1 Service de Biologie de la Reproduction SIHCUS-CMCO, 19 rue Louis Pasteur BP120, 67303 Schiltigheim, 2 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, and 3 Service de Chirurgie Urologique HUS, Hôpital Civil, 1 place de l'Hôpital, BP 426, F-67091 Strasbourg Cedex, France
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Abstract |
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Key words: congenital absence of the vas deferens/fluorescent in-situ hybridization/histological analysis/ICSI/testicular sperm extraction
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Introduction |
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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.
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Materials and methods |
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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/2xsodium chloride/sodium citrate (SSC) for use at a final concentration of 12 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 2xSSC 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, 2xSSC, 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 12 h. Post hybridization washes included 5 min in 60% formamide/2xSSC and 5 min in 2xSSC at 42°C, followed by two 5 min washes in 4xSSC/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
).
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Results |
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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, 72.598.2% 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 14% (Guttenbach et al., 1997
).
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Discussion |
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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.
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Notes |
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References |
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Bonduelle, M., Legein, J., Buysse, A. et al. (1996a) Prospective follow-up study of 423 children born after intracytoplasmic sperm injection. Hum. Reprod., 11, 15581564.
Bonduelle, M., Wilikens, A., Buysse, A. et al. (1996b) Prospective follow-up study of 877 children born after intracytoplasmic sperm injection (ICSI), with ejaculated epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum. Reprod., 11 (Suppl. 4), 131155; discussion 156159.[ISI][Medline]
Bonduelle, M., Aytoz, A., Van Assche, E. et al. (1998) Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. [Editorial]. Hum. Reprod., 13, 781782.
Boucher, D., Creveaux, I., Grizard, G. et al. (1999) Screening for cystic fibrosis transmembrane conductance regulator gene mutations in men included in an intracytoplasmic sperm injection programme. Mol. Hum. Reprod., 5, 587593.
Chillon, M., Casals, T., Mercier, B. et al. (1995) Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N. Engl. J. Med., 332, 14751480.
De Braekeleer, M. and Ferec, C. (1996) Mutations in the cystic fibrosis gene in men with congenital bilateral absence of the vas deferens. Mol. Hum. Reprod., 2, 669677.[Abstract]
Engel, W., Murphy, D. and Schmid, M. (1996) Are there genetic risks associated with microassisted reproduction? Hum. Reprod., 11, 23592370.[Abstract]
Govaerts, I., Koenig, I., Van den Bergh, M. et al. (1996) Is intracytoplasmic sperm injection (ICSI) a safe procedure? What do we learn from early pregnancy data about ICSI? Hum. Reprod., 11, 440443.[Abstract]
Guttenbach, M., Engel, W. and Schmid, M. (1997) Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations. A review. Hum. Genet., 100, 121.[ISI][Medline]
Hewitson, L., Dominko, T., Takahashi, D. et al. (1999) Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys [see comments]. Nature Med., 5, 431433.[ISI][Medline]
Hopman, A.H., Ramaekers, F.C., Raap, A.K. et al. (1988) In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors. Histochemistry, 89, 307316.[ISI][Medline]
In't Veld, P., Brandenburg, H., Verhoeff, A. et al. (1995) Sex chromosomal abnormalities and intracytoplasmic sperm injection. [Letter.] Lancet, 346, 773.[ISI][Medline]
In't Veld, P. A., Broekmans, F. J., de France, H.F. et al. (1997) Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa. Hum. Reprod., 12, 752754.[Abstract]
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, 12301234.[Abstract]
Johnsen, S.G. (1970) Testicular biopsy score counta method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones, 1, 225.[Medline]
Lamb, D.J. (1999) Debate: is ICSI a genetic time bomb? Yes [comment]. J. Androl., 20, 2333.
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. Reprod., 11 (Suppl. 4), 5578; discussion 7980.[Abstract]
Luetjens, C.M., Payne, C. and Schatten, G. (1999) Non-random chromosome positioning in human sperm and sex chromosome anomalies following intracytoplasmic sperm injection. [Letter.] Lancet, 353, 1240.[ISI][Medline]
Martin, R. H., Ko, E. and Chan, K. (1993) Detection of aneuploidy in human interphase spermatozoa by fluorescence in situ hybridization (FISH) Cytogenet. Cell Genet., 64, 2326.[ISI][Medline]
Martin, R.H., Spriggs, E., Ko, E. and Rademaker, A.W. (1995) The relationship between paternal age, sex ratios, and aneuploidy frequencies in human sperm, as assessed by multicolor FISH. Am. J. Hum. Genet., 57, 13951399.[Medline]
Meschede, D. and Horst, J. (1997) Genetic counselling for infertile male patients. Int. J. Androl., 20 (Suppl. 3), 2030.[ISI][Medline]
Meyer, J.M., Roos, M. and Rumpler, Y. (1988) Statistical study of a semiquantitative evaluation of testicular biopsies. Arch. Androl., 20, 7179.[ISI][Medline]
Nagy, Z.P., Joris, H., Verheyen, G. et al. (1998) Correlation between motility of testicular spermatozoa, testicular histology and the outcome of intracytoplasmic sperm injection. Hum. Reprod., 13, 890895.[Abstract]
Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A.C. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 1718.[ISI][Medline]
Pauer, H.U., Hinney, B., Michelmann, H.W. et al. (1997) Relevance of genetic counselling in couples prior to intracytoplasmic sperm injection. Hum. Reprod., 12, 19091912.[Abstract]
Schlegel, P.N. (1999) Debate: is ICSI a genetic time bomb? No: ICSI is safe and effective [see comments]. J. Androl., 20, 1822.
Silber, S.J., Van Steirteghem, A.C., Liu, J. et al. (1995) High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum. Reprod., 10, 148152.[Abstract]
Tournaye, H., Liu, J., Nagy, P.Z. et al. (1996) Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa [see comments]. Hum. Reprod., 11, 127132.[Abstract]
Valette, J., Grimaud, J.A., Lansac, J. and Cognat, M. (1976) [Attempted standardized interpretation of testicular histopathology and male fertility]. Gynecologie, 27, 219224.[Medline]
Viville, S., Messaddeq, N. and Gerlinger, P. (1997) Towards the preparation of preimplantation genetic diagnosis (PGD) practice in France. J. Assist. Reprod. Genet., 14, 478.
Yoshida, A., Miura, K. and Shirai, M. (1997) Evaluation of seminiferous tubule scores obtained through testicular biopsy examinations of nonobstructive azoospermic men [see comments]. Fertil. Steril., 68, 514518.[ISI][Medline]
Yurov, Y.B., Saias, M.J., Vorsanova, S.G. et al. (1996) Rapid chromosomal analysis of germ-line cells by FISH: an investigation of an infertile male with large-headed spermatozoa. Mol. Hum. Reprod., 2, 665668.[Abstract]
Submitted on January 11, 1999; accepted on April 7, 2000.