Human Reproduction, Vol. 15, No. 5, 1136-1139,
May 2000
© 2000 European Society of Human Reproduction and Embryology
Chromosome analysis of human spermatozoa from an oligoasthenozoospermic carrier for a 13;14 Robertsonian translocation by their injection into mouse oocytes
1 Department of Obstetrics and Gynecology, Jichi Medical School, Minamikawachimachi, Tochigi, 329-0498 and 2 Institute for central clinic, Minamikawachimachi, Tochigi, 329-0431 Japan
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Abstract |
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We present a case of a 46,XY der(13;14) Robertsonian translocation carrier whose spermatozoa were karyotyped after injection into mouse oocytes. Fresh semen samples as well as recovered samples were used. There was no significant difference in the survival rate of mouse oocytes (fresh: 78.1% versus frozen: 81.7%), activation rate (fresh: 84.0% versus frozen: 90.6%), fertilization rate (fresh: 72.0% versus thawing of frozen: 76.5%) between fresh or frozen spermatozoa. Metaphase chromosome spreads from 45 spermatozoa were analysed. The frequency of spermatozoa that were chromosomally unbalanced with respect to the translocation was 8.9%, and the frequency of abnormalities unrelated to translocation was 4.4%. An excess of spermatozoa with balanced chromosomes was observed: compared with normal, 23 (51.1%) versus 16 (35.6%) respectively; but this segregation difference was not statistically significant (
2 = 0.9, P > 0.3). After genetic counselling with the carrier and his partner, intracytoplasmic sperm injection treatment was performed. Healthy female and male infants were delivered at 36 weeks gestation via a Caesarean section. Both babies were carriers for the balanced Robertsonian translocations detected for prenatal diagnosis at 16 weeks gestation. The present study demonstrates that patients can be given further information about the proportion of the spermatozoa which carry a chromosomal abnormality.
Key words: ICSI/karyotype/oligoasthenozoospermia/Robertsonian translocation/spermatozoa
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Introduction |
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Since normal offspring were born following intracytoplasmic sperm injection (ICSI) (Palermo et al., 1992
), the procedure has become the method of choice for treating severe male factor infertility (Van Steirteghem et al., 1993; Nagy et al., 1995). ICSI bypasses sperm–oocyte membrane fusion, permitting conception with spermatozoa carrying chromosomal or other genetic defects. Infertile males presenting with severe oligozoospermia and azoospermia might carry a chromosomal abnormality detected by lymphocyte karyotype analysis (Mau et al., 1997; Yoshida et al., 1997; Meschede et al., 1998). The chromosomal constitution of human spermatozoa has been studied following the technique of in-vitro penetration of zona-free hamster oocytes (Rudak et al., 1978; Kamiguchi and Mikamo, 1986; Martin et al., 1991). Since that system requires millions of motile and acrosome-reacted spermatozoa, the hamster test is inappropriate for the patients with severe male factor. Recently, cytogenetic analysis of spermatozoa using fluorescent in-situ hybridization (FISH) has proven useful (Han et al., 1992; Holmes and Martin, 1993; Rousseaux et al., 1995). However, FISH is suitable for analysis of a few specific chromosomal sites simultaneously, and it is difficult to examine the detail of an abnormal site without a specific probe. The incidences of major chromosomal abnormalities were 6.9% for the patients with severe oligozoospermia (Yoshida et al., 1997). Almost all such chromosomal abnormalities were balanced, such as reciprocal translocations, Robertsonian translocations and inversions (Mau et al., 1997; Yoshida et al., 1997; Meschede et al., 1998). Up to now, chromosome analysis of spermatozoa from a male heterozygous for a Robertsonian translocation has been elucidated using human sperm hamster oocyte assay (Balkan and Martin, 1983; Pellestor et al., 1987; Martin, 1988; Pellestor, 1990; Martin et al., 1992; Syme and Martin, 1992). However, all subjects had normal semen parameters. Rybouchkin et al. demonstrated the use of mouse oocytes for analysis of the oocyte-activating capacity and karyotyping of human spermatozoa (Rybouchkin et al., 1995). The current report is the first to analyse the karyotype of human spermatozoa from a severe oligoasthenozoospermic carrier for a 13;14 Robertsonian translocation by ICSI into mouse oocytes. |
Materials and methods |
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Semen characteristics and preparation for injection
A 32 year old man was found to be heterozygous for a Robertsonian translocation, 45,XY, der(13;14), (q10;q10),inv(9), following a 2 year history of infertility associated with oligoasthenozoospermia (concentration, 2.0–6.0 x106/ml; motile, 19–35%). The spermatozoa that were injected into mouse oocytes were recovered from either fresh or cryopreserved samples. Cryopreservation of the spermatozoa was performed according to Prins and Weidel (1986) using TEST–yolk buffer (Irvine Scientific, Santa. Ana, USA) as the cryoprotectant. After washing in 80% Percoll and M2 medium (Sigma Chemical Co., St Louis, MO, USA), fresh or frozen–thawed spermatozoa were transferred to a drop of M2 medium containing 5% polyvinylpyrrolidone (PVP) (360 kDa; Sigma) under mineral oil immediately before injection.
Preparation of oocytes
B6D2F1 female mice, 8–12 weeks old, were induced to superovulate by s.c. injection of 8 IU of pregnant mare's serum gonadotrophin followed by i.p. injection of 8 IU human chorionic gonadotrophin (HCG) 48 h later. Oocytes were collected from the oviducts about 16 h after HCG injection. They were freed from cumulus cells by 5 min incubation in M2 medium containing 100 IU/ml hyaluronidase (Sigma). The oocytes were rinsed thoroughly in M2 medium and kept in M16 medium (Sigma) for up to 1 h at 37°C under 5% CO2 in air.
Sperm injection into oocytes
The injection of mouse oocytes with human spermatozoa was performed at 17–23 h post-HCG administration. Oocytes that had extruded their polar body (metaphase II) were transferred into a small drop of M2 medium under mineral oil at 17°C for 5 min before injection by ICSI at 17°C. The injection pipette had an inner diameter of 4.5–5.0 µm, and the holding pipette had an inner and outer diameter of 8 and 130 µm respectively. The patient's motile spermatozoa were immobilized by two to three piezo-pulses (Kimura and Yanagimachi, 1995) before injection into mouse oocytes using a Piezo-driven micromanipulator (Prima Meat Packers, Tsuchiura-city, Ibaraki-ken, Japan). After injection, the oocytes were kept in M2 medium at room temperature (24–25°C) for ~20 min before transfer to M16 medium at 37°C under 5% CO2 in air.
Cytological analysis
Following culture, the oocytes with one pronucleus or two pronuclei were transferred in M2 medium with 0.2 µg/ml nocodazole (Sigma) to prevent mouse and human pronuclei fusing and forming the common metaphase plate. They were checked every 30 min and were incubated until 1 h after the observation of pronuclear disappearance. The oocytes were fixed using a modification of the air-drying method for chromosome preparation from mouse eggs described by Tarkowski (1966). Fertilized oocytes were placed in a 1% sodium citrate hypotonic solution for 90 s. The swollen zygotes were transferred into a solution of 5:1:4 methanol:acetic acid:distilled water for a few seconds and transferred to the centre of a slide. Two to three drops of 3:3:1 methanol:acetic acid:distilled water solution were dropped onto the swollen zygotes until the oolemma was broken and the oocyte cytoplasm was dispersed. These procedures were done under direct visual control using a dissecting microscope. As soon as the ooplasm dispersed, the slide was dehydrated. Unfertilized oocytes were also fixed at 30 h after ICSI.
G-banding of chromosomes
Fresh metaphase spreads were incubated at 50°C in air for 48 h to age the chromosomes. The slides were treated with a 0.125% trypsin solution in a Ca2+- and Mg2+-free phosphate buffer solution at pH 6.8 for 40 s at room temperature (24–25°C). The slides were washed with a Ca2+- and Mg2+-free phosphate buffer solution in 50% ethanol, and treated with 5% Gimsa stain solution for 4 min.
Statistics
The differences in survival and activation rates between the two categories of spermatozoa injected were statistically analysed using the 2-test.
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Results |
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Mouse oocyte survival, activation and fertilization rates are summarized in Table I
. Of 25 oocytes which survived the injection with fresh motile spermatozoa, 21 (84.0%) were activated. Eighteen (72%) of activated oocytes formed the 2-pronuclear stage (2PN). Of the 85 oocytes that survived the injection with frozen–thawed spermatozoa, 77 (90.6%) were activated, and 65 (76.5%) activated formed 2PN. There was no significant difference in survival, activation and fertilization rates. Forty-five metaphase chromosome spreads could be analysed from the fertilized oocytes. Figure 1 shows a G-banding karyotype pattern from human spermatozoa decondensed within the mouse ooplasm. The types of meiotic segregations that were observed are presented in Table II. The overall sex ratio was 23X:21Y, which is not significantly different from the expected 1:1 segregation (2 = 0.09, P > 0.9). An excess of spermatozoa with a balanced translocation was observed: 23 with `balanced' (51.1%) versus 16 with normal (35.6%)
(Figure 2A); but this difference in segregation was not statistically significant (2 = 0.9, P > 0.3). The proportion of unbalanced spermatozoa was 8.9% (four out of 45). Three spermatozoa displayed missing chromosome 13 and one spermatozoon an extra chromosome 14
(Figure 2B). Abnormalities unrelated to the translocation were identified at a rate of 4.4% (two out of 45).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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With an incidence of about 1:1000, Robertsonian translocations are the most common structural chromosome anomalies in man, and have been identified at a rate of 3.4% of the patients with oligozoospermia (Tharapel et al., 1985
). Carriers of Robertsonian translocations are phenotypically normal, but are at an increased risk for spontaneous abortions and chromosomally unbalanced offspring. It is difficult to establish the meiotic segregation in translocation carriers based on information from live births and prenatal diagnosis. Up to now, 550 sperm chromosome complements from six carriers of Robertsonian translocations have been analysed by the hamster egg–human sperm fusion technique (Balkan and Martin, 1983; Pellestor et al., 1987; Martin, 1988; Pellestor, 1990; Martin et al., 1992; Syme and Martin., 1992), and 1116 spermatozoa chromosome complements from one carrier were analysed by two-colour FISH (Rousseaux et al., 1995).
FISH is performed easier and faster for large numbers of spermatozoa. However, it is difficult to reveal the details of some chromosomal abnormalities, i.e. reciprocal carriers, inversions etc. The present method is certainly worth applying to these patients because of yielding G-banding of chromosomes without using any specific probes. The majority of spermatozoa displayed alternate chromosome segregation. Similarly, analysis of meiotic prophase cells in heterozygous carriers of different Robertsonian translocations have a preferential cis-configuration of the meiotic trivalent structure (Vidal et al., 1982; Luciani et al., 1984; Templado et al., 1984; Rosenmann et al., 1985). This predominance of cis-configuration supports alternate segregation. In the present study, the incidence of adjacent chromosome segregation was observed in 8.9% of the spermatozoa, and that of abnormalities unrelated to the translocation was 4.4%. These findings agree with those of meiotic and epidemiological studies. The incidence of unbalanced constellations determined by sperm chromosome analyses is distinctly higher than that observed in prenatal diagnosis and neonates (Daniel et al., 1980; Boue and Gallano, 1984). The hypothesis of a 1:1 segregation ratio of normal and balanced gametes was based on family studies of carriers for Robertsonian translocations between D-group chromosomes der (D;D) (Evans et al., 1978). With one exception (Balkman and Martin, 1983), normal and balanced sperm chromosome complements were found in almost equal numbers in all studies, utilizing the hamster egg–human sperm fusion assay. However, in couples with paternal carriers for a (13;14) Robertsonian translocation there is an excess of offspring with balanced karyotypes compared to those with normal karyotypes (Boue and Gallano, 1984). In the present study, an excess of balanced spermatozoa was observed, but this difference was not a significant deviation from a 1:1 segregation ratio (2 = 0.9, P > 0.3). Therefore, the present study suggests that normal and balanced sperm chromosome complements may not always be equal. After consultation with the patients, ICSI treatment was performed. Five oocytes were retrieved and four of them fertilized and cleaved. The three highest quality embryos were transferred and two implanted. Both fetuses were carriers of a balanced Robertsonian translocation detected by prenatal diagnosis at 16 weeks gestation. The pregnancy was uneventful and a healthy female infant with birthweight of 2335 g and a healthy male infant with birthweight of 2420 g were delivered at 36 weeks gestation by Caesarean section. In specific patients chromosomal abnormalities are thought to be a major contributor to the genetic risks of infertility treatment by ICSI (Mau et al., 1997; Yoshida et al., 1997; Meschede et al., 1998). In terms of genetic counselling prior to ICSI treatment, the chromosomal analysis of human spermatozoa is thought to be feasible for such patients.
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Acknowledgments |
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The authors would like to thank Dr Simon Fishel for his advice during the preparation of this paper.
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Notes |
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3 To whom correspondence should be addressed
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References |
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Submitted on November 24, 1999; accepted on February 7, 2000.
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