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| Correspondence to: M R Guichaoua, Laboratoire de Biologie de la Reproduction, Hôpital de la Conception, 147 Bd Baille 13005 Marseilles, France (e-mail: mguichaoua{at}ap-hm.fr). |
| Received for publication October 3, 2006; accepted for publication March 21, 2007. |
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Abstract |
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Key words: Chromosomal abnormalities, meiosis, FISH, nuclear surface
Aside from patients exhibiting 100% MNSs, infertile patients exhibiting both large-headed spermatozoa (32% to 64%) and various nuclear sizes have been reported (Yurov et al, 1996). In all cases, total teratozoospermia was always detected. Until now, patients with mild percentages of MNSs have been poorly explored. Viville et al (2000) and Vicari et al (2003) reported that the presence of MNSs is associated with an increased incidence of chromosomal abnormalities. Here, we report a detailed semen analysis of 4 infertile human males undergoing an ICSI program and showing various percentages of sperm abnormality. One of them had a predominance of regular large-headed spermatozoa and a family history of reproductive failure. The spermatozoa of all patients were investigated by FISH and semiautomated analysis of nuclear surface. ICSI was performed with selected normal-sized and typical spermatozoa.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Patients 4, 5, 6, and 11 were 27, 36, 36, and 37 years old, respectively. In all cases, the men presented with problems of infertility (Table 1). No direct consanguinity was observed between the relatives of the men. Analysis of the pedigree of patient 11 showed that 5 siblings died during the first days of life from unknown causes. Interestingly, a brother of the patient also suffered from primary infertility but refused to submit to semen analysis. Three patients (patients 4, 5, 6) had a normal karyotype. For patient 11, 50 mitoses were analysed, 2 had the karyotype 47,XY,+21, 1 mitosis was 47,XXY, and 1 mitosis was 45,X. Complementary study of 20 mitosis showed chromosomal loss on 4 mitoses (10-, 15-, 18-, 21-). Interphase FISH with specific probes for chromosomes X, 8, 13, 18, and 21 showed that 5% of the nuclei presented with either a monosomy for chromosome 18 or a trisomy for chromosome 21. This percentage was considered nonsignificantly increased by cytogeneticists, and a constitutional mosaic was eliminated.
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All women had normal menstrual cycles, hysterosalpingography, and hormonal assessment. The women's ages were 24, 37, 38, and 35 for patients 4, 5, 6, and 11, respectively.
Semen Analysis
Sperm concentration and motility were evaluated according to the World
Health Organization guidelines
(1999). One hundred
spermatozoa were evaluated for each patient according to strict criteria
described by Kruger et al
(1986).
Semiautomated Analysis of Sperm Nuclear Surface and its Statistical Treatment
Analysis of sperm nuclear surface was performed for the patients, 3 healthy
controls, and a control patient with 100% MNSs (control 100%). Healthy
controls were selected among fertile men showing normal spermiograms.
Papanicoloaou-stained sperm were analysed using a Zeiss Axioplan microscope
(63x magnification; Zeiss, Oberkocher, Germany). Images were digitized
with a Sony 3CCD XC077 camera (Sony, Tokyo, Japan) and analyzed with the IPS
32 software (Samba Technologies, Grenoble, France). A specific macro was
designed: a constant greytone threshold distinguished sperm heads from
background, and the operator controlled the limit between head base and
flagellum. The nuclear surface was automatically calculated in
µm2. Measures were analyzed with an analytical software (STAT
2005; Samba) to fit the maximum amount of measurements in a normal
distribution. Results for this operation are given as mean, SD, and percentage
of measures that fit in the model.
FISH on Sperm
Written informed consents were obtained for each patient before analysis.
Cryopreserved semen samples from the patients were thawed and washed twice in
0.01 mol/L Tris, pH 8, for 10 minutes at 600 x g. The in vitro
decondensation procedure was performed as previously described
(Yurov et al, 1996). In brief,
the sperm preparations were treated with 2N NaOH for 2 minutes at room
temperature to induce sperm nuclear decondensation.
Three-color and dual-color FISH experiments were performed using Qbiogen classical satellite probe (Qbiogen, Illkirch, France). Three-color FISH experiments were performed with specific probes for chromosomes 9, X, and Y (Chromosome X Alpha Satellite DXZ1 Probe/Red, Chromosome Y Classical Satellite DYZ1 Probe/Green, Chromosome 9 Classical Satellite Probe/Green and Red; Abbott Molecular Inc, Des Plaines, Ill). Dual-color FISH experiments were performed with specific probes for chromosomes 8 and 18 (Chromosome 8 Alpha Satellite D8Z2 Probe/Green, Chromosome 18 Alpha Satellite D18Z1 Probe/Red; Abbott). FISH was performed according to the protocol recommended by the manufacturer. Preparations were counterstained with 4,6-diamino-2-phenylindole (DAPI). Slides were screened using a 100x objective on a Zeiss Axioskop microscope equipped with a fluorescein isothiocyanate (FITC)/rhodamine/DAPI triple band-pass filter. Signals of a specific color were considered multiple only when separated by at least 1 signal diameter. The efficiency of hybridization was evaluated from the DAPI-stained spermatozoa without FISH signals.
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Statistical Analysis
Sperm Head Surface Analysis—
To determine whether multiple sperm subpopulations (according to nuclear
size) coexisted within an ejaculate, a maximum threshold of 20
µm2 was applied and the remaining population was tested for
normal distribution by Shapiro-Wilk W, Anderson-Darling, Martinez-Iglewicz,
and Kolmogorov-Smirnov tests. Whenever the remaining population showed a
normal distribution, the existence of 2 sperm populations was assumed.
FISH on Spermatozoa—
To compare the difference between sex chromosome and autosome
nondisjunction, a 
2 test was used.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Sperm Head Surfaces Analysis
Characteristics of measured spermatozoal surfaces for each control/patient
are presented in Table 2. Means
of healthy controls (1, 2, and 3) were 13.5, 11.9, and 13.4 µm2
with low dispersion (ie, SDs were 2.8, 1.9, and 1.9 µm2,
respectively). Measurements for control 100% had an elevated mean (30.4
µm2) and a widely spread distribution (SD, 9.8
µm2). We observed that the distributions of the measured
surfaces for the 4 patients had overall characteristics that were intermediate
between those of normal and 100% macronuclear controls. The means were 16,
18.4, 18.7, and 11.8 µm2 for patients 4, 5, 6, and 11,
respectively, with SDs of 5.9, 6.1, 6.3, and 4.2 µm2. The
observation of the frequency histograms for each surface
(Figure 2) shows a shift toward
elevated values for patients 5 and 11 but not for healthy controls. Indeed,
the percentages of spermatozoa whose surfaces were higher than 20
µm2 represented only 2.7% (10/366), 0.5% (2/416), and 0% (0/277)
of the total for controls 1, 2, and 3 whereas they accounted for 20% (44/220),
32.2% (55/171), 35.2% (38/108), and 4.4% (42/946) in patients 4, 5, 6, and 11,
respectively. The presence of a subpopulation of MNSs for 2 patients was
assessed in the following manner. Measures for controls showed a normal
distribution whereas measures for the patients did not (the normality tests
used are listed in "Material and Methods"). However, after
suppressing values above 20 µm2, the remaining populations
showed normal distribution for patients 4 and 5, with means of 13.7 ± 3
µm2 and 15 ± 2.5 µm.2 This was not observed
in patients 6 and 11 because of the low number of measures above the
threshold. Therefore, we concluded that, for patients 4 and 5, 2 populations
of spermatozoa coexisted (normal and oversized).
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FISH on Sperm
The mean efficiency of hybridization was 99.2% (range, 96.4% to 100%). The
percentage of chromosomal abnormalities was first evaluated on all spermatozoa
despite their size and then on apparently normal-sized spermatozoa
(nonmacronuclear). Totals of 1430, 2811, 1955, and 3981 sperm nuclei were
scored from patients 4, 5, 6, and 11, respectively.
Three-Color FISH— High percentages of chromosomal abnormalities were found in the 4 patients (51.4%, 43.6%, 71.7%, and 25.6% in patients 4, 5, 6, and 11, respectively) (Table 3). These values are greater than the percentages observed in 3 controls (total abnormalities, 0.9%, 0.3%, and 0.5%, respectively). A great heterogeneity in the distribution of the chromosomes was found in the 4 patients. Nevertheless, diploidy was the prevalent abnormality in patient 11 (64.5% of the chromosomal abnormalities vs 33.2%, 23.6%, and 25.3%, for patients 4, 5, and 6, respectively) whereas complex aneuploidies prevailed in other patients.
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Various types of diploidies were present with respect to the sex chromosomes (ie, either 99XY, 99XX, or 99YY). Nevertheless, for patients 4, 5, and 6, nondisjunction at the first meiotic division (99XY) (12.4%, 7.3%, and 14.1% for patients 4, 5, and 6, respectively) prevailed over nondisjunction at the second meiotic division (99XX or 99YY) (4.7%, 2.9%, and 4.0% for patients 4, 5, and 6, respectively). Concerning patient 11, nondisjunction was prevalent at the second meiotic division (9.9% vs 6.6% of nondisjunction at the first meiotic division).
In the 4 patients, the total percentage of chromosome abnormalities with probes specific for chromosomes X,Y and 9 was higher than the mean percentage of MNSs (Tables 1 and 4): 51.4% vs 29.5%, 43.6% vs 22%, 71.7% vs 49.7%, and 25.6% vs 19% for patients 4, 5, 6, and 11, respectively. The mean percentage of aneuploid normal-sized spermatozoa in patients was significantly higher than those observed in the controls (3.5% vs 0.5%, P < .001); patient 11 had particularly few aneuploid normal-sized spermatozoa (1.1% in patient 11 vs 12.3%, 4.5%, and 3.7% in patients 4, 5, and 6, respectively).
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Dual-color FISH— High percentages of chromosomal abnormalities were found (54.5%, 37.6%, 61.8%, and 11.1% for patients 4, 5, 6, and 11, respectively) (Table 4). These values were greater than the percentages observed in the 3 controls (0.7%, 0.5%, and 0.8%, respectively). As with 3-color FISH, few spermatozoa had more than 46 chromosomes in patient 11 compared with the 3 others. The mean rate of aneuploid normal-sized spermatozoa was significantly higher than those observed in the controls (2.8% vs 0.7%, P < .001); this rate is normal in patient 11 (0.4% vs 5.3%, 3.1%, and 2.4% in patients 4, 5, and 6, respectively).
ICSI Cycles
Seven ICSIs were performed for these 4 patients. Thirty-two metaphase II
oocytes were retrieved and injected, and 21 diploid zygotes were obtained. The
mean fertilization rate (diploid zygotes/metaphase II oocytes) was not
different from the normal values obtained in our laboratory (65.6% vs 36%).
Notably, however, this rate was lower for patients 4, 5, and 6 (50%, 40%, and
50%, respectively) than for patient 11 (83.3%). The cleavage and transfer
rates (diploid embryos/diploid zygotes) were 90.5% (19/21) and 100% (7/7),
respectively. The mean number of transferred embryos per transfer was not
different from usual values of our laboratory (1.7 [12/7] vs 1.8). One
clinical pregnancy was obtained for patient 4, leading to the delivery of a
healthy girl. The clinical pregnancy rate per transfer was lower (14.3%)
compared with the mean rate of our center (30%). Nevertheless, the number of
patients is too limited for a statistical analysis.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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FISH on sperm also revealed in all patients high frequencies of chromosomal abnormalities (Tables 3 and 4) compared with the frequencies of MNSs (Table 1), suggesting that apparently normal-sized spermatozoa presented aneuploidy. Indeed, evaluation of the aneuploidy rate of apparently normal-sized spermatozoa (see "Results") showed that these spermatozoa, which could be used for ICSI, have significantly higher abnormal karyotype than controls but with interindividual variations, leading to a high risk of chromosomally abnormal embryos. We compared the results of FISH with semiautomated analysis of the sperm nuclear surface. This last approach enabled the visualization of an increase of the mean nuclear sperm surface for patients 4, 5, and 6 and above all in the SD of the 4 patients with either a spreading of the histogram toward the great value (patients 6 and 11) or a second subpopulation of oversized spermatozoa (patients 4 and 5) (Figure 2). This method also shows the existence of various sizes of spermatozoa from the apparently normal-sized to fourfold increased nuclear spermatozoa (Figure 2). Consequently, several spermatozoa with moderate size increase may be classified as normal sized during the assessment of sperm morphology by light microscopy analysis or during the selection of spermatozoa for ICSI.
The present study points out that mild rates of MNSs significantly impair fertility and constitute a risk for embryos. Nevertheless, ICSI was performed on selected typical spermatozoa in the 4 patients. Although the mean fertilization rate was normal in comparison with the normal values of our center (65.6% vs 63%), only patient 11 had a normal fertilization rate (83.3%). The other 3 patients displayed relatively low fertilization rates (50%, 40%, and 50%, for patients 4, 5, and 6, respectively). This discrepancy could be related to the low rate of chromosomal abnormalities in the apparently normal-sized spermatozoa in patient 11 (1.1% and 0.4% with 3-color and dual-color FISH, respectively). Moreover, in this limited experience with mild levels of MNSs, the implantation and ongoing pregnancy rates were low (14.3%). Only 1 pregnancy was obtained, in the couple in which the wife was the youngest (24 years old versus 35, 36, and 37 years old) (Table 1). Further studies will be necessary to confirm the low fertilization rate in these couples.
In conclusion, this study emphasizes that mild phenotypes of MNSs reduce the fertility and may have consequences on the embryos. Low fertilization and pregnancy rates may be due to the high incidence of aneuploidy in the apparently normal-sized spermatozoa that may be used for ICSI (patients 4, 5 and 6). Yet, chromosomal imbalance in the embryos could directly result from the gene mutation (patient 11). Indeed, a mutation in a gene that plays a role in the function and regulation of both mitotic and meiotic cycles could lead to the failure of chromosomal segregation in an embryo that initially has a normal karyotype, leading to embryo developmental arrest or perinatal death. Although the factor involved in this syndrome is unknown, we suggest conducting FISH on spermatozoa and genetic counseling before ICSI for infertile couples when the percentage of MNSs reaches 20% in at least 1 spermiogram. The monitoring of possible pregnancies is difficult to define. We think that several ultrasound examinations must be recommended in case of ongoing pregnancy, whereas PGD is arguable. Indeed, Kahraman et al (1999, 2004) reported a possible beneficial effect of eliminating chromosomal abnormal embryos with PGD on abortion rates in patients with high percentages of MNSs whereas the success of PGD has yet to be proven in aneuploidy screening (Shahine and Cedars, 2006).
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Acknowledgments |
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Footnotes |
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