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Comparison of the sperm aneuploidy rate in severe oligozoospermic and oligozoospermic men and its relation to intracytoplasmic sperm injection outcome
Comparison of the sperm aneuploidy rate in severe oligozoospermic and oligozoospermic men and its relation to intracytoplasmic sperm injection outcome Punam Nagvenkar, M.Sc.,a,b Kusum Zaveri, M.D.,b and Indira Hinduja, M.D., Ph.D.a,b a
Jaslok Hospital and Research Centre, and b Inkus IVF Centre, Mehta Bhavan, Mumbai, India
Objective: To determine the incidence of disomy and diploidy for chromosomes 18, X, and Y in the sperm samples of severe oligozoospermic (⬍5 ⫻ 106 spermatozoa/mL) and oligozoospermic (5–20 ⫻ 106 spermatozoa/ mL) men undergoing intracytoplasmic sperm injection (ICSI) and to evaluate the influence of sperm aneuploidy on pregnancy outcome. Design: Prospective study. Setting: Infertility clinic and genetic laboratory. Patient(s): Fifteen patients with severe oligozoospermia, 15 patients with oligozoospermia, and 10 normal fertile donors. Intervention(s): Fluorescence in-situ hybridization (FISH) performed on sperm samples. Main Outcome Measure(s): The frequency of disomy and diploidy for chromosomes 18, X, and Y was analyzed using FISH, and the clinical outcome after ICSI was correlated. Result(s): Significantly greater frequencies of XY, YY disomy and diploidy were observed in severe oligozoospermic men compared with oligozoospermic and normozoospermic men. Although the fertilization rate was similar, the pregnancy rate was higher in the group with oligozoospermia versus severe oligozoospermia. Conclusion(s): This study demonstrated the presence of an elevated sperm aneuploidy rate in patients with low semen quality. Additionally, the data show a negative influence of sperm chromosome abnormalities on ICSI outcome. (Fertil Steril威 2005;84:925–31. ©2005 by American Society for Reproductive Medicine.) Key Words: Severe oligozoospermia, FISH, sperm aneuploidy, ICSI, pregnancy rate
Infertility affects about 15% of couples attempting pregnancy, with male factor infertility identified in approximately 50% of the cases (1). Intracytoplasmic sperm injection (ICSI) has been a boon to many childless couples, and it has revolutionized the treatment of male infertility (2) by allowing infertile men with severely compromised semen parameters to achieve fatherhood. However, concerns have been raised regarding the safety of this technique, as it bypasses the process of natural selection (3). Although a spermatozoon of normal morphologic features is selected and injected in the oocyte, normal morphologic features are not an absolute indicator of genetically normal sperm (4, 5). Thus, the chance of transmission of genetic abnormalities to the offspring is raised. Aberrant chromosome constitutions, such as reciprocal translocation, Robertsonian translocation, or Klinefelter syndrome, are known to be associated with male infertility (6). Infertile men with such abnormal chromosome complement are thus expected to have an increased frequency of abnormal spermatozoa and offspring (7). Recent reports demonstrate the presence of an increased frequency of abnormal Received January 19, 2005; revised and accepted April 19, 2005. Supported by Jaslok Hospital and Research Centre, project no. 336, Mumbai, India. Reprint requests: Indira Hinduja, M.D., Ph.D., Inkus IVF Centre, Mehta Bhavan, 311, Charni Road, Mumbai, 400 004, India (FAX: 91-2223862054; E-mail: [email protected]).
0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2005.04.048
chromosomal constitution in spermatozoa of infertile men in spite of a normal blood karyotype (8 –10). Several studies also describe an inverse association between the sperm aneuploidy rate and sperm concentration (11–13), motility (12), morphology (14 –17), and maturity (18). A review of the literature reveals the birth of chromosomally abnormal fetuses and children to men with an elevated frequency of sperm chromosome anomalies (19, 20). Moreover, an increased incidence of chromosomal abnormalities of paternal origin (21) in children conceived after ICSI compared with the general population has been reported (22–24). We undertook this study to investigate sperm aneuploidy in Indian males and its consequential effect on oocyte fertilization and/or embryonic development and pregnancy outcome. We report on the comparison of the incidence of aneuploidy frequencies in spermatozoa from severe oligozoospermic (sperm concentration ⬍5 ⫻ 106 /mL) and oligozoospermic (sperm concentration 5–20 ⫻ 106/mL) men. We also present an analysis of ICSI outcome in these infertile men. MATERIALS AND METHODS Thirty oligozoospermic patients participating in an ICSI program at Inkus IVF Centre, Mumbai, were prospectively recruited for evaluation of sperm aneuploidy by fluorescence in-situ hybridization (FISH). Informed consent was obtained
Fertility and Sterility姞 Vol. 84, No. 4, October 2005 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.
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from all the patients and donors before the sperm FISH analysis and ICSI. Institutional review board approval was obtained for this study. The patients were selected based on severe male factor infertility and past history of infertility for more than 2 years. The semen analysis was performed according to World Health Organization recommendations and standards (25). The patients were sorted according to the degree of oligozoospermia into two groups: 15 patients with severe oligozoospermia with a sperm count of ⬍5 ⫻ 106/mL and 15 patients with oligozoospermia with a sperm count of 5–20 ⫻ 106/mL. The age of the patients ranged from 30 to 42 years; mean ages were 33.4 years in the former group and 34.9 years in the latter. Ten normozoospermic male donors with mean age of 35 years (range: 32 to 41 years), normal semen parameters (sperm count ⬎20 ⫻ 106/mL), and proven fertility with children of ⬎2 years of age were included as controls. All the patients and controls had a normal 46,XY karyotype, and none had any past history of childhood disease, environmental exposure, radiation exposure, prescription drug usage, or presence of varicoceles that could account for their infertility. Ovarian Stimulation and ICSI Procedure Ovulation was induced in all the female partners by injecting 20 units of gonadotropin-releasing hormone analog (500 g; Lupride 4; Sun Pharmaceutical, Mumbai, India) subcutaneously from day 21 or 22 of the menstrual cycle. This was followed by administration of human menopausal gonadotropin (hMG; Pergonal 75; Serono SA, Geneva, Switzerland), 3 ampules or 4 ampules in women above 35 years of age from day 2 or day 3 of the menstrual cycle until optimal ovarian response was observed. When three or more follicles were ⬎18 mm in diameter, 10,000 IU of human chorionic gonadotropin (hCG; Profasi; Serono) was administered. Oocyte retrieval was carried out 34 to 36 hours after hCG administration with the help of vaginal ultrasonographyguided ovum pick-up. The surrounding cumulus and corona cells were removed by enzymatic hyaluronidase treatment, followed by mechanical denudation. The denuded oocytes were examined to assess the nuclear maturation, and only metaphase II oocytes with an extruded polar body were selected for microinjection. The ICSI procedure was carried out under Olympus IM70 inverted microscope (Olympus, Tokyo, Japan) at ⫻400 magnification using Hoffmann modulation optics equipped with manipulators (Narishige, Tokyo, Japan). Immediately before injection, sperm suspension was loaded next to the 10% polyvinylpyrrolidone (PVP; Medicult, Copenhagen, Denmark) droplet. Injection of oocytes was performed in microdroplets of HEPES-buffered medium (COOK IVF, Queensland, Australia). All the microdroplets were covered with lightweight paraffin oil. A single motile spermatozoon with apparently normal morphology was aspirated into the injection pipette from the sperm drop then transferred to the PVP 926
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droplet. The sperm was then immobilized by touching its tail with the injection pipette and then aspirated into the injection pipette. After securing the oocyte at 9 o’clock (with the polar body at 6 or 12 o’clock) with the holding pipette, the injection pipette was pushed through the zona pellucida into the ooplasm at the 3 o’clock position. A small amount of the cytoplasm was aspirated into the injection pipette and then expelled along with the spermatozoon. The injection pipette was withdrawn gently, and the oocyte was released from the holding pipette. After washing of the injected oocytes, they were cultured in microdrops of culture medium (COOK IVF) under lightweight paraffin oil. Fertilization was assessed after 16 to 18 hours by the observation of two distinct pronuclei. Embryo cleavage was evaluated after a further 24 and 48 hours of culture. A total of 30 ICSI cycles were performed in 30 patients, and the embryo transfer was done on day 3. The luteal phase was supplemented with progesterone (50 mg/mL IM, Gestone; Ferring Pharmaceuticals Pvt Ltd, Mumbai, India) from the day of ultrasonography-guided ovum pick-up. Pregnancy was confirmed by measuring serum -hCG concentration 2 weeks after the embryo transfer. The ICSI outcomes, including fertilization and pregnancy rates, were compared between the two groups. Sperm Fixation and Sperm Head Decondensation Semen samples were collected by masturbation after 3 days of abstinence on the day of ovum pick-up. Following liquefaction, an aliquot of the sperm samples were washed three times in phosphate buffered saline (PBS), pH 7.2, and centrifuged at 280 ⫻ g for 10 minutes; the sediment was then fixed in methanol/acetic acid (3:1). The fixed specimens were stored at ⫺20°C until further processing. Methanol/ acetic acid-fixed spermatozoa were spread on slides. The slides were washed in 2⫻ standard saline citrate (SSC; Sigma Chemical, St. Louis, MO) and incubated for 5 minutes at room temperature in 1 M Tris buffer, pH 9.5, containing 25 mM dithiothreitol (DTT; Sigma) (26). After decondensation, the slides were washed once in 2⫻ SSC, once in 1⫻ PBS and finally dehydrated through an ethanol series (70%, 85%, and 100%). FISH Procedure The chromosome enumeration DNA probes (CEP) (Vysis, Inc., Downers Grove, IL) used for this study were satellite probes for the centromeric regions for chromosomes 18 (D18Z1) and X (DXZ1), and a satellite probe for the heterochromatic region Yq12 (DYZ1) directly labeled with spectrum aqua, spectrum green, and spectrum orange fluorophores, respectively. The probe mixture consisted of 7 L of CEP hybridization buffer, 1 L of CEP 18-spectrum aqua, 1 L of CEP X-spectrum green, and 1 L of CEP Yspectrum orange. The FISH procedure was performed according to the protocol recommended by Vysis for directly labeled probes. That is, 10 L of the probe mixture was Vol. 84, No. 4, October 2005
applied to the specimen target area and covered with a coverslip. The coverslip was sealed with rubber cement, placed in the HYBrite machine (Vysis), and denatured at 75°C for 3 minutes, followed by hybridization at 37°C overnight. For the posthybridization washing, coverslips were removed, and the slides were immersed immediately in 0.4⫻ SSC/0.3% NP-40 (Igepal CA-630; Sigma) solution at 73 ⫾ 1°C for 2 minutes and then in 2⫻ SSC/0.1% NP-40 solution for 1 minute at room temperature. Finally, the slides were mounted in antifade medium containing 4=,6=-diamidino-2-phenylindole (DAPI) counterstain. Microscopy and Scoring Criteria The FISH preparations were evaluated using an Olympus BX60 epifluorescence microscope (Olympus) at ⫻1000 magnification equipped with appropriate filter sets (Vysis): single band pass filter DAPI, single-band pass filter green, single-band pass filter orange, triple-band pass filter DAPI/ green/orange, and single-band pass filter aqua. The images were captured using Cytovision chromosome and FISH analysis system (Applied Imaging Inc., San Jose, CA). A minimum of 2000 sperm nuclei per patient were scored. Scoring of sperm nuclei was done according to the criteria described by Mateizel et al. (27). 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, except for Y signals, which had to be separated by only half of the diameter of the signal because of the large size of the Y signal. Sperm nuclei were scored as disomic for a specific chromosome if two distinct signals were shown for that chromosome and one each of the simultaneously probed chromosomes. Sperm nuclei were scored as nullisomic if they showed no signal for a given chromosome, provided that the signal of the other chromosome tested was present. Sperm nuclei were considered diploid when an extra X or Y chromosome signal and two chromosome 18 signals were present. Statistical Analysis The frequency of disomy and diploidy for the analyzed chromosomes in men with severe oligozoospermia, oligozoospermia, and normozoospermia was compared using chisquare test. For the comparison of the fertilization and pregnancy rates after ICSI in the severe oligozoospermic and oligozoospermic men, the chi-square test was applied. P⬍.05 was considered statistically significant. Fertility and Sterility姞
RESULTS Triple-color FISH was performed in 15 men with severe oligozoospermia, 15 men with oligozoospermia, and 10 men with normozoospermia. A total of 90,700 spermatozoa were analyzed from 40 men included in the study. The results of the FISH analysis using DNA probes for chromosomes 18, X, and Y (Fig. 1) are shown in Table 1. A statistically significantly higher frequency of XY disomy was found in the severe oligozoospermia group (0.60%) than in the oligozoospermia group (0.22%, P⬍.001) or the control group (0.18%, P⬍.001). There was no statistically significant difference in the frequency of XY disomy between the oligozoospermia group and the control group. A more elevated frequency of YY disomy was observed in the severe oligozoospermic males (0.35%) than in the oligozoospermic males (0.15%, P⬍.001) or normozoospermic males (0.12%, P⬍.001). Oligozoospermic males and normozoospermic males did not show such statistically significant differences in the frequency. The frequency of XX disomy was 0.13%, 0.13%, 0.11% in patients with severe oligozoospermia, oligozoospermia, and normozoospermia, respectively. The frequency of 18 disomy was similar in all three groups (0.03%). Statistically significant differences in the mean frequency were not observed for XX disomy or 18 disomy among the three groups. The difference in the frequency of total diploidy was statistically significant in severe oligozoospermics (0.45%) compared with oligozoospermics (0.25%, P⬍.001) or normozoospermics (0.19%, P⬍.001). However, no statistically significant difference was observed in the frequency for oligozoospermics and normozoospermics. Fifteen ICSI cycles were performed in 15 couples each in the severe oligozoospermia and oligozoospermia group. The results of assisted reproductive procedures are detailed in Table 2. Although there was no statistically significant difference in the fertilization rate, the pregnancy rate was higher in the couples with oligozoospermic male partners versus severe oligozoospermic male partners (P⬍.05). There were four healthy live births in the group with oligozoospermic male partners and two in the group with severe oligozoospermic male partners; the remaining pregnancies are still ongoing. All the live births have been regularly observed by the pediatrician. DISCUSSION In the present study, we analyzed the relationship between the sperm aneuploidy rate and assisted reproduction outcome. To accomplish this, we performed triple-color FISH for chromosomes 18, X, and Y on sperm retrieved from 15 men with severe oligozoospermia, 15 men with oligozoospermia, and 10 donors with proven fertility. We found a statistically significantly higher frequency for XY disomy in spermatozoa from males with severe oligozoospermia compared with men with oligozoospermia and 927
FIGURE 1 Sperm FISH with probes for chromosome 18 (aqua), X (green), and Y (orange) showing XY disomic sperm (white arrow), YY disomic sperm (red arrow), XX disomic sperm (green arrow), diploid sperm (yellow arrow), and normal sperm (blue arrow).
Nagvenkar. Sperm aneuploidy and ICSI results. Fertil Steril 2005.
normozoospermia. Similarly, the frequency of YY disomy was higher in severe oligozoospermic men than in oligozoospermic and normozoospermic men. However, no statistically significant differences in the frequencies of XX disomy and 18 disomy were observed among the three groups. A statistically significant increase in the frequency for diploidy was seen in patients with severe oligozoospermia compared with oligozoospermia and normozoospermia. Thus, an inverse correlation between sperm aneuploidy rate and sperm count was observed. Several studies have reported similar relationship between sperm concentration and sperm chromosome abnormalities. Rives et al. (28) observed the mean frequency of YY and XY disomy was increased in spermatozoa of patients with low semen quality parameters more than in controls of proven fertility. Vegetti et al. (12) found increased incidence of XX, YY, and XY disomy and diploidy in 15 infertile men with 928
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abnormal semen parameters than in men with normal semen parameters. A study of 19 oligoasthenoteratozoospermia (OAT) patients using multicolor FISH for chromosomes 8, 12, 18, X, and Y showed that the rate of aneuploidy in the spermatozoa negatively correlated with sperm concentration (13). Ohashi et al. (10) detected significantly higher frequencies of XY disomy and diploidy in the severe oligozoospermic group compared with the oligozoospermic and normozoospermic groups. The study by Martin et al. (29) comparing the frequency of sperm chromosome abnormalities in men with mild, moderate, and severe oligozoospermia demonstrated a significant inverse correlation between the frequency of sperm chromosome abnormalities and sperm concentration. An increased frequency of pairing disruptions during meiosis resulting in meiotic arrest in infertile men was reported by Egozcue et al. (30). Disruption of spermatogenesis due to Vol. 84, No. 4, October 2005
TABLE 1 FISH results for chromosomes 18, X, and Y in men with normozoospermia (n ⴝ 10), severe oligozoospermia (n ⴝ 15), and oligozoospermia (n ⴝ 15).
Haploid X Haploid Y Nullisomy X/Y XY disomy XX disomy YY disomy Nullisomy 18 Disomy 18 Diploidy Total
Controls no. (%)
Severe oligozoospermic no. (%)
14,976 (49.5) 15,037 (49.7) 37 (0.12) 56 (0.18) 33 (0.11) 38 (0.12) 0 (0.00) 10 (0.03) 57 (0.19) 30,244
14,852 (49.1) 14,858 (49.1) 45 (0.15) 183 (0.60) 39 (0.13) 105 (0.35) 3 (0.01) 9 (0.03) 136 (0.45) 30,230
P value NS NS NS ⬍.001 NS ⬍.001 NS NS ⬍.001
Oligozoospermic no. (%) 14,966 (49.5) 14,979 (49.5) 42 (0.14) 68 (0.22) 39 (0.13) 46 (0.15) 1 (0.00) 9 (0.03) 76 (0.25) 30,226
P value NS NS NS NS NS NS NS NS NS
Note: Statistical analysis using 2 test. NS ⫽ not statistically significant. Nagvenkar. Sperm aneuploidy and ICSI results. Fertil Steril 2005.
meiotic arrest thus results in oligozoospermia or azoospermia. In particular, the sex chromosome bivalent is predisposed to pairing abnormalities as there is only one crossover in the pseudoautosomal region (31). We further evaluated the influence of sperm aneuploidy rate on the ICSI performance. To accomplish this, we divided the patients into two groups, patients with severe oligozoospermia and oligozoospermia, and studied the resulting ICSI outcome in these groups. There was no statistically significant difference in the fertilization rate among the patients in both the groups. However, aneuploidy affected the pregnancy rates as we observed a statistically significant higher pregnancy rate in the oligozoospermic group (with lower sperm aneuploidy rate) compared with the
severe oligozoospermic group (with higher sperm aneuploidy rate). A total aneuploidy rate of 33% to 74% in nine OAT patients undergoing ICSI was reported by Pang et al. (11). ICSI performed in 5 patients failed to establish any ongoing pregnancies. A study conducted by Pfeffer et al. (32) observed higher rates of diploidy and aneuploidy in 10 patients with OAT. Although the overall fertilization was 70%, a pregnancy rate of 20% was observed. Van Dyk et al. (33) found that a high incidence of sperm aneuploidy was associated with failure to achieve pregnancy through ICSI. An elevated sperm aneuploidy rate among the infertile couples with pregnancy failure after ICSI was observed by Calogero et al. (34). The study by Rubio et al. (35) demonstrated an
TABLE 2 ICSI outcome using spermatozoa from patients with severe oligozoospermia and oligozoospermia.
No. of patients No. of cycles No. oocytes retrieved No. of oocytes injected Fertilization rate No. of embryo transfers Pregnancy rate No. of abortions Ongoing pregnancy rate No. of live births
Severe oligozoospermia
Oligozoospermia
15 15 243 200 143/200 (71.5%) 15 5/15 (33.3%) 1 4/15 (26.7%)a 2
15 15 135 105 78/105 (74.3%) 15 11/15 (73.3%) 1 10/15 (66.7%) 4
Note: Statistical analysis using 2 test. a P⬍.05 versus oligozoospermia. Nagvenkar. Sperm aneuploidy and ICSI results. Fertil Steril 2005.
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association between low semen quality and decreased pregnancy and implantation rates and increased miscarriage rates. Furthermore, Burello et al. (36) showed that chromosomally abnormal sperm have a negative impact on ICSI outcome. The results of our study are in agreement with the previous studies. Intracytoplasmic sperm injection involves the selection of motile spermatozoa with normal morphologic characteristics. The possibility of the embryologist selecting a sperm with abnormal morphologic features would be low. However, Pfeffer et al. (32) demonstrated that the swim-up procedure does not completely eliminate genetically abnormal sperm. In addition, the study by Ryu et al. (4) suggested that sperm with normal morphologic features may not always have a normal complement of chromosomes. Other investigators have similarly suggested that normally shaped spermatozoa have an increased aneuploidy rate (5, 16). Thus, in the present study although selection of morphologically normal spermatozoon was implemented for micromanipulation, a higher total sperm aneuploidy rate could have been the cause for a lower pregnancy rate. The results of the current study indicate that, in spite of a normal blood karyotype, men with suboptimal semen quality have a high occurrence of sperm chromosome aneuploidy. Furthermore, an inverse relationship exists between pregnancy outcome and sperm aneuploidy rate. Thus, genetic evaluation of spermatozoa in infertile men undergoing assisted reproduction is highly recommended. In addition, proper genetic counseling regarding the possible risk of transmission of chromosomal abnormality to the offspring should be provided to the patients, and meticulous follow-up of babies born after ICSI is of utmost importance. REFERENCES 1. Bhasin S, De Kretser DM, Baker HWG. Clinical review 64: pathophysiology and natural history of male infertility. J Clin Endocrinol Metab 1994;79:1525–9. 2. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17– 8. 3. Chandley AC, Hargreave TB. Genetic anomaly and ICSI. Hum Reprod 1996;11:930 –2. 4. Ryu HM, Lin WW, Lamb DJ, Chuang W, Lipschultz LI, Bischoff FZ. Increased chromosome X, Y, and 18 nondisjunction in sperm from infertile patients that were identified as normal by strict morphology: implication for intracytoplasmic sperm injection. Fertil Steril 2001;76: 879 – 83. 5. Burrello N, Arcidiacono G, Vicari E, Asero P, Di Benedetto D, De Palma A, et al. Morphologically normal spermatozoa of patients with secretory oligo-astheno-teratozoospermia have an increased aneuploidy rate. Hum Reprod 2004;19:2298 –302. 6. Johnson MD. Genetic risks of intracytoplasmic sperm injection in the treatment of male infertility: recommendations for genetic counseling and screening. Fertil Steril 1998;70:397– 411. 7. Shi Q, Martin RH. Aneuploidy in human spermatozoa: FISH analysis in men with constitutional chromosomal abnormalities, and in infertile men. Reproduction 2001;121:655– 66. 8. McInnes B, Rademaker A, Greene CA, Ko E, Barclay L, Martin RH. Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men. Hum Reprod 1998;13:2787–90.
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et al. Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection. Fertil Steril 1999;72:472– 8. Van Dyk Q, Lanzendorf S, Kolm P, Hodgen GD, Mahony MC. Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound. Hum Reprod 2000;15:1529 –36. Calogero AE, De Palma A, Grazioso C, Barone N, Burrello N, Palermo I, et al. High sperm aneuploidy rate in unselected infertile patients and its relationship with intracytoplasmic sperm injection outcome. Hum Reprod 2001;16:1433–9. Rubio C, Gil-Salom M, Simon C, Vidal F, Rodrigo L, Minguez Y, et al. Incidence of sperm chromosomal abnormalities in a risk population: relationship with sperm quality and ICSI outcome. Hum Reprod 2001; 16:2084 –92. Burrello N, Vicari E, Shin P, Agarwal A, De Palma A, Grazioso C, et al. Lower sperm aneuploidy frequency is associated with high pregnancy rates in ICSI programmes. Hum Reprod 2003;18:1371– 6.
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Jaslok Hospital and Research Centre, and b Inkus IVF Centre, Mehta Bhavan, Mumbai, India
Objective: To determine the incidence of disomy and diploidy for chromosomes 18, X, and Y in the sperm samples of severe oligozoospermic (⬍5 ⫻ 106 spermatozoa/mL) and oligozoospermic (5–20 ⫻ 106 spermatozoa/ mL) men undergoing intracytoplasmic sperm injection (ICSI) and to evaluate the influence of sperm aneuploidy on pregnancy outcome. Design: Prospective study. Setting: Infertility clinic and genetic laboratory. Patient(s): Fifteen patients with severe oligozoospermia, 15 patients with oligozoospermia, and 10 normal fertile donors. Intervention(s): Fluorescence in-situ hybridization (FISH) performed on sperm samples. Main Outcome Measure(s): The frequency of disomy and diploidy for chromosomes 18, X, and Y was analyzed using FISH, and the clinical outcome after ICSI was correlated. Result(s): Significantly greater frequencies of XY, YY disomy and diploidy were observed in severe oligozoospermic men compared with oligozoospermic and normozoospermic men. Although the fertilization rate was similar, the pregnancy rate was higher in the group with oligozoospermia versus severe oligozoospermia. Conclusion(s): This study demonstrated the presence of an elevated sperm aneuploidy rate in patients with low semen quality. Additionally, the data show a negative influence of sperm chromosome abnormalities on ICSI outcome. (Fertil Steril威 2005;84:925–31. ©2005 by American Society for Reproductive Medicine.) Key Words: Severe oligozoospermia, FISH, sperm aneuploidy, ICSI, pregnancy rate
Infertility affects about 15% of couples attempting pregnancy, with male factor infertility identified in approximately 50% of the cases (1). Intracytoplasmic sperm injection (ICSI) has been a boon to many childless couples, and it has revolutionized the treatment of male infertility (2) by allowing infertile men with severely compromised semen parameters to achieve fatherhood. However, concerns have been raised regarding the safety of this technique, as it bypasses the process of natural selection (3). Although a spermatozoon of normal morphologic features is selected and injected in the oocyte, normal morphologic features are not an absolute indicator of genetically normal sperm (4, 5). Thus, the chance of transmission of genetic abnormalities to the offspring is raised. Aberrant chromosome constitutions, such as reciprocal translocation, Robertsonian translocation, or Klinefelter syndrome, are known to be associated with male infertility (6). Infertile men with such abnormal chromosome complement are thus expected to have an increased frequency of abnormal spermatozoa and offspring (7). Recent reports demonstrate the presence of an increased frequency of abnormal Received January 19, 2005; revised and accepted April 19, 2005. Supported by Jaslok Hospital and Research Centre, project no. 336, Mumbai, India. Reprint requests: Indira Hinduja, M.D., Ph.D., Inkus IVF Centre, Mehta Bhavan, 311, Charni Road, Mumbai, 400 004, India (FAX: 91-2223862054; E-mail: [email protected]).
0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2005.04.048
chromosomal constitution in spermatozoa of infertile men in spite of a normal blood karyotype (8 –10). Several studies also describe an inverse association between the sperm aneuploidy rate and sperm concentration (11–13), motility (12), morphology (14 –17), and maturity (18). A review of the literature reveals the birth of chromosomally abnormal fetuses and children to men with an elevated frequency of sperm chromosome anomalies (19, 20). Moreover, an increased incidence of chromosomal abnormalities of paternal origin (21) in children conceived after ICSI compared with the general population has been reported (22–24). We undertook this study to investigate sperm aneuploidy in Indian males and its consequential effect on oocyte fertilization and/or embryonic development and pregnancy outcome. We report on the comparison of the incidence of aneuploidy frequencies in spermatozoa from severe oligozoospermic (sperm concentration ⬍5 ⫻ 106 /mL) and oligozoospermic (sperm concentration 5–20 ⫻ 106/mL) men. We also present an analysis of ICSI outcome in these infertile men. MATERIALS AND METHODS Thirty oligozoospermic patients participating in an ICSI program at Inkus IVF Centre, Mumbai, were prospectively recruited for evaluation of sperm aneuploidy by fluorescence in-situ hybridization (FISH). Informed consent was obtained
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from all the patients and donors before the sperm FISH analysis and ICSI. Institutional review board approval was obtained for this study. The patients were selected based on severe male factor infertility and past history of infertility for more than 2 years. The semen analysis was performed according to World Health Organization recommendations and standards (25). The patients were sorted according to the degree of oligozoospermia into two groups: 15 patients with severe oligozoospermia with a sperm count of ⬍5 ⫻ 106/mL and 15 patients with oligozoospermia with a sperm count of 5–20 ⫻ 106/mL. The age of the patients ranged from 30 to 42 years; mean ages were 33.4 years in the former group and 34.9 years in the latter. Ten normozoospermic male donors with mean age of 35 years (range: 32 to 41 years), normal semen parameters (sperm count ⬎20 ⫻ 106/mL), and proven fertility with children of ⬎2 years of age were included as controls. All the patients and controls had a normal 46,XY karyotype, and none had any past history of childhood disease, environmental exposure, radiation exposure, prescription drug usage, or presence of varicoceles that could account for their infertility. Ovarian Stimulation and ICSI Procedure Ovulation was induced in all the female partners by injecting 20 units of gonadotropin-releasing hormone analog (500 g; Lupride 4; Sun Pharmaceutical, Mumbai, India) subcutaneously from day 21 or 22 of the menstrual cycle. This was followed by administration of human menopausal gonadotropin (hMG; Pergonal 75; Serono SA, Geneva, Switzerland), 3 ampules or 4 ampules in women above 35 years of age from day 2 or day 3 of the menstrual cycle until optimal ovarian response was observed. When three or more follicles were ⬎18 mm in diameter, 10,000 IU of human chorionic gonadotropin (hCG; Profasi; Serono) was administered. Oocyte retrieval was carried out 34 to 36 hours after hCG administration with the help of vaginal ultrasonographyguided ovum pick-up. The surrounding cumulus and corona cells were removed by enzymatic hyaluronidase treatment, followed by mechanical denudation. The denuded oocytes were examined to assess the nuclear maturation, and only metaphase II oocytes with an extruded polar body were selected for microinjection. The ICSI procedure was carried out under Olympus IM70 inverted microscope (Olympus, Tokyo, Japan) at ⫻400 magnification using Hoffmann modulation optics equipped with manipulators (Narishige, Tokyo, Japan). Immediately before injection, sperm suspension was loaded next to the 10% polyvinylpyrrolidone (PVP; Medicult, Copenhagen, Denmark) droplet. Injection of oocytes was performed in microdroplets of HEPES-buffered medium (COOK IVF, Queensland, Australia). All the microdroplets were covered with lightweight paraffin oil. A single motile spermatozoon with apparently normal morphology was aspirated into the injection pipette from the sperm drop then transferred to the PVP 926
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droplet. The sperm was then immobilized by touching its tail with the injection pipette and then aspirated into the injection pipette. After securing the oocyte at 9 o’clock (with the polar body at 6 or 12 o’clock) with the holding pipette, the injection pipette was pushed through the zona pellucida into the ooplasm at the 3 o’clock position. A small amount of the cytoplasm was aspirated into the injection pipette and then expelled along with the spermatozoon. The injection pipette was withdrawn gently, and the oocyte was released from the holding pipette. After washing of the injected oocytes, they were cultured in microdrops of culture medium (COOK IVF) under lightweight paraffin oil. Fertilization was assessed after 16 to 18 hours by the observation of two distinct pronuclei. Embryo cleavage was evaluated after a further 24 and 48 hours of culture. A total of 30 ICSI cycles were performed in 30 patients, and the embryo transfer was done on day 3. The luteal phase was supplemented with progesterone (50 mg/mL IM, Gestone; Ferring Pharmaceuticals Pvt Ltd, Mumbai, India) from the day of ultrasonography-guided ovum pick-up. Pregnancy was confirmed by measuring serum -hCG concentration 2 weeks after the embryo transfer. The ICSI outcomes, including fertilization and pregnancy rates, were compared between the two groups. Sperm Fixation and Sperm Head Decondensation Semen samples were collected by masturbation after 3 days of abstinence on the day of ovum pick-up. Following liquefaction, an aliquot of the sperm samples were washed three times in phosphate buffered saline (PBS), pH 7.2, and centrifuged at 280 ⫻ g for 10 minutes; the sediment was then fixed in methanol/acetic acid (3:1). The fixed specimens were stored at ⫺20°C until further processing. Methanol/ acetic acid-fixed spermatozoa were spread on slides. The slides were washed in 2⫻ standard saline citrate (SSC; Sigma Chemical, St. Louis, MO) and incubated for 5 minutes at room temperature in 1 M Tris buffer, pH 9.5, containing 25 mM dithiothreitol (DTT; Sigma) (26). After decondensation, the slides were washed once in 2⫻ SSC, once in 1⫻ PBS and finally dehydrated through an ethanol series (70%, 85%, and 100%). FISH Procedure The chromosome enumeration DNA probes (CEP) (Vysis, Inc., Downers Grove, IL) used for this study were satellite probes for the centromeric regions for chromosomes 18 (D18Z1) and X (DXZ1), and a satellite probe for the heterochromatic region Yq12 (DYZ1) directly labeled with spectrum aqua, spectrum green, and spectrum orange fluorophores, respectively. The probe mixture consisted of 7 L of CEP hybridization buffer, 1 L of CEP 18-spectrum aqua, 1 L of CEP X-spectrum green, and 1 L of CEP Yspectrum orange. The FISH procedure was performed according to the protocol recommended by Vysis for directly labeled probes. That is, 10 L of the probe mixture was Vol. 84, No. 4, October 2005
applied to the specimen target area and covered with a coverslip. The coverslip was sealed with rubber cement, placed in the HYBrite machine (Vysis), and denatured at 75°C for 3 minutes, followed by hybridization at 37°C overnight. For the posthybridization washing, coverslips were removed, and the slides were immersed immediately in 0.4⫻ SSC/0.3% NP-40 (Igepal CA-630; Sigma) solution at 73 ⫾ 1°C for 2 minutes and then in 2⫻ SSC/0.1% NP-40 solution for 1 minute at room temperature. Finally, the slides were mounted in antifade medium containing 4=,6=-diamidino-2-phenylindole (DAPI) counterstain. Microscopy and Scoring Criteria The FISH preparations were evaluated using an Olympus BX60 epifluorescence microscope (Olympus) at ⫻1000 magnification equipped with appropriate filter sets (Vysis): single band pass filter DAPI, single-band pass filter green, single-band pass filter orange, triple-band pass filter DAPI/ green/orange, and single-band pass filter aqua. The images were captured using Cytovision chromosome and FISH analysis system (Applied Imaging Inc., San Jose, CA). A minimum of 2000 sperm nuclei per patient were scored. Scoring of sperm nuclei was done according to the criteria described by Mateizel et al. (27). 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, except for Y signals, which had to be separated by only half of the diameter of the signal because of the large size of the Y signal. Sperm nuclei were scored as disomic for a specific chromosome if two distinct signals were shown for that chromosome and one each of the simultaneously probed chromosomes. Sperm nuclei were scored as nullisomic if they showed no signal for a given chromosome, provided that the signal of the other chromosome tested was present. Sperm nuclei were considered diploid when an extra X or Y chromosome signal and two chromosome 18 signals were present. Statistical Analysis The frequency of disomy and diploidy for the analyzed chromosomes in men with severe oligozoospermia, oligozoospermia, and normozoospermia was compared using chisquare test. For the comparison of the fertilization and pregnancy rates after ICSI in the severe oligozoospermic and oligozoospermic men, the chi-square test was applied. P⬍.05 was considered statistically significant. Fertility and Sterility姞
RESULTS Triple-color FISH was performed in 15 men with severe oligozoospermia, 15 men with oligozoospermia, and 10 men with normozoospermia. A total of 90,700 spermatozoa were analyzed from 40 men included in the study. The results of the FISH analysis using DNA probes for chromosomes 18, X, and Y (Fig. 1) are shown in Table 1. A statistically significantly higher frequency of XY disomy was found in the severe oligozoospermia group (0.60%) than in the oligozoospermia group (0.22%, P⬍.001) or the control group (0.18%, P⬍.001). There was no statistically significant difference in the frequency of XY disomy between the oligozoospermia group and the control group. A more elevated frequency of YY disomy was observed in the severe oligozoospermic males (0.35%) than in the oligozoospermic males (0.15%, P⬍.001) or normozoospermic males (0.12%, P⬍.001). Oligozoospermic males and normozoospermic males did not show such statistically significant differences in the frequency. The frequency of XX disomy was 0.13%, 0.13%, 0.11% in patients with severe oligozoospermia, oligozoospermia, and normozoospermia, respectively. The frequency of 18 disomy was similar in all three groups (0.03%). Statistically significant differences in the mean frequency were not observed for XX disomy or 18 disomy among the three groups. The difference in the frequency of total diploidy was statistically significant in severe oligozoospermics (0.45%) compared with oligozoospermics (0.25%, P⬍.001) or normozoospermics (0.19%, P⬍.001). However, no statistically significant difference was observed in the frequency for oligozoospermics and normozoospermics. Fifteen ICSI cycles were performed in 15 couples each in the severe oligozoospermia and oligozoospermia group. The results of assisted reproductive procedures are detailed in Table 2. Although there was no statistically significant difference in the fertilization rate, the pregnancy rate was higher in the couples with oligozoospermic male partners versus severe oligozoospermic male partners (P⬍.05). There were four healthy live births in the group with oligozoospermic male partners and two in the group with severe oligozoospermic male partners; the remaining pregnancies are still ongoing. All the live births have been regularly observed by the pediatrician. DISCUSSION In the present study, we analyzed the relationship between the sperm aneuploidy rate and assisted reproduction outcome. To accomplish this, we performed triple-color FISH for chromosomes 18, X, and Y on sperm retrieved from 15 men with severe oligozoospermia, 15 men with oligozoospermia, and 10 donors with proven fertility. We found a statistically significantly higher frequency for XY disomy in spermatozoa from males with severe oligozoospermia compared with men with oligozoospermia and 927
FIGURE 1 Sperm FISH with probes for chromosome 18 (aqua), X (green), and Y (orange) showing XY disomic sperm (white arrow), YY disomic sperm (red arrow), XX disomic sperm (green arrow), diploid sperm (yellow arrow), and normal sperm (blue arrow).
Nagvenkar. Sperm aneuploidy and ICSI results. Fertil Steril 2005.
normozoospermia. Similarly, the frequency of YY disomy was higher in severe oligozoospermic men than in oligozoospermic and normozoospermic men. However, no statistically significant differences in the frequencies of XX disomy and 18 disomy were observed among the three groups. A statistically significant increase in the frequency for diploidy was seen in patients with severe oligozoospermia compared with oligozoospermia and normozoospermia. Thus, an inverse correlation between sperm aneuploidy rate and sperm count was observed. Several studies have reported similar relationship between sperm concentration and sperm chromosome abnormalities. Rives et al. (28) observed the mean frequency of YY and XY disomy was increased in spermatozoa of patients with low semen quality parameters more than in controls of proven fertility. Vegetti et al. (12) found increased incidence of XX, YY, and XY disomy and diploidy in 15 infertile men with 928
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abnormal semen parameters than in men with normal semen parameters. A study of 19 oligoasthenoteratozoospermia (OAT) patients using multicolor FISH for chromosomes 8, 12, 18, X, and Y showed that the rate of aneuploidy in the spermatozoa negatively correlated with sperm concentration (13). Ohashi et al. (10) detected significantly higher frequencies of XY disomy and diploidy in the severe oligozoospermic group compared with the oligozoospermic and normozoospermic groups. The study by Martin et al. (29) comparing the frequency of sperm chromosome abnormalities in men with mild, moderate, and severe oligozoospermia demonstrated a significant inverse correlation between the frequency of sperm chromosome abnormalities and sperm concentration. An increased frequency of pairing disruptions during meiosis resulting in meiotic arrest in infertile men was reported by Egozcue et al. (30). Disruption of spermatogenesis due to Vol. 84, No. 4, October 2005
TABLE 1 FISH results for chromosomes 18, X, and Y in men with normozoospermia (n ⴝ 10), severe oligozoospermia (n ⴝ 15), and oligozoospermia (n ⴝ 15).
Haploid X Haploid Y Nullisomy X/Y XY disomy XX disomy YY disomy Nullisomy 18 Disomy 18 Diploidy Total
Controls no. (%)
Severe oligozoospermic no. (%)
14,976 (49.5) 15,037 (49.7) 37 (0.12) 56 (0.18) 33 (0.11) 38 (0.12) 0 (0.00) 10 (0.03) 57 (0.19) 30,244
14,852 (49.1) 14,858 (49.1) 45 (0.15) 183 (0.60) 39 (0.13) 105 (0.35) 3 (0.01) 9 (0.03) 136 (0.45) 30,230
P value NS NS NS ⬍.001 NS ⬍.001 NS NS ⬍.001
Oligozoospermic no. (%) 14,966 (49.5) 14,979 (49.5) 42 (0.14) 68 (0.22) 39 (0.13) 46 (0.15) 1 (0.00) 9 (0.03) 76 (0.25) 30,226
P value NS NS NS NS NS NS NS NS NS
Note: Statistical analysis using 2 test. NS ⫽ not statistically significant. Nagvenkar. Sperm aneuploidy and ICSI results. Fertil Steril 2005.
meiotic arrest thus results in oligozoospermia or azoospermia. In particular, the sex chromosome bivalent is predisposed to pairing abnormalities as there is only one crossover in the pseudoautosomal region (31). We further evaluated the influence of sperm aneuploidy rate on the ICSI performance. To accomplish this, we divided the patients into two groups, patients with severe oligozoospermia and oligozoospermia, and studied the resulting ICSI outcome in these groups. There was no statistically significant difference in the fertilization rate among the patients in both the groups. However, aneuploidy affected the pregnancy rates as we observed a statistically significant higher pregnancy rate in the oligozoospermic group (with lower sperm aneuploidy rate) compared with the
severe oligozoospermic group (with higher sperm aneuploidy rate). A total aneuploidy rate of 33% to 74% in nine OAT patients undergoing ICSI was reported by Pang et al. (11). ICSI performed in 5 patients failed to establish any ongoing pregnancies. A study conducted by Pfeffer et al. (32) observed higher rates of diploidy and aneuploidy in 10 patients with OAT. Although the overall fertilization was 70%, a pregnancy rate of 20% was observed. Van Dyk et al. (33) found that a high incidence of sperm aneuploidy was associated with failure to achieve pregnancy through ICSI. An elevated sperm aneuploidy rate among the infertile couples with pregnancy failure after ICSI was observed by Calogero et al. (34). The study by Rubio et al. (35) demonstrated an
TABLE 2 ICSI outcome using spermatozoa from patients with severe oligozoospermia and oligozoospermia.
No. of patients No. of cycles No. oocytes retrieved No. of oocytes injected Fertilization rate No. of embryo transfers Pregnancy rate No. of abortions Ongoing pregnancy rate No. of live births
Severe oligozoospermia
Oligozoospermia
15 15 243 200 143/200 (71.5%) 15 5/15 (33.3%) 1 4/15 (26.7%)a 2
15 15 135 105 78/105 (74.3%) 15 11/15 (73.3%) 1 10/15 (66.7%) 4
Note: Statistical analysis using 2 test. a P⬍.05 versus oligozoospermia. Nagvenkar. Sperm aneuploidy and ICSI results. Fertil Steril 2005.
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association between low semen quality and decreased pregnancy and implantation rates and increased miscarriage rates. Furthermore, Burello et al. (36) showed that chromosomally abnormal sperm have a negative impact on ICSI outcome. The results of our study are in agreement with the previous studies. Intracytoplasmic sperm injection involves the selection of motile spermatozoa with normal morphologic characteristics. The possibility of the embryologist selecting a sperm with abnormal morphologic features would be low. However, Pfeffer et al. (32) demonstrated that the swim-up procedure does not completely eliminate genetically abnormal sperm. In addition, the study by Ryu et al. (4) suggested that sperm with normal morphologic features may not always have a normal complement of chromosomes. Other investigators have similarly suggested that normally shaped spermatozoa have an increased aneuploidy rate (5, 16). Thus, in the present study although selection of morphologically normal spermatozoon was implemented for micromanipulation, a higher total sperm aneuploidy rate could have been the cause for a lower pregnancy rate. The results of the current study indicate that, in spite of a normal blood karyotype, men with suboptimal semen quality have a high occurrence of sperm chromosome aneuploidy. Furthermore, an inverse relationship exists between pregnancy outcome and sperm aneuploidy rate. Thus, genetic evaluation of spermatozoa in infertile men undergoing assisted reproduction is highly recommended. In addition, proper genetic counseling regarding the possible risk of transmission of chromosomal abnormality to the offspring should be provided to the patients, and meticulous follow-up of babies born after ICSI is of utmost importance. REFERENCES 1. Bhasin S, De Kretser DM, Baker HWG. Clinical review 64: pathophysiology and natural history of male infertility. J Clin Endocrinol Metab 1994;79:1525–9. 2. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17– 8. 3. Chandley AC, Hargreave TB. Genetic anomaly and ICSI. Hum Reprod 1996;11:930 –2. 4. Ryu HM, Lin WW, Lamb DJ, Chuang W, Lipschultz LI, Bischoff FZ. Increased chromosome X, Y, and 18 nondisjunction in sperm from infertile patients that were identified as normal by strict morphology: implication for intracytoplasmic sperm injection. Fertil Steril 2001;76: 879 – 83. 5. Burrello N, Arcidiacono G, Vicari E, Asero P, Di Benedetto D, De Palma A, et al. Morphologically normal spermatozoa of patients with secretory oligo-astheno-teratozoospermia have an increased aneuploidy rate. Hum Reprod 2004;19:2298 –302. 6. Johnson MD. Genetic risks of intracytoplasmic sperm injection in the treatment of male infertility: recommendations for genetic counseling and screening. Fertil Steril 1998;70:397– 411. 7. Shi Q, Martin RH. Aneuploidy in human spermatozoa: FISH analysis in men with constitutional chromosomal abnormalities, and in infertile men. Reproduction 2001;121:655– 66. 8. McInnes B, Rademaker A, Greene CA, Ko E, Barclay L, Martin RH. Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men. Hum Reprod 1998;13:2787–90.
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et al. Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection. Fertil Steril 1999;72:472– 8. Van Dyk Q, Lanzendorf S, Kolm P, Hodgen GD, Mahony MC. Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound. Hum Reprod 2000;15:1529 –36. Calogero AE, De Palma A, Grazioso C, Barone N, Burrello N, Palermo I, et al. High sperm aneuploidy rate in unselected infertile patients and its relationship with intracytoplasmic sperm injection outcome. Hum Reprod 2001;16:1433–9. Rubio C, Gil-Salom M, Simon C, Vidal F, Rodrigo L, Minguez Y, et al. Incidence of sperm chromosomal abnormalities in a risk population: relationship with sperm quality and ICSI outcome. Hum Reprod 2001; 16:2084 –92. Burrello N, Vicari E, Shin P, Agarwal A, De Palma A, Grazioso C, et al. Lower sperm aneuploidy frequency is associated with high pregnancy rates in ICSI programmes. Hum Reprod 2003;18:1371– 6.
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