  27.5  6  109  109 (100)  47 (43.1)  60 (55.0)  0  2 (1.8)  0  0  0  0  0  1.8  7  68  68 (100)  39 (57.4)  20 (29.4)  0  3 (4.4)  0  5 (7.4)  1 (1.5)  0  0  13.2  8  72  63 (87.5)  17 (27.0)  41 (65.1)  0  4 (6.3)  1 (1.6)  0  0  0  0  7.9  9  195  183 (93.8)  89 (48.6)  72 (39.3)  0  7 (3.8)  11 (6.0)  2 (1.1)  1 (0.5)  1 (0.5)  0  12.0  10  188  188 (100)  63 (33.5)  110 (58.5)  0  2 (1.1)  12 (6.4)  0  1 (0.5)  0  0  7.9  Patient No.  No. analysed  No. with interpretable FISH signals (%)  FISH results  Total % of Aneuploidy        Normal haploid  Diploid  Aneuploid          X,1(%)  Y,1(%)  X,Y,1,1(%)  X,Y,1(%)  1(%)  X,1,1(%)  Y,1,1(%)  Y,Y,1(%)  Y(%)    1  699  677 (96.8)  257 (38.0)  396 (58.5)  0  4 (0.6)  19 (2.8)  0  0  0  1 (0.1)  3.5  2  444  433 (97.5)  194 (44.8)  226 (52.2)  3 (0.7)  3 (0.7)  6 (1.4)  0  0  0  1 (0.2)  3.0  3  112  104 (92.8)  49 (47.1)  48 (46.2)  0  0  6 (5.8)  1 (1.0)  0  0  0  6.7  4  297  281 (94.6)  150 (53.4)  116 (41.3)  0  5 (1.8)  9 (3.2)  0  0  1 (0.4)  0  5.3  5  64  40 (62.5)  12 (30.0)  17 (42.5)  0  2 (5.0)  9 (22.5)  0  0  0  0  27.5  6  109  109 (100)  47 (43.1)  60 (55.0)  0  2 (1.8)  0  0  0  0  0  1.8  7  68  68 (100)  39 (57.4)  20 (29.4)  0  3 (4.4)  0  5 (7.4)  1 (1.5)  0  0  13.2  8  72  63 (87.5)  17 (27.0)  41 (65.1)  0  4 (6.3)  1 (1.6)  0  0  0  0  7.9  9  195  183 (93.8)  89 (48.6)  72 (39.3)  0  7 (3.8)  11 (6.0)  2 (1.1)  1 (0.5)  1 (0.5)  0  12.0  10  188  188 (100)  63 (33.5)  110 (58.5)  0  2 (1.1)  12 (6.4)  0  1 (0.5)  0  0  7.9  View Large Figure 1. View largeDownload slide Histopathological aspect of testicular tubules in different cases of congenital bilteral absence of the vas deferens (CBVAD). (A) Patient 2; normal healthy seminiferous epithelium with regular distribution of the germ cells. (B) Patient 4; healthy seminiferous epithelium quite similar to A, but with thinning of the tunica propria (arrows) inducing hernia of the epithelium. (C) Patient 7; impairment of the seminiferous epithelium showing figures of cytolysis. However, the spermiogenesis is efficient with numerous round and elongating spermatids. (D) Patient 10; the germ cells are irregularly distributed and dissociated. The tunica propria is thickened with the beginning of a hyalinization process. Spermiogenesis is maintained. Figure 1. View largeDownload slide Histopathological aspect of testicular tubules in different cases of congenital bilteral absence of the vas deferens (CBVAD). (A) Patient 2; normal healthy seminiferous epithelium with regular distribution of the germ cells. (B) Patient 4; healthy seminiferous epithelium quite similar to A, but with thinning of the tunica propria (arrows) inducing hernia of the epithelium. (C) Patient 7; impairment of the seminiferous epithelium showing figures of cytolysis. However, the spermiogenesis is efficient with numerous round and elongating spermatids. (D) Patient 10; the germ cells are irregularly distributed and dissociated. The tunica propria is thickened with the beginning of a hyalinization process. Spermiogenesis is maintained. 4 To whom correspondence should be addressed at: Service de Biologie de la Reproduction SIHCUS-CMCO, 19 rue Louis Pasteur BP120, 67303 Schiltigheim, France.E-mail: viville@igbmc.u-strasbg.fr References Bischoff, F.Z., Nguyen, D.D., Burt, K.J. and Shaffer, L.G. (1994) Estimates of aneuploidy using multicolor fluorescence in situ hybridization on human sperm. Cytogenet. 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( 1997) Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa. Hum. Reprod.  , 12, 752–754. Google Scholar Jezek, D., Knuth, U.A. and Schulze, W. ( 1998) Successful testicular sperm extraction (TESE) in spite of high serum follicle stimulating hormone and azoospermia: correlation between testicular morphology, TESE results, semen analysis and serum hormone values in 103 infertile men. Hum. Reprod.  , 13, 1230–1234. Google Scholar Johnsen, S.G. ( 1970) Testicular biopsy score count—a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones  , 1, 2–25. Google Scholar Lamb, D.J. ( 1999) Debate: is ICSI a genetic time bomb? Yes [comment]. J. Androl.  , 20, 23–33. Google Scholar Lissens, W., Mercier, B., Tournaye, H. et al. ( 1996) Cystic fibrosis and infertility caused by congenital bilateral absence of the vas deferens and related clinical entities. Hum. 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( 1996) Rapid chromosomal analysis of germ-line cells by FISH: an investigation of an infertile male with large-headed spermatozoa. Mol. Hum. Reprod.  , 2, 665–668. Google Scholar © European Society of Human Reproduction and Embryology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Reproduction Oxford University Press

Histological and genetic analysis and risk assessment for chromosomal aberration after ICSI for patients presenting with CBAVD

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Oxford University Press
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© European Society of Human Reproduction and Embryology
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0268-1161
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1460-2350
DOI
10.1093/humrep/15.7.1613
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Abstract

Abstract Intracytoplasmic sperm injection (ICSI) has opened a new field in the treatment of male infertility, leading to a debate concerning its genetic safety. In this study we present an analysis of 11 patients presenting congenital bilateral absence of the vas deferens (CBAVD). In all 11 cases, genetic counselling, histological analysis of testicular biopsies, cystic fibrosis transmembrane conductance regulator (CFTR) mutation screenings of both partners and spermatozoa three-colour fluorescent in-situ hybridization (FISH) analysis were performed. A total of 31 CFTR mutations were screened and mutations were found in eight out of 11 cases, with ΔF508 being the most common mutation found. Histological analyses showed that seven out of 11 patients had normal tubule/membrane/interstitium (TMI) and Johnsen scores, while the remaining four patients had mild impairment of testicular parenchyma. The average aneuploidy rate was 6.8 ± 3.9% compared with two control subjects with 4.4 and 5.4% aneuploidy rates respectively, using FISH analysis. After ICSI, the fertilization and pregnancy rates were 66.2 and 22.7% respectively. Thus, in our case of CBAVD, the risk of chromosomal aberration following ICSI, in the absence of a CFTR mutation in the male patient and/or in his partner, was not higher than in normal fertile men. Furthermore, the pregnancy success rate following ICSI of these CBAVD patients was comparable to the general ICSI population, even when histological analysis showed limited spermatogenesis. congenital absence of the vas deferens, fluorescent in-situ hybridization, histological analysis, ICSI, testicular sperm extraction Introduction The successful development of intracytoplasmic sperm injection (ICSI) for assisted reproduction (Palermo et al., 1992) represents a milestone in the treatment of male infertility. ICSI is now offered to couples either suffering from an andrological subfertility or having experienced repeated fertility failures following conventional IVF procedures. ICSI pregnancies have thus been achieved with ejaculated, epididymal and testicular spermatozoa. Since its introduction, however, there have been major safety concerns regarding the violation, by the microinjection pipette, of the natural barriers to fertilization (Lamb, 1999; Schlegel, 1999). Further, although ICSI does not appear to result in an increase in congenital malformations, some controversy concerning the genetic risks of such an invasive technique remains (Bonduelle et al., 1996a,b; Engel et al., 1996; Govaerts et al., 1996). In a short series of prenatal diagnostic tests, a high incidence of sex-chromosome aberrations was found after ICSI (In't Veld et al., 1995). In a more recent and larger study comprising 1082 prenatal tests performed upon pregnancies obtained after ICSI, a six-fold increase in sex chromosomal aberrations and a two-fold increase in autosomal aberrations was reported (Bonduelle et al., 1998). This difference in incidence between sex chromosomal and autosomal aberrations may be explained by the fact that autosomal aberrations often lead to early termination which is not the case with sex chromosomal aberrations, the latter being more compatible with full term pregnancies. Although the reason for the increase in chromosomal aberrations following ICSI remains unclear, many hypotheses have been proposed. Bonduelle et al. (1998) suggested that it may result from the selection of spermatozoa which would otherwise be unable to naturally fertilize an oocyte. Two recent reports, however, equally offer alternative explanations (Hewitson et al., 1999; Luetjens et al., 1999). The latter authors explained that the increase in sex chromosome aberrations may be due to non-random chromosome positioning and differential nuclear decondensation following the injection of non-acrosome-reacted spermatozoa. In agreement with this, another study (Hewitson et al., 1999) showed that, at least in the rhesus monkey, ICSI resulted in abnormal sperm decondensation, uncharacteristic vesicle-associated membrane protein and perinuclear theca retention and nuclear mitotic apparatus exclusion from the decondensing nuclear apex. Furthermore, they showed a time delay in the replication of both parental genomes and variable positioning of the first polar body relative to the meiotic spindle. Microinjection targeting, therefore, using the first polar body as reference may result in direct contact between the micropipette and the meiotic spindle thus resulting in its destruction and a subsequent embryonic lethality. There is general agreement for prerequisite genetic counselling, karyotyping and, in cases of a congenital bilateral absence of the vas deferens (CBAVD), screening for cystic fibrosis transmembrane conductance regulator (CFTR) mutations before ICSI can be performed (Meschede and Horst, 1997; Pauer et al., 1997). A high incidence of CFTR mutations has been associated with CBAVD (Chillon et al., 1995; De Braekeleer and Ferec, 1996; Lissens et al., 1996; Boucher et al., 1999). Recently, studies of ICSI patients have been performed to assess spermatozoa chromosomal content and thus evaluate any potential genetic risk (Yurov et al., 1996; In't Veld et al., 1997). An overview of structural and numerical abnormalities in spermatozoa of normal men and carriers of chromosome aberrations has been recently published (Guttenbach et al., 1997). In the context of our ICSI programme, and when possible, we systematically perform fluorescent in-situ hybridization (FISH) analysis in all cases of testicular sperm extraction (TESE) in order to establish the genetic risks, according to the specific type of azoospermia. Our goal is to both evaluate the chances of a successful pregnancy and further be in a position to inform couples of potential embryonic genetic risks linked to the procedure. Here we present an analysis of 11 cases of azoospermia resulting from CBAVD. In all 11 cases, genetic counselling, histological analyses of testicular biopsies, CFTR mutation screenings of both partners and spermatozoa three-colour FISH analyses were performed. Materials and methods Patients Included in this study were 11 couples in which the male partner presented with obstructive azoospermia due to CBAVD. No renal malformations were observed in these patients. ICSI with spermatozoa recovered by open testicular biopsy was carried out as previously described (Silber et al., 1995). Testicular tissues Testicular tissues were obtained on the same day as oocytes were retrieved from the female partner. Biopsy samples were placed in G1.2 medium (IVF Science Scandinavia; Gothenburg, Sweden) and immediately squashed between two microscopic slides. Spermatozoa were recovered from all biopsies. Surplus spermatozoa were frozen to avoid a second biopsy for any future ICSI. Remaining spermatozoa used during the injection procedure and thus not suitable for the freezing procedure were analysed by FISH. These samples were washed once in phosphate-buffered saline (PBS) and placed in an area previously delimited with a diamond pen on superfrosted slides (CML, France). Slides were air-dried at room temperature, washed once in PBS, dehydrated in an ethanol series (70, 90, 100%), air-dried and stored at –20°C. Histological analysis of testicular biopsies Diagnostic testicular biopsies were performed in parallel at the urological surgery ward. Recovered tissue was fixed for 1 h in a 0.1 mol/l cacodylate buffered 2% glutaraldehyde solution at +4°C. Fragments were rinsed for 30 min in the same buffer and post-fixed for 1 h in 1% OsO4 (osmic acid) After dehydratation, the samples were embedded in epoxy resin. Semi-thin sections (1 μm) were cut and stained with Toluidine Blue. DNA probes Probes specific for chromosomes X (pBamX5), Y (pCY98) and 1 (pUC1.77) were directly labelled, as described previously (Viville et al., 1997), by nick translation (Boehringer, Mannheim, Germany) with (i) rhodamine-4-dUTP (Amersham, Les Ullis, France); (ii) fluorescent isothiocyanate (FITC)-12-dUTP (Boehringer); and (iii) a mixture of (1:1) FITC-12dUTP and rhodamine-4-dUTP respectively. Probes were dissolved in 60% formamide/2×sodium chloride/sodium citrate (SSC) for use at a final concentration of 1–2 ng/μl. The use of two sex chromosomal probes and an autosomal specific probe allowed the determination of ploidy status of each spermatozoa analysed. FISH Sperm nuclei decondensation was performed by a 5 min incubation in 1 mol/l Tris buffer at pH 9.5 containing 25 mmol/l dithiothreitol (DTT) (Martin et al., 1995). Slides were then washed for 5 min in 2×SSC and 5 min in PBS, dehydrated through an ethanol series and air-dried. Slides were then treated with pepsin (100 mg/ml) in 0.01 N HCl for 20 min at 37°C, rinsed twice in bi-distilled water, fixed in methanol/acid acetic (3:1) for 10 min at 4°C, rinsed twice in bi-distilled water and dehydrated through an ethanol series. The hybridization solution (60% formamide, 2×SSC, 10% dextran) containing the three probes was applied to the slides under a coverslip. The probes and the nuclei were denatured at 78°C for 3 min. Hybridization was performed in a dark, moist chamber at 37°C for 1–2 h. Post hybridization washes included 5 min in 60% formamide/2×SSC and 5 min in 2×SSC at 42°C, followed by two 5 min washes in 4×SSC/0.05% Tween 20 at room temperature. After dehydratation through an ethanol series, slides were counterstained with 4′,6 diaminidino-2-phenylindol (DAPI) in antifading solution (Vector, Burlingame, USA) Signal analysis was performed on a Zeiss microscope according to scoring criteria described previously (Hopman et al., 1988). Results Patients In seven of the 11 cases, azoospermia represented the only apparent reason for infertility whereas in the remaining four a female factor (endocrinological or tubal) was also implicated. In total, 31 different CFTR mutations, including ΔF508, R117H and the variant IVS5T, which represent the more common mutations found in CBVAD patients (Chillon et al., 1995), responsible for 86% of all cystic fibrosis cases in the Caucasian population were investigated. Eight patients (73%) presented at least one mutation, three patients (27%) presented only the ΔF508 mutation and five (45%) presented compound heterozygosity for ΔF508/R347H, ΔF508/R117C, ΔF508/5T, ΔF508/D443Y and R117H–7T/5T (see Table I) As expected the ΔF508 mutation was the most frequently found (64%). For three patients no mutations, according to our criteria, were detectable, although one patient demonstrated a positive sweat test. For four patients (patients 2, 3, 4 and 5), in addition to CFTR mutation screenings, karyotypic analyses were also performed. All four were normal. For three of the patients (patients 4, 6 and 10) presenting a CFTR mutation, the families accepted genetic counselling and genetic screening revealed a familial origin of the defect. In two of these families, siblings had also consulted the fertility clinic regarding a CBAVD reproductive problem. All patients reported in this study presented normal serum concentrations of FSH. Histopathological analyses In all 11 cases, histopathological analysis of the testis demonstrated spermatogenesis characterized by elongated spermatids and spermatozoa in the majority of tubule sections. Seven of the patients had normal tubule/membrane/interstitium (TMI) (Valette et al., 1976) and Johnsen scores (Johnsen, 1970; Yoshida et al., 1997) see Table I. For four patients, histological analysis revealed a mild impairment of the testicular parenchyma, with irregularities in tubule diameter, decreases in germ cell density, exfoliation and necrobiosis of the seminiferous epithelia, hernia, tubule breaks and moderate thickening of the tunica propria (Figure 1). Table I shows that semi-quantitative evaluation using the TMI score is more discriminative than the Johnsen score, in agreement with a previous study (Meyer et al., 1988). TESE results Sufficient motile spermatozoa were recovered from the testicular biopsies of all 11 patients. Table II presents an overview of the TESE results. A total of 109 oocytes were collected (mean 9.9 per woman, range 0–18) following ovarian stimulation. After removal of cumulus and corona cells 74 oocytes exhibited the first polar body and were thus chosen for sperm injection. ICSI was carried out only in 10 couples, as no oocytes could be recovered from one patient. Fertilization occurred in 49 cases, thus giving a fertilization rate of 66.2% (range 36–100% for each couple). Embryo transfer took place in all 10 couples. Five clinical pregnancies, including one twin pregnancy resulted, from which five healthy babies were born. One case of spontaneous abortion occurred (patient 11). The pregnancy rate per embryo transferred was 22.7% (5/22). FISH results Considering the relative scarcity of samples which results from the biopsy procedure when compared to conventional IVF procedures which use ejaculated spermatozoa, priority was given to the freezing protocol. From the 11 biopsies tested, one gave no FISH signal (patient 11). Of the remaining 10, the number of spermatozoa analysed varied between 64 and 699. The results are shown in Table III. We present the results with aneuploidy rate as the cumulative frequency of disomy, diploidy and nullisomy signals. Statistical analysis was not possible due to the limited number of spermatozoa available. In each of the 10 cases, 72.5–98.2% of spermatozoa presented a normal X,1 or Y,1 signal. In patient number 5, 25% spermatozoa presented a nullisomy for the sex chromosomes (0, 1 signal). Furthermore, only 64 spermatozoa from patient 5 could be analysed of which 40 gave an interpretable FISH signal and a distorted sex chromosome ratio in favour of the Y chromosome (42.5% of Y1 signals versus 30.0% of X1 signals). Therefore, not considering patient 5, the average aneuploidy rate of our patients was 6.8 ± 3.9% (Table III) and was thus largely comparable with our fertile control subjects (two patients: 4.4 and 5.4%) and to the average general population rate, which was 1–4% (Guttenbach et al., 1997). Discussion We present here the results of a study employing a three-colour FISH analysis of testicular spermatozoa chromosomal content from patients presenting CBAVD. Our purpose was to establish a correlation between (i) the quality of testicular function, and (ii) the genetic safety of the TESE technique, as judged by histological criteria and FISH analysis, and success of the TESE/ICSI procedure. Because of the high prevalence of CFTR mutations in CBVAD patients (Chillon et al., 1995; De Braekeleer and Ferec, 1996; Lissens et al., 1996; Boucher et al., 1999) a screening for 31 different CFTR mutations covering 86% of all known cases was performed for all patients. For eight patients, at least one mutation (73%) was found, five patients were detected as compound heterozygotes (45%) and one patient for which no mutation found had a positive sweat test. These results are in accordance with previously published data (Lissens et al., 1996). With the exception of patient 5, and bearing in mind the limited number of spermatozoa available for analysis, patients presenting with obstructive subfertility due to CBAVD did not show an elevated spermatic aneuploidy rate overall, in comparison with normal fertile controls. This is in agreement with previously published results (Martin et al., 1993; Bischoff et al., 1994; for review see, Guttenbach et al., 1997). We presented the aneuploidy rate as the cumulative frequency of disomy, diploidy and nullisomy which may represent an over-estimation of the aneuploidy rate since some nullisomy certainly results from probe non-hybridization. Therefore, the rate of aneuploidy for these patients is within an acceptable range. Thus, although only a limited number of spermatozoa could be analysed, these results suggest that the genetic risks for children conceived by TESE from patients presenting with CBAVD are not increased. The higher level of chromosomal abnormalities found in patient 5 is difficult to explain. Indeed, of 64 spermatozoa obtained only 40 gave sufficient FISH signals for analysis. It must be mentioned that sperm FISH signals obtained by testicular biopsy are of a poorer quality than signals obtained from the ejaculated spermatozoa of fertile males (C.Falquet and S.Viville personal observations) For this patient, therefore, it is difficult to conclude whether the high level of aneuploidy is real or due to a technical artefact limited to this specific case. Nevertheless 72.5% of this patient's spermatozoa did present a normal chromosomal composition, at least for the chromosomes analysed. We believe, therefore, that we can still safely offer a TESE to this couple. It is interesting to note that the incidence of aneuploidy did not correlate with the presence of a CFTR mutation and furthermore, that the success rate of IVF following TESE/ICSI was independent of the quality of spermatogenesis, as judged by TMI and Johnsen scores. This latter result is in agreement with previous reports (Tournaye et al., 1996; Jezek et al., 1998; Nagy et al., 1998). For these couples where the male partner presents with CBAVD, a pregnancy can thus be expected as soon as spermatozoa are recovered. In conclusion, considering the three chromosomes analysed during this study and according to the pregnancy rate (22.7% per embryo transferred) obtained, we believe it is appropriate to propose this TESE/ICSI technique to patients with CBAVD. This implies genetic counselling and a search for CFTR mutations in order to establish the risk of having a child suffering from cystic fibrosis. If mutations are found in both partners then we offer either prenatal or preimplantation genetic diagnosis. Furthermore, according to this present study spermatozoa recovered from patients with CBAVD do not present an elevated aneuploidy rate compared with normal fertile males and lastly the TESE/ICSI pregnancy rate is as high as that observed for classical IVF or ICSI techniques. This study is currently being extended in order to aid geneticists to establish counselling programmes for infertile males presenting CBAVD. Table I. Patients' history Patient no.  TMI score  Johnsen score  CF mutation screening  Sweat test  Karyotype  Family history  TMI = tubule/membrane/interstitium; CF = cystic fibrosis; ND = not determined; Neg = negative; Pos = positive; NF = not found in 31 screened mutations, including ΔF508, R117H and the variant IVS5T.  1  1  12  NF  ND  ND  No  2  1  12  ΔF508/NF  ND  Normal  No  3  3  11  R117H-7T/5T  ND  Normal  No  4  3  11  NF  ND  Normal  Yes  5  1  11  ΔF508/NF  ND  Normal  No  6  2  11  ΔF508/R347H  ND  ND  Yes  7  4  10  ΔF508/5T  ND  ND  No  8  3  10  ΔF508/NF  Neg  ND  No  9  1  11  NF  Pos  ND  No  10  2  11  ΔF508/R117C  Pos  ND  Yes  11  2  11  ΔF508/D443Y  Pos  ND  No  Patient no.  TMI score  Johnsen score  CF mutation screening  Sweat test  Karyotype  Family history  TMI = tubule/membrane/interstitium; CF = cystic fibrosis; ND = not determined; Neg = negative; Pos = positive; NF = not found in 31 screened mutations, including ΔF508, R117H and the variant IVS5T.  1  1  12  NF  ND  ND  No  2  1  12  ΔF508/NF  ND  Normal  No  3  3  11  R117H-7T/5T  ND  Normal  No  4  3  11  NF  ND  Normal  Yes  5  1  11  ΔF508/NF  ND  Normal  No  6  2  11  ΔF508/R347H  ND  ND  Yes  7  4  10  ΔF508/5T  ND  ND  No  8  3  10  ΔF508/NF  Neg  ND  No  9  1  11  NF  Pos  ND  No  10  2  11  ΔF508/R117C  Pos  ND  Yes  11  2  11  ΔF508/D443Y  Pos  ND  No  View Large Table II. testicular sperm extraction Patient no.  Female partner  Fertilization per oocyte injected (%)  No. of day 2 embryos  No. of day 3 embryos  No. of embryos transferred  Pregnancy  Birth  1  Tubal factor  7/7 (100)  6  2  2  0  0  D2 and D3 = days 2 and 3 post-fertilization respectively; N = none.  aSpontaneous abortion.  2  N  4/10 (40)  4  3  3  0  0  3  Endocrinological  3/4 (75)  3  2  2  0  0  4  Endocrinological  6/6 (100)  6  6  2  0  0  5  N  0  0  0  0  0  0  6  N  5/7 (71)  4  4  2  1 (twin)  2  7  N  2/2 (100)  2  2  2  1  1  8  Endocrinological  3/6 (50)  3  2  2  0  0  9  N  4/11 (36)  4  2  2  1  1  10  N  7/13 (54)  6  5  2  1  1  11  N  8/10 (80)  7  6  3  1  0a  Total    54/83 (65.1)  50  39  22  5  5  Patient no.  Female partner  Fertilization per oocyte injected (%)  No. of day 2 embryos  No. of day 3 embryos  No. of embryos transferred  Pregnancy  Birth  1  Tubal factor  7/7 (100)  6  2  2  0  0  D2 and D3 = days 2 and 3 post-fertilization respectively; N = none.  aSpontaneous abortion.  2  N  4/10 (40)  4  3  3  0  0  3  Endocrinological  3/4 (75)  3  2  2  0  0  4  Endocrinological  6/6 (100)  6  6  2  0  0  5  N  0  0  0  0  0  0  6  N  5/7 (71)  4  4  2  1 (twin)  2  7  N  2/2 (100)  2  2  2  1  1  8  Endocrinological  3/6 (50)  3  2  2  0  0  9  N  4/11 (36)  4  2  2  1  1  10  N  7/13 (54)  6  5  2  1  1  11  N  8/10 (80)  7  6  3  1  0a  Total    54/83 (65.1)  50  39  22  5  5  View Large Table III. Fluorescent in-situ hybridization (FISH) results in spermatozoa Patient No.  No. analysed  No. with interpretable FISH signals (%)  FISH results  Total % of Aneuploidy        Normal haploid  Diploid  Aneuploid          X,1(%)  Y,1(%)  X,Y,1,1(%)  X,Y,1(%)  1(%)  X,1,1(%)  Y,1,1(%)  Y,Y,1(%)  Y(%)    1  699  677 (96.8)  257 (38.0)  396 (58.5)  0  4 (0.6)  19 (2.8)  0  0  0  1 (0.1)  3.5  2  444  433 (97.5)  194 (44.8)  226 (52.2)  3 (0.7)  3 (0.7)  6 (1.4)  0  0  0  1 (0.2)  3.0  3  112  104 (92.8)  49 (47.1)  48 (46.2)  0  0  6 (5.8)  1 (1.0)  0  0  0  6.7  4  297  281 (94.6)  150 (53.4)  116 (41.3)  0  5 (1.8)  9 (3.2)  0  0  1 (0.4)  0  5.3  5  64  40 (62.5)  12 (30.0)  17 (42.5)  0  2 (5.0)  9 (22.5)  0  0  0  0  27.5  6  109  109 (100)  47 (43.1)  60 (55.0)  0  2 (1.8)  0  0  0  0  0  1.8  7  68  68 (100)  39 (57.4)  20 (29.4)  0  3 (4.4)  0  5 (7.4)  1 (1.5)  0  0  13.2  8  72  63 (87.5)  17 (27.0)  41 (65.1)  0  4 (6.3)  1 (1.6)  0  0  0  0  7.9  9  195  183 (93.8)  89 (48.6)  72 (39.3)  0  7 (3.8)  11 (6.0)  2 (1.1)  1 (0.5)  1 (0.5)  0  12.0  10  188  188 (100)  63 (33.5)  110 (58.5)  0  2 (1.1)  12 (6.4)  0  1 (0.5)  0  0  7.9  Patient No.  No. analysed  No. with interpretable FISH signals (%)  FISH results  Total % of Aneuploidy        Normal haploid  Diploid  Aneuploid          X,1(%)  Y,1(%)  X,Y,1,1(%)  X,Y,1(%)  1(%)  X,1,1(%)  Y,1,1(%)  Y,Y,1(%)  Y(%)    1  699  677 (96.8)  257 (38.0)  396 (58.5)  0  4 (0.6)  19 (2.8)  0  0  0  1 (0.1)  3.5  2  444  433 (97.5)  194 (44.8)  226 (52.2)  3 (0.7)  3 (0.7)  6 (1.4)  0  0  0  1 (0.2)  3.0  3  112  104 (92.8)  49 (47.1)  48 (46.2)  0  0  6 (5.8)  1 (1.0)  0  0  0  6.7  4  297  281 (94.6)  150 (53.4)  116 (41.3)  0  5 (1.8)  9 (3.2)  0  0  1 (0.4)  0  5.3  5  64  40 (62.5)  12 (30.0)  17 (42.5)  0  2 (5.0)  9 (22.5)  0  0  0  0  27.5  6  109  109 (100)  47 (43.1)  60 (55.0)  0  2 (1.8)  0  0  0  0  0  1.8  7  68  68 (100)  39 (57.4)  20 (29.4)  0  3 (4.4)  0  5 (7.4)  1 (1.5)  0  0  13.2  8  72  63 (87.5)  17 (27.0)  41 (65.1)  0  4 (6.3)  1 (1.6)  0  0  0  0  7.9  9  195  183 (93.8)  89 (48.6)  72 (39.3)  0  7 (3.8)  11 (6.0)  2 (1.1)  1 (0.5)  1 (0.5)  0  12.0  10  188  188 (100)  63 (33.5)  110 (58.5)  0  2 (1.1)  12 (6.4)  0  1 (0.5)  0  0  7.9  View Large Figure 1. View largeDownload slide Histopathological aspect of testicular tubules in different cases of congenital bilteral absence of the vas deferens (CBVAD). (A) Patient 2; normal healthy seminiferous epithelium with regular distribution of the germ cells. (B) Patient 4; healthy seminiferous epithelium quite similar to A, but with thinning of the tunica propria (arrows) inducing hernia of the epithelium. (C) Patient 7; impairment of the seminiferous epithelium showing figures of cytolysis. However, the spermiogenesis is efficient with numerous round and elongating spermatids. (D) Patient 10; the germ cells are irregularly distributed and dissociated. The tunica propria is thickened with the beginning of a hyalinization process. Spermiogenesis is maintained. Figure 1. View largeDownload slide Histopathological aspect of testicular tubules in different cases of congenital bilteral absence of the vas deferens (CBVAD). (A) Patient 2; normal healthy seminiferous epithelium with regular distribution of the germ cells. (B) Patient 4; healthy seminiferous epithelium quite similar to A, but with thinning of the tunica propria (arrows) inducing hernia of the epithelium. (C) Patient 7; impairment of the seminiferous epithelium showing figures of cytolysis. However, the spermiogenesis is efficient with numerous round and elongating spermatids. (D) Patient 10; the germ cells are irregularly distributed and dissociated. The tunica propria is thickened with the beginning of a hyalinization process. Spermiogenesis is maintained. 4 To whom correspondence should be addressed at: Service de Biologie de la Reproduction SIHCUS-CMCO, 19 rue Louis Pasteur BP120, 67303 Schiltigheim, France.E-mail: viville@igbmc.u-strasbg.fr References Bischoff, F.Z., Nguyen, D.D., Burt, K.J. and Shaffer, L.G. (1994) Estimates of aneuploidy using multicolor fluorescence in situ hybridization on human sperm. Cytogenet. 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Journal

Human ReproductionOxford University Press

Published: Jul 1, 2000

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