1 Centre for Reproductive Medicine and 2 Centre for Medical Genetics, University Hospital, Dutch-speaking Free University of Brussels, Laarbeeklaan 101, 1090 Brussels, Belgium
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: azoospermia/chromosomal aneuploidy/FISH/testicular sperm
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Moreover, there is concern as to whether ICSI, the most invasive assisted reproduction technique, might lead to an increase in the proportion of children with de-novo chromosomal abnormalities or gene defects, or to inheritance of the father's infertility problem. Indeed, the results of several studies have shown an increase in de-novo chromosomal aberrations in fetuses and children born after ICSI (In't Veld et al., 1995; Liebaers et al., 1995
; Bonduelle et al., 2002
). An overview of seven studies on prenatal diagnosis after ICSI (Testart et al., 1996
; Van Opstal et al., 1997
; Govaerts et al., 1998
; Loft et al., 1999
; Van Golde et al., 1999
; Wennerholm et al., 2000
; Bonduelle et al., 2002
) reported 73 abnormal fetal karyotypes out of 2139 analysed (Van Steirteghem et al., 2002
). Forty-two abnormal karyotypes were due to de-novo chromosomal aberrations (of which 16 were due to sex chromosome aberrations and 26 to autosome aberrations) and 31 were due to inherited structural aberrations.
The most relevant study was conducted at the Centre for Medical Genetics and Centre for Reproductive Medicine, University Hospital, Dutch-speaking Free University of Brussels (Bonduelle et al., 2002). The authors' data on 1437 karyotypes determined by prenatal diagnosis show a statistically significant increase in de-novo sex chromosomal aneuploidy (0.6%) and structural autosomal abnormalities (0.4%) as compared with the general neonatal population (0.2 and 0.07% respectively). Concerning the major congenital malformation rate, the same study reports similar rates between children born after ICSI with ejaculated (3.4%) and non-ejaculated (epididymal or testicular) sperm (3.2%). No statistical difference was observed in major malformations after use of testicular (2.91%) or epididymal sperm (3.8%). The higher incidence of chromosomal aberrations may be explained by the higher frequency of chromosomal abnormalities reported in sperm from men with fertility problems, who are commonly the ICSI candidates (Moosani et al., 1995
; Bernardini et al., 1997
; Aran et al., 1999
; Pang et al., 1999
; Rives et al., 1999
; Ushijima et al., 2000
; Vegetti et al., 2000
; Calogero et al., 2001b
). Meiotic disorders are frequent in infertile patients and are responsible for autosomal and sex-chromosome disomies and diploidy in sperm (Egozcue et al., 2000
).
These observations emphasise the need to assess the frequency of chromosomal abnormalities in testicular sperm from patients with spermatogenic failure. Therefore, the aim of the present study was to analyse the frequency of numeric abnormalities for chromosomes X, Y and 18 by fluorescence in-situ hybridization (FISH) in testicular sperm from patients with spermatogenic failure, and to compare the findings with the frequency of chromosomal abnormalities of testicular sperm from patients with normal spermatogenesis.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Testicular sperm
Testicular biopsies were excised under local or general anaesthesia, and were collected in a Petri dish (Falcon Plastics, type 1006; Becton-Dickinson, Aalst, Belgium) filled with 3 ml modified HEPES-buffered Earle's medium supplemented with 2.25% human serum albumin (HSA) (Tournaye et al., 1995). One small tissue specimen per testis was fixed in Bouin's for histological examination. The other testicular biopsy specimens were shredded using two sterile microscopic slides in order to obtain a testicular cell suspension for therapeutic or diagnostic purposes (Verheyen et al., 1995
). Microscopic examination of the wet preparation was carried out at 400x magnification under an inverted microscope with Hoffman Modulation Contrast System (Modulation Optics Inc., Greenvale, NY, USA). Where no sperm were observed after 1 h of initial searching, enzymatic treatment of the testicular tissue with 100 IU/ml collagenase type IV (Sigma) was carried out in order to digest the tissue and release the few sperm that might be present (Crabbé et al., 1998
).
Testicular biopsy suspensions were prepared either freshly on the day of surgical intervention (n = 25) or after thawing a fraction of a previously frozen suspension (n = 18).
Fresh testicular suspensions were prepared as follows: the shredded samples were allowed to sediment in a tube. The supernatant was decanted and centrifuged at 300 g for 5 min (Nagy et al., 1995) and the supernatant discarded. The pellet was resuspended in a small volume of modified HEPES-buffered Earle's medium supplemented with 2.25% HSA. Drops of 5 µl of pellet were smeared on the bottom of an ICSI dish, diluted with 5 µl of Earle's medium and covered with 3.5 ml of sterile paraffin oil.
Frozen testicular suspensions were prepared as follows: one or more straws of frozen material were thawed and washed twice with 5 ml of HEPES-buffered Earle's medium supplemented with 2.25% HSA. The pellet was resuspended in a minimum volume of medium and droplets of 5 µl were smeared on the bottom of an ICSI dish, diluted with 5 µl of Earle's medium and covered with 3.5 ml paraffin oil.
The ICSI procedure was first performed with selected sperm. The sperm left in the dish after the ICSI procedure were retrieved individually from the droplets and placed on a microscope slide using the micromanipulator. Only mature-looking sperm or elongated spermatids (type Sd2) (Clermont, 1963) were selected and individually aspirated with the micromanipulator from testicular suspensions, irrespective of their motility and morphological quality.
The fresh testicular tissue and suspension left in the tubes after ICSI were frozen for the patient's use in a subsequent cycle if pregnancy did not occur, or for a subsequent pregnancy.
Sperm retrieval
The retrieval of individual sperm from testicular suspensions was carried out under an inverted microscope (Diaphot; Nikon Corporation, Tokyo, Japan) at 400x magnification using the Hoffman Modulation Contrast System (Modulation Optics Inc., Greenvale, NY, USA). The microscope was equipped with a coarse positioning manipulator (3D Motor Driven Coarse control Manipulator MM-188; Narishige) and with a three-dimensional hydraulic remote-control micromanipulator (Joystick Hydraulic Micromanipulator MO-88; Narishige). The aspiration pipette had a diameter of 10 µm and the tip was ground and bent to an angle of
30° by gentle heating (Joris et al., 1998
).
Another dish was prepared with 8 drops of 5 µl of 10% polyvinylpyrrolidone (PVP) solution, 8 drops of PVP diluted with medium and 8 drops of medium alone, and covered with sterile paraffin oil. In the first step, the pipette was filled with PVP. The sperm were then aspirated one by one using the micromanipulator from the dish containing the testicular suspension and replaced in the drop of diluted PVP in the second dish, in order to remove the debris and other cells that might be aspirated along with sperm and/or which might stick to the sperm during micromanipulation. If the drop become too contaminated by other cells and debris, the sperm were aspirated together and replaced into a clean drop with medium only. The last step consisted of replacing groups of sperm from the clean medium droplet into a minimum volume of phosphate-buffered saline (PBS), previously delineated with a diamond pen, on a SuperFrost/Plus slide. The droplet was allowed to dry.
The sperm on the slide were fixed with cold fixative (methanol:acetic acid, 3:1) and stored at 20°C.
Nuclei decondensation of testicular sperm
On the day of the FISH procedure, the slides were washed in 2x standard saline citrate (SSC) solution to remove excess fixative and were then incubated at room temperature for 5 min in 1 mol/l TrisHCl buffer (pH 9.5) containing 25 mmol/l dithiothreitol in order to allow DNA decondensation (Martini et al., 1995). Slides were then washed in 2xSSC, 1xPBS, dehydrated in an ethanol series (709696100100%) and air-dried.
FISH procedure
Prior to hybridization, the slides were incubated for 15 min at 37°C in a pretreatment solution (0.01 mol/l HCl containing 50 µl pepsin) and washed twice in purified water and twice in 1xPBS, followed by 10 min fixation in methanol:acetic acid (3:1) at 4°C. After two washing steps with 1xPBS and two with purified water, the slides were dehydrated in an ethanol series (7090100100%).
The DNA probes (Vysis, Inc., Downers Grove, IL, USA) used for this study were centromeric DNA probes (chromosome enumeration probe, CEP) for chromosomes X, Y and 18, directly labelled with Spectrum Green, Spectrum Orange and Spectrum Aqua fluorophores respectively. The probe mixture consisted of 7 µl CEP buffer, 1 µl CEP X-Spectrum Green, 1 µl CEP Y-Spectrum Orange and 1 µl CEP 18-Spectrum Aqua.
The FISH procedure was performed according to the protocol recommended by Vysis for directly-labelled probes: the probe mixture was applied to the specimen target area, covered with a glass coverslip and denatured at 75°C on a hot plate for 3 min. The coverslips were sealed with rubber cement and hybridization took place overnight in a humidified chamber at 37°C.
For the post-hybridization washing, coverslips were removed and the slides were immersed immediately in 0.4xSSC solution at 73 ± 1°C for 2 min and then washed at room temperature for 1 min with 2xSSC/0.1% NP40 (Tergitol NP40; Sigma) solution. Finally, the slides were mounted in Vectashield antifade medium (Vector Laboratories Inc., Burlingame, CA, USA) containing 4',6'-diamidino-2-phenylindole (DAPI) counterstain.
In order to test the efficiency of the procedure, peripheral blood lymphocytes from a healthy donor with a normal (46,XY) karyotype was used as a control for each FISH procedure on testicular sperm.
Microscopy and scoring criteria
Slides were examined using a Zeiss-Axioplan fluorescence microscope with the appropriate filter sets (Vysis): single band pass filter DAPI, single band pass filter orange, single band pass filter green, triple band pass filter DAPI/orange/green and a single band pass filter Aqua.
A sperm nucleus was scored only if it was intact and did not overlap with other nuclei. An X or Y chromosome in a sperm nucleus was recognized by a green or orange fluorescent spot respectively. Chromosome 18 was recognized by the presence of an aqua fluorescent spot in the sperm nucleus.
Only clear hybridization signals, comparable in brightness and size and separated from each other by at least one diameter, were taken into consideration. Sperm showing no signal for chromosome X, Y or 18 were not included in the study, as this outcome may reflect unsuccessful hybridization due to inadequate nuclear decondensation.
Sperm nuclei were scored as disomic for sex chromosomes when two distinct X or Y signals or a X and a Y signal and a single fluorescent spot for chromosome 18 were clearly visible within the nucleus (Van Dyk et al., 2000). Sperm nuclei with only a single visible chromosome 18 signal were scored as nullisomic for sex chromosomes. Sperm nuclei with no aqua fluorescent signal detected in the presence of any sex chromosome signal were scored as nullisomic for chromosome 18. Sperm nuclei were considered diploid when two sex chromosome signals and two chromosome 18 signals were present. Sperm nuclei were considered disomic for chromosome 18 when two aqua fluorescent signals and a green or an orange fluorescent spot was present in the sperm nucleus.
Statistical analysis
In order to compare different parameters (age, hormonal values and testicular volume) in the patients with normal spermatogenesis and those with spermatogenic failure, the MannWhitney U-test was applied.
The 2-test was used to analyse: (i) the differences in frequency of aneuploidy for sex chromosomes and for chromosome 18 among and between the groups showing normal spermatogenesis and spermatogenic failure; and (ii) the proportion of normal haploid sperm bearing an X or Y chromosome between those two groups.
The statistical analysis was performed using MedCalc software (Ghent, Belgium). All tests were performed at the 5% level of significance.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The median age for patients with spermatogenic failure (30.0 years, range 2254) was significantly lower than in cases with normal spermatogenesis (39.0 years, range 2955; P = 0.0022).
Significantly higher values of serum FSH (normal values: 1.512.4 mIU/ml) were found in cases with spermatogenic failure (median 23.7 mIU/ml, range 1.960.8) than in cases with normal spermatogenesis (median 5.1 mIU/ml, range 2.127.3; P < 0.01). Similarly, higher serum LH levels (normal values: 1.78.6 mIU/ml) were found in patients with spermatogenic failure (median 7 mIU/ml, range 2.715.5) than in patients with normal spermatogenesis (median 4.2 mIU/ml, range 2.88.8; P < 0.05). No significant difference was observed in testosterone levels (normal values: 2.89.9 µg/l) of patients with normal spermatogenesis (median 3.5 µg/l, range 2.54.6) as compared with patients with spermatogenic failure (median 3.5 µg/l, range 2.77.5). In terms of testicular volume, a significantly higher volume was observed in patients with normal spermatogenesis (median 20 ml, range 1235) than in patients with spermatogenic failure (median 10 ml, range 620; P < 0.01).
FISH results
Sperm retrieval
A total number of 1839 sperm cells were analysed by FISH and 1697 of these provided interpretable results, giving a total FISH efficiency of 92.2% (1697/1839). The number of testicular sperm successfully analysed in both groups of patients are presented in Table I. Due to the limited number of cells, statistical analysis between SCOS and MA groups was considered inappropriate.
|
|
|
The frequency of chromosomal abnormalities (considered as the cumulative frequencies of sperm showing disomy, nullisomy or diploidy for the sex chromosomes or for chromosome 18) was 5.6% (range 018.2) in the group with normal spermatogenesis and 8.2% (range 025) in the group with spermatogenic failure. After pooling the data of all groups, 6.3% of testicular sperm were found to be abnormal. In all groups, the frequency of numeric chromosomal abnormalities showed a wide variation among patients (Table II).
Comparison of chromosomal abnormalities in testicular sperm derived from patients with normal spermatogenesis and from patients with spermatogenic failure
Comparison of the distribution of numerical chromosomal abnormalities in testicular sperm derived from patients with normal spermatogenesis and from patients with spermatogenic failure is reported in Table III.
|
The frequency of total disomy, defined as disomy for the sex chromosomes (XX, XY, YY) or for chromosome 18 (1818), was 2.5% in sperm from the group with normal spermatogenesis and 3.7% in sperm from the group with spermatogenic failure (not significant). The frequency of sex chromosome disomy in the group characterized by normal spermatogenesis was 2.2%, similar to the frequency of 2.4% in the group characterized by spermatogenic failure. A higher frequency of disomy for chromosome 18 was observed in testicular sperm derived from patients with spermatogenic failure (1.3%) than in that retrieved from patients with normal spermatogenesis (0.3%; P = 0.05).
The frequency of total nullisomy, defined as the absence of any sex chromosome or of chromosome 18, did not differ between the two groups of patients. Neither for the sex chromosome nullisomy rate, nor for chromosome 18 nullisomy was a difference observed between the group with normal spermatogenesis and the group with spermatogenic failure (Table III).
The frequency of diploidy, defined as XX1818, XY1818 or YY1818, was also similar in the two groups.
Distribution of chromosomal abnormalities in testicular sperm of patients with normal spermatogenesis
Among testicular sperm derived from patients with normal spermatogenesis, there was a significantly higher frequency of sex chromosomal aneuploidy (4.5%) than in the group with chromosome 18 aneuploidy (1.3%; P < 0.001). The incidence of disomy and the incidence of nullisomy for the sex chromosomes (2.2 and 2.1% respectively) were higher (P < 0.001 and P = 0.007) than for chromosome 18 (0.3 and 0.7% respectively) (Table IV).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study aimed to analyse testicular sperm from this specific group of infertile patients by FISH. The purpose was to evaluate chromosomal aneuploidy in the testicular sperm retrieved from patients with normal spermatogenesis and from patients with spermatogenic failure, in order to estimate the risk for chromosomal abnormalities in the offspring of these two groups of patients. Due to practical problems and limited cell numbers, it was inappropriate to group the retrieved testicular sperm according to their morphology and/or motility. Consequently, our results provide the overall aneuploidy rate of all mature-looking testicular sperm and not only of a subgroup presenting good morphology and motility. Immotile sperm or sperm with poor morphology are indeed sometimes used in clinical ICSI practice for cases of severe testicular failure.
So far, few studies on chromosomal abnormalities in testicular sperm have been reported. Moreover, the data are controversial, which may relate to differences in patient selection, cell selection and/or methodology. In these studies, the patients were mostly characterized on the basis of their clinical diagnosis and were grouped together as obstructive azoospermia and non-obstructive azoospermia, but the result of the histological examination was not always provided (Martin et al., 2000). The obstructive azoospermia cases showed congenital absence of the vas deferens (Viville et al., 2000b
; Levron et al., 2001
) with presumed normal spermatogenesis. In cases of non-obstructive azoospermia, Martin et al. did not provide any histological result (Martin et al., 2000
). Although thousands of sperm had been observed, their patients were defined as non-obstructive. Our criteria for classifying patients was based on histological diagnosis, which is considered a most reliable parameter.
Considering the cell selection and methodology used, testicular sperm were analysed either after smearing the testicular suspensions on slides (Bernardini et al., 2000), or after aspirating and transferring sperm on slides with the micromanipulator (Martin et al., 2000
; Viville et al., 2000b
; Levron et al. 2001
). In our study, as in that by Levron et al. the sperm were individually aspirated from testicular suspensions left in the ICSI dish after microinjection (Levron et al., 2001
). By this technique, which most resembles the procedure applied in clinical ICSI practice, we allowed a proper identification of the sperm cell and its maturational stage. In this way any possible confusion was avoided with the immature stages and/or with other cells from the testicular suspension that might have led to a misinterpretation of the FISH signals.
In the present study, despite a reasonable number of patients (n = 43) being involved, the number of cells analysed in the three groups was limited: 1232 in the group with normal spermatogenesis and 465 in the group with spermatogenic failure. Only the sperm remaining in the droplets under oil after completion of ICSI were accessible for the study while the remaining tissue suspension was frozen for the patient. In the cases with spermatogenic failure, however, it repeatedly happened that the entire suspension, sometimes after enzymatic digestion, was used to carry out ICSI, after which exploration of the very few remaining sperm cells for FISH was carried out.
The frequency of chromosomal abnormalities in testicular sperm from azoospermic patients with normal spermatogenesis (5.6%) and spermatogenic failure (8.2%), or from both groups together (6.3%) suggest a slight increase in the frequency of chromosomal abnormalities compared with the reported data in ejaculated sperm of fertile control donors, which is <2% (Aran et al., 1999; Colombero et al., 1999
; Bernardini et al., 2000
; Ushijima et al., 2000
; Vegetti et al., 2000
; Calogero et al., 2001a
; Levron et al., 2001
). Considering the group of patients with normal spermatogenesis, the present result of 5.6% of chromosome abnormalities in testicular sperm is comparable with the 6.8% presented by Viville et al. (Viville et al., 2000b
) and with the 8.2% presented by Levron et al. for obstructive cases (Levron et al., 2001
). Our results on testicular sperm from patients with normal spermatogenesis, as well as the results of Levron et al. from obstructive cases, show a significantly higher frequency in sex chromosome aneuploidy than in aneuploidy for chromosome 18 (Levron et al., 2001
). This higher frequency is manifested in a significant increase in both the disomy and the nullisomy frequencies for sex chromosomes as compared with chromosome 18. This suggests that during male meiosis, the sex chromosomes are more susceptible to non-disjunction than the autosomes (Downie et al., 1997
).
In the group of patients with spermatogenic failure, an overall frequency of chromosomal abnormalities of 8.2% was obtained. No difference between sex chromosomes and chromosome 18 with respect to disomy or nullisomy was observed. Our frequency of chromosomal abnormalities (8.2%) deviates from three other studies carried out on testicular sperm from non-obstructive patients: 2% for chromosomes X,Y,13,21 in the study by Martin et al.,
44% for chromosomes 1, 17 and
56% for sex chromosomes in the study by Bernardini et al., and 19.6% in the study by Levron et al. (Bernardini et al., 2000
; Martin et al., 2000
; Levron et al., 2001
). Bernardini et al. found very high frequencies of aneuploidy, which may be related to a difference in methodology (cytospun TESE suspension) and/or scoring criteria (Bernardini et al., 2000
). Cells without any signal were scored as nullisomic in this study. The frequency of nullisomy rose to 23.5% for sex chromosomes and to 15% for chromosomes 1/17. In our study, cells without any FISH signal were considered a FISH failure and were not taken into account for analysis. However, if we consider the cells in our study without any signal (only DAPI signal) as being nullisomic, the frequency of chromosomal abnormalities would be 7.2% in the group with normal spermatogenesis and 9.9% in the group with spermatogenic failure, which does not affect the conclusions of our study. Martin et al. were able to collect high numbers of sperm per patient (>1000 for two patients and >400 for the third) (Martin et al., 2000
). Their patients showing normal FSH levels were considered non-obstructive cases in the absence of a histological diagnosis. All of this suggests at least partial obstruction in these patients. In our hands, patients with a histological diagnosis indicating non-obstructive azoospermia suffer from severe testicular failure and several hours may be spent looking for the limited sperm numbers required to inject the oocytes.
Comparing the frequencies of chromosomal abnormalities in testicular sperm from patients with normal spermatogenesis (5.6%) and with spermatogenic failure (8.2%), our results suggest no overall difference between these two groups. Our findings are in contradiction to the study by Levron et al. whose results showed a higher aneuploidy rate in the non-obstructive (19.6%) than in the obstructive group (8.2%) (Levron et al., 2001). In our study, a significant difference was observed only in the frequency of disomy for chromosome 18, which was highest in the group with spermatogenic failure. One possible explanation for this contradiction might be the difference between the number of cells analysed in our study (465 and 1232) and in the study by Levron et al. (153 and 367 respectively).
There is no clear explanation for the elevated frequency of chromosomal abnormalities (tested for chromosomes X, Y and 18) in testicular sperm. Autosomal disomy can arise from non-disjunction in both meiosis I and meiosis II (Asada et al., 2000). Abnormal segregation of the sex chromosomes leading to nullisomy (only chromosome 18 present) or different arrangements of disomy (XY, XX, YY) suggests non-disjunction at either the mitotic stages in diploid spermatogonial cells, or at meiotic (I and/or II) division stages (Huang et al., 1999
). In sex chromosomes, non-disjunction during meiosis I results in an XY disomy, whereas non-disjunction during meiosis II results in an XX or YY disomy (Asada et al., 2000
). Nevertheless, the increased frequency of chromosomal abnormalities in testicular sperm from patients with normal spermatogenesis, as compared with ejaculated sperm, is hard to explain. It may be hypothesized that there is a mechanism involved in sequestration of abnormal sperm during epididymal transit, resulting in a lower aneuploidy rate in ejaculated sperm (Levron et al., 2001
).
Studies on chromosomal aneuploidy in testicular sperm are scarce. Moreover, the cell numbers are small and the patient selection and methodology differs greatly. This may explain the high variability in aneuploidy frequencies in testicular sperm from azoospermic patients among different studies. For our study, the availability of the cells was limited, as cryopreservation of supernumerary testicular suspension was considered to have priority. This suggests that the study might be prone to a type II (ß) error. These findings imply the need for assessment of a higher number of testicular cells in future studies.
FISH on ejaculated sperm is easier and more reliable (high cell numbers) than FISH on testicular sperm. In contrast to ejaculated sperm, testicular sperm show different degrees of sperm nuclei decondensation, leading to differences in the access of the probes to the DNA of the sperm. This may partly explain the lower hybridization efficiency in testicular than in ejaculated sperm reported in the literature (Bernardini et al., 2000; 80%) (Viville et al., 2000b
; 95.4%) as well as in our study (92.2%). Additional factors, such as variation in signal scoring criteria, fluorescence microscope configuration, DNA cross-hybridization and patient-to-patient variation may contribute to this variability in numeric chromosome abnormalities.
In conclusion, the results of the present study suggest an increased frequency of chromosome abnormalities in testicular sperm from azoospermic patients as compared with the reported data on ejaculated sperm. So far, no difference in the frequency of chromosome abnormality has been observed for testicular sperm of patients with normal spermatogenesis and patients with spermatogenic failure. However, the frequency of aneuploidy for chromosome 18 was higher in the group with spermatogenic failure than in the group with normal spermatogenesis. Overall, sex-chromosome aneuploidies seemed to occur more frequently than aneuploidy for chromosome 18.
The wide range of individual frequency of chromosomal aneuploidy detected in testicular sperm from both groups of patients considered in this study might lead to the idea, also suggested by Martin et al., that some azoospermic patients may have a higher risk of carrying chromosomal abnormalities in meiotic cells than others (Martin et al., 2000). Additional FISH studies on testicular sperm and data from preimplantation genetic diagnosis by aneuploidy screening or prenatal diagnosis in cases where ICSI was carried out with testicular sperm are necessary in order to give better genetic counselling to couples referred for ICSI.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asada, H., Sueoka, K., Hashiba, T., Kuroshima, M., Kobayashi, N. and Yoshimura, Y. (2000) The effect of age and abnormal sperm count on the non-disjunction of spermatozoa. J. Assist. Reprod. Genet., 17, 5159.[ISI][Medline]
Bernardini, L., Martini, L., Geraedts, J.P.M., Hopman, A.H.N., Lanteri, S., Conte, N. and Capitanio, G.L. (1997) Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in-situ hybridization. Mol. Hum. Reprod., 3, 431438.[Abstract]
Bernardini, L., Borini, A., Preti, S., Conte, N., Flamigni, C., Capitanio, G.L. and Venturini, P.L. (1998) Study of aneuploidy in normal and abnormal germ cells from semen of fertile and infertile men. Hum. Reprod., 13, 34063413.[Abstract]
Bernardini, L., Gianaroli, L., Fortini, D., Conte, N., Magli, C., Cavani, S., Gaggero, G., Tindiglia, C., Ragni, N. and Venturini, P.L. (2000) Frequency of hyper-, hypohaploidy and diploidy in ejaculate, epididymal and testicular germ cells of infertile patients. Hum. Reprod., 15, 21652172.
Bonduelle, M., Liebaers, I., Deketelaere, V., Derde, M-P., Camus, M., Devroey, P. and Van Steirteghem, A. (2002) Neonatal data on a cohort of 2889 infants born after intracytoplasmic sperm injection (ICSI) (19911999) and of 2995 infants born after in vitro fertilization (IVF) (19831999). Hum. Reprod., 17, 671694.
Calogero, A.E., De Palma, A., Grazioso, C., Barone, N., Romeo, R., Rappazzo, G. and D'Agata, R. (2001a) Aneuploidy rate in spermatozoa of selected men with abnormal semen parameters. Hum. Reprod., 16, 11721179.
Calogero, A.E., De Palma, A., Grazioso, C., Barone, N., Burrello, N., Palermo, I., Gulisano, A., Pafumi, C. and D'Agata, R. (2001b) High sperm aneuploidy rate in unselected infertile patients and its relationship with intracytoplasmic sperm injection outcome. Hum. Reprod., 16, 14331439.
Clermont, Y. (1963) The cycle of the seminiferous pithelium in men. Am. J. Anat., 112, 3552.[ISI]
Colombero, L.T., Hariprashad, J.J., Ming, C.T., Rosenwaks, Z. and Palermo, G.D. (1999) Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome. Fertil. Steril., 72, 9096.[ISI][Medline]
Crabbé, E., Verheyen, G., Silber, S., Tournaye, H., Van de Helde, H., Goosens, A. and Van Steirteghem, A. (1998) Enzymatic digestion of testicular tissue may rescue the intracytoplasmic sperm injection cycle in some patients with non-obstructive azoospermia. Hum. Reprod., 13, 27912796.
Craft, I., Bennet, V. and Nicholson, N. (1993) Fertilising ability of testicular spermatozoa. Lancet, 342, 864868.
De Croo, I., Van der Elst, J., Everaert, K., De Sutter, P. and Dhont, M. (2000) Fertilization, pregnancy and embryo implantation rates after ICSI in cases of obstructive and non-obstructive azoospermia. Hum. Reprod., 15, 13831388.
Downie, S.E., Flaherty, S.P. and Matthews, C.D. (1997) Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridisation. Mol. Hum. Reprod., 3, 585598.[Abstract]
Egozcue, S., Blanco, J., Vendrell, J.M., Garcia, F., Veiga, A., Aran, B., Barri, P.N., Vidal, F. and Egozcue, J. (2000) Human male infertility: chromosome anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion. Hum. Reprod. Update, 6, 93105.
Fahmy, I., Mansour, R., Aboulghar, M., Serour, G., Kamal, A., Tawab, N.A., Ramzy, A.M. and Amin, Y. (1997) Intracytoplasmic sperm injection using surgically retrieved epididymal and testicular spermatozoa in cases of obstructive and non-obstructive azoospermia. Int. J. Androl., 20, 3744.[ISI][Medline]
Govaerts, I., Devreker, F., Koenig, I., Place, I., Van den Bergh, M. and Englert, Y. (1998) Comparison of pregnancy outcome after intracytoplasmic sperm injection and in-vitro fertilization. Hum. Reprod., 13, 15141518.[Abstract]
Huang, W.J., Lamb, D., Kim, E.D., de Lara, J., Lin, W. W., Lipshultz, L.I. and Bischoff, F.Z. (1999) Germ-cell non disjunction in testes biopsies of men with idiopathic infertility. Am. J. Hum. Genet., 64, 16381645.[ISI][Medline]
In't Veld, P.A., Brandenburg, H., Verhoeff, A., Dhont, M. and Los, F. (1995) Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet, 346, 773.[ISI][Medline]
In't Veld, P.A., Broekmans, F.J.M., deFrance, H.F., Pearson, P.L., Pieters, M.H.E.C. and van Kooij, R.J. (1997) Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa. Hum. Reprod., 12, 752754.[Abstract]
Joris, H., Nagy, Z., Van de Velde, H., De Vos, A. and Van Steirteghem, A. (1998) Intracytoplasmic sperm injection: laboratory set-up and injection procedure. Hum. Reprod., 13 (Suppl. 1), 7686.
Kovanci, E., Kovacs, T., Moretti, E., Vigue, L., Bray-Ward, P., Ward, D.C. and Huszar, G. (2001) FISH assessment of aneuploidy frequencies in immature human spermatozoa classified by the absence or presence of the cytoplasmic retention. Hum. Reprod., 16, 12091217.
Levron, J., Aviram-Goldring, A., Madgar, I., Raviv, G., Barkai, G. and Dor, J. (2001) Sperm chromosome abnormalities in men with severe male factor infertility who are undergoing in vitro fertilization with intracytoplasmic sperm injection. Fertil. Steril., 76, 479484.[ISI][Medline]
Liebaers, I., Bonduelle, M. and Van Assche, E. (1995) sex chromosome abnormalities after intracytoplasmic sperm injection [letter]. Lancet, 346, 1095.
Loft, A., Petersen, K., Erb, K., Mikkelsen, A.L., Grinsted, J., Hald, F., Hindkjaer, J., Nielsen, K.M., Lundstrøm, P., Gabrielsen, A. et al. (1999) A Danish national cohort of 730 infants born after intracytoplasmic sperm injection (ICSI) 19941997. Hum. Reprod., 14, 21432148.
Mansour, R., Kamal, A., Fahmy, I., Tawab, N., Serour, G.I. and Aboulghar, M.A. (1997) Intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia. Hum. Reprod., 12, 19741979.[Abstract]
Martin, R.H., Greene, C., Rademaker, A., Barclay, L., Ko, E. and Chernos, J. (2000) Chromosome analysis of spermatozoa extracted from testes of men with non-obstructive azoospermia. Hum. Reprod., 15, 11211124.
Martini, E., Speel, E.J.M., Geraedts, J.P.M., Ramaekers, F.C.S. and Hopman, A.H.N. (1995) Application of different in-situ hybridisation detection methods for human sperm analysis. Hum. Reprod., 10, 855861.[Abstract]
Moosani, N., Pattinson, H.A., Carter, M.D., Cox, D.M., Rademaker, A.W. and Martin, R.H. (1995) Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in-situ hybridization. Fertil. Steril., 64, 811817.[ISI][Medline]
Nagy, P., Liu, J., Cecile, J., Silber, S., Devroey, P. and Van Steirteghem, A. (1995) Using ejaculated, fresh, and frozenthawed epididymal and testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection. Fertil. Steril., 63, 808815.[ISI][Medline]
Nagy, Z.P., Joris, H., Verheyen, G., Tournaye, H., Devroey, P. and Van Steirteghem, A.C. (1998) Correlation between motility of testicular spermatozoa, testicular histology and the outcome of intracytoplasmic sperm injection. Hum. Reprod., 13, 890895.[Abstract]
Palermo, G.D., Schlegel, P.N., Hariprashad, J.J., Ergün, B., Mielnik, A., Zaninovic, N., Veeck, L.L. and Rosenwaks, Z. (1999) Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men. Hum. Reprod., 3, 741748.[Abstract]
Pang, M.G., Hoegerman, S.F., Cuticchia, A.J., Moon, S.Y., Doncel, G.F., Acosta, A.A. and Kearns, W.G. (1999) Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridisation in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum. Reprod., 14, 12661273.
Rives, N., Saint Clair, A., Mazurier, S., Sibert, L., Simeon, N., Joly, G. and Mace, B. (1999) Relationship between clinical phenotype, semen parameters and aneuploidy frequency in sperm nuclei of 50 infertile males. Hum. Genet., 105, 266272.[ISI][Medline]
Schoysman, R., Vanderzwalmen, P., Nijs, M., Segal, L., Segal-Bertin, G., Geerts, L., Van Roosendaal, E. and Schoysman-Deboeck, A. (1993) Pregnancy after fertilization of human testicular sperm. Lancet, 342, 1237.
Testart, J., Gautier, E., Brami, C., Rolet, F., Sedbon, E. and Thebault, A. (1996) Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities. Hum. Reprod., 11, 26092612.[Abstract]
Tournaye, H., Camus, M., Goossens, A., Liu, J., Nagy, P., Silber, S., Van Steirteghem, A.C. and Devroey, P. (1995) Recent concepts in the treatment of infertility because of non-obstructive azoospermia. Hum. Reprod., 10 (Suppl. 1), 115119.[ISI][Medline]
Tournaye, H., Liu, J., Nagy, P.Z., Camus, M., Goossens, A., Silber, S., Van Steirteghem, A.C. and Devroey, P. (1996) Correlations between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa. Hum. Reprod., 11, 127132.[Abstract]
Ushijima, C., Kumasako, Y., Kihaile, P.E., Hirotsuru, K. and Utsunomiya, T. (2000) Analysis of chromosomal abnormalities in human spermatozoa using multi-colour fluorescence in-situ hybridisation. Hum. Reprod., 15, 11071111.
Van Dyk, Q., Lanzendorf, S., Kolm, P., Hodgen, G.D. and Mahony, M.C. (2000) Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound. Hum. Reprod., 15, 15291536.
Van Golde, R., Boada, M., Veiga, A., Evers, J., Geraedts, J. and Barri, P. (1999) A retrospective follow-up study on intracytoplasmic sperm injection. J. Assisted Reprod. Genet., 16, 227232.[ISI][Medline]
Van Opstal, D., Los, F.J., Ramlakhan, S., Van Hemel, J.O., Van Den Ouweland, A.M.W., Brandenburg, H., Pieters, M.H.E.C., Verhoeff, A., Vermeer, M.C.S., Dhont, M. et al. (1997) Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection. Hum. Reprod., 12, 682686.[Abstract]
Van Steirteghem, A., Bonduelle, M., Devroey, P. and Liebaers, I. (2002) Follow-up of children born after ICSI. Hum. Reprod. Update, 8, 111116.
Vegetti, W., Van Assche, E., Frias, A., Verheyen, G., Bianchi, M.M., Bonduelle, M., Liebaers, I. and Van Steirteghem, A. (2000) Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridisation in infertile men. Hum. Reprod., 15, 351365.
Verheyen, G, De Croo, I., Tournaye, H., Pletincx, I., Devroey, P. and Van Steirteghem, A. (1995) Comparison of four mechanical methods to retrieve spermatozoa from testicular tissue. Hum. Reprod., 10, 29562959.[Abstract]
Viville, S., Mollard, R., Bach, M.L., Falquet, C., Gerlinger, P. and Warter, S. (2000a) Do morphological anomalies reflect chromosomal aneuploidies? Hum. Reprod., 15, 25632566.
Viville, S., Warter, S., Meyer, J.M., Wittemer, C., Loriot, M., Mollard, R. and Jacqmin, D. (2000b) Histological and genetic analysis and risk assessment for chromosomal aberration after ICSI for patients presenting with CBAVD. Hum. Reprod., 15, 16131618.
Wennerholm, U.B., Bergh, C., Hamberger, L., Westlander, G., Wikland, M. and Wood, M. (2000) Obstetric outcome of pregnancies following ICSI, classified according to sperm origin and quality. Hum. Reprod., 15, 11891194.
Submitted on February 20, 2002; accepted on May 14, 2002.