Cytogenetic investigation of fetuses andinfants conceived through intracytoplasmicsperm injection

Ryan Lam, B.Sc.,a Sai Ma, Ph.D,a Wendy P. Robinson, Ph.D.,b Theresa Chan, B.Sc.,a

and Basil Ho Yuen, M.B.a

Vancouver Hospital and Health Sciences Centre and the British Columbia Research Institute for Children andWomen’s Health, Vancouver, British Columbia, Canada

Objective: To determine the incidence of aneuploidy among fetuses and infants conceived through intracy-toplasmic sperm injection (ICSI) in our clinic using umbilical cord blood samples.

Design: Follow-up study of the cytogenetic outcome of ICSI pregnancies.

Setting: University-based IVF clinic.

Patient(s): Forty-six couples who underwent ICSI and conceived.

Intervention(s): Umbilical cord blood was taken after delivery of the infant for analysis. Samples ofchorionic villi and chorion were taken for studies on the spontaneous abortuses. Amniocentesis was performedfor couples that chose prenatal diagnosis.

Main Outcome Measure(s): The cytogenetic chromosomal status of the pregnancy outcome.

Result(s): Fifty pregnancies and 55 live births were recorded, with nine spontaneous abortions. Of 43separate umbilical cord blood samples analyzed, 1 abnormality (2%) was found, 45, XX,�21. Nine birthswent through prenatal diagnosis alone, with four accepting both forms of analysis—no abnormalities werefound. Origin of abnormality was established in two spontaneous abortion cases (45, XO and 45, XY,�21),and the maternal chromosome was lost in both cases.

Conclusion(s): Using umbilical cord blood obtained after birth, we obtained karyotype results from 78% ofthe ICSI population in our clinic. Combined with results from five additional cases that underwent prenataldiagnosis but not umbilical cord blood sampling, a chromosomal result was obtained in 87% of our ICSIpopulation. The use of umbilical cord blood for cytogenetic analysis substantially improves the ability todetermine rates of chromosomal abnormalities in newborns produced via ICSI clinics. (Fertil Steril� 2001;76:1272–5. ©2001 by American Society for Reproductive Medicine.)

Key Words: Intracytoplasmic sperm injection (ICSI), umbilical cord blood, incidence of chromosomalabnormality, spontaneous abortions, origin of chromosomal abnormality

Since the birth of the first baby derived fromintracytoplasmic sperm injection (ICSI) by Pal-ermo et al. in 1992 (1), there has been muchconcern over the safety of this technique. In-jecting a single spermatozoon through the pro-tective barriers of the oocyte and depositing itdirectly into the cytoplasm to achieve fertiliza-tion could contribute to the increased rates ofchromosomal abnormality seen in studies per-formed around the world. The publication ofthe successful use of immature sperm in theICSI procedure to achieve fertilization and vi-able embryos has added to this concern. Onepossibility for an increased risk in this popula-tion is the recent findings that severely infertile

men have an elevated incidence of aneuploidyin their sperm. Many studies have addressedthis concern, with most showing no increase inmajor congenital malformations comparedwith that found in the general population.There was, however, an increase in chromo-somal abnormalities, most often involving thesex chromosomes (1).

Earlier studies evaluating ICSI usually re-quired the participants to undergo mandatoryprenatal diagnosis (1). However, it has beenobserved that given a choice, most couples inICSI decline prenatal diagnosis because of theincreased risk of losing their pregnancy (1). Todate, there have been no studies that have used

Received January 29,2001; revised andaccepted June 4, 2001.Reprint requests: Sai Ma,Ph.D., Department ofObstetrics andGynecology, Room 313,Willow Pavilion, 855 W12th Avenue, Vancouver,British Columbia, CanadaV5Z 1M9 (FAX: 604-875-5124; E-mail:sai@interchange.ubc.ca).a Department of Obstetricsand Gynecology, Universityof British Columbia,Vancouver, BritishColumbia, Canada.b Department of MedicalGenetics, University ofBritish Columbia,Vancouver, BritishColumbia, Canada.

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umbilical cord blood to measure the rate of abnormalitiesamong ICSI babies. We feel that this would more accuratelyrepresent the population of infants derived from ICSI andwould complement the data that have already been reportedin the literature using data from prenatal diagnosis.

Furthermore, an additional route of study is to determinethe origin of an abnormality in the infants or the products ofconception in the event of a spontaneous loss. We recentlydemonstrated a maternal origin for the abnormality in twospontaneous abortuses. These findings indicate that morpho-logically normal oocytes used in assisted fertilization proce-dures may also harbor significant abnormalities. By bypass-ing the natural selection barriers that may prevent anabnormal oocyte from being fertilized, genetic abnormalitiesassociated with ICSI may have a maternal as well as apaternal origin. Only through larger studies can it be trulyestablished whether an increase of both paternal and mater-nally derived abnormalities may be present in this popula-tion.

In this study, we closely observed all pregnancies estab-lished through ICSI in terms of the cytogenetic outcome.This objective was achieved using three methods of analysis:[1] prenatal diagnosis, [2] cytogenetic study of postnatalumbilical cord blood samples, and [3] cytogenetic study ofspontaneous abortuses. If any chromosomal abnormality wasdetected, further investigation regarding the origin of theabnormality was conducted.

MATERIALS AND METHODS

After the ICSI program was established in December1995, couples who suffered from male factor infertility, hadprevious failed IVF cycles, or had IVF cycles with lowfertilization rates were referred to our ICSI clinic. Universityof British Columbia Ethics Committee approval was ob-tained before initiating this study. At this clinic, the couplesare informed of several options for analyzing the cytogeneticstatus of their child should they become pregnant through theICSI program: prenatal diagnosis, postnatal umbilical cordblood analysis, and spontaneous abortion analysis (should amiscarriage occur). If an abnormality is found, the patientsare contacted and presented with the option of donating theirblood to determine the origin of the abnormality. Coupleswho chose to have amniocentesis done are also offered theoption of having a cytogenetic analysis of the umbilical cordblood done after the infant is born. All cytogenetic analyseswere performed using standard techniques.

Amniocentesis was performed on any couple who pro-vided informed consent to have this procedure done. Thesecouples were referred to the British Columbia Women’sHospital, where the amniocentesis was performed accordingto standard protocols. The sample was then sent to theBritish Columbia Women’s Hospital Cytogenetics Depart-ment for culturing and G-band karyotyping. Umbilical cord

blood samples (2 mL) were collected postnatally by theattending physician. Harvesting and G-banding was thenperformed on the sample to analyze the cytogenetic consti-tution of the sample. In the case of spontaneous abortion,samples of the chorion and chorionic villi were sent to theclinical cytogenetics lab for culturing and determination ofthe karyotype. For molecular analysis of spontaneous abor-tions, DNA from peripheral blood samples of both parentswas extracted for analysis. Cytogenetic examination of theabortus employed samples of chorionic villi and chorion.DNA typing of parents, villi, and chorion was done usingPCR amplification of highly polymorphic microsatellitemarkers. Primers were obtained from Research Genetics Inc.(Huntsville, AL). Each sample was run on a 0.4-mm-thick6% polyacrylamide/50% urea gel and visualized by silverstaining.

RESULTS

The ICSI program in our clinic was established in De-cember 1995 and since then has shown very promisingresults. The data are summarized in Table 1. There were 50established pregnancies that resulted from the program dur-ing the study period (1997–1999; Table 1). A total of 55infants were born, including 29 singletons, 10 twins, and 2triplets, whereas 9 pregnancies ended in miscarriage. Themean age of the mothers in the study was 35.3 years (range,24–43 years), which is within the age range of mothersusing standard IVF procedures. The sex ratio of the infantswas 29:25 (male:female)—one birth was of unknown sexbecause the parents could not be contacted; however, thisdifference was not statistically significant. Two of the chil-dren among the live births were born with major congenitalmalformations. One was born with a renal malformation(hydroureter, hydronephrosis), and one was born with con-genital heart malformations (pulmonary atresia, hypoplasticright heart). Subsequent cytogenetic analysis of these twochildren revealed normal karyotypes.

Nine couples opted for prenatal diagnosis by amniocen-tesis; four of these couples decided also to have postnatalcord blood samples analyzed to ensure accuracy of thekaryotype. No abnormalities were found in this group, andfive male and four female infants resulted. Although only afew couples chose prenatal diagnosis, there were 43 individ-ual postnatal umbilical cord blood samples collected andanalyzed from the 55 live births. Within the group of um-bilical cord blood samples, there were 23 male and 20 femaleinfants. One abnormality, a cytogenetic constitution of 47,XX,�21, was found in this group.

There were nine spontaneous losses during the studyinterval. The mean age of the mothers experiencing a losswas 36 (range, 32–41 years). Five of the abortuses under-went cytogenetic study, and two were found to have anabnormal karyotype; the first abortus had a 45, XO consti-

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tution, and the second abortus had a 45, XY,�21 constitu-tion. Subsequent analysis of these two cases revealed a lossof a maternal chromosome in each case. In the case of themonosomy X, using the markers AR and FMR-1 clearlydemonstrated a lack of a maternal X allele. The geneticmarkers DXS102, DXS1226, and DXS543 were also tested,but the results were uninformative. With the monosomy 21case, the markers D21S11, D21S212, and D21S259 allowedidentification of the missing maternal 21 alleles, whereasD21S269, D21S120, D21S1899, and D21S265 gave unin-formative results.

DISCUSSION

This study employs a more representative method ofdetermining incidence of chromosomal abnormalities inchildren born through ICSI with the use of primarily post-natal umbilical cord blood samples to represent the ICSIpopulation. Cord blood samples could be obtained in 78% ofthe population undergoing the procedure, which is muchhigher than the percentage of cord blood samples obtained instudies relying only on prenatal diagnoses (54.5%) (1). Cou-ples who chose ICSI to treat their infertility problems willfrequently refuse prenatal diagnosis because of the increasein risk of losing their child. The lack of enthusiasm towardprenatal diagnosis seems to follow a trend reported in othercenters (1). In our study, only nine couples (16%) acceptedprenatal diagnosis by amniocentesis to detect potential ab-normalities in their unborn child. The use of umbilical cordblood, in addition to prenatal results, thus yields a betterestimate for the incidence of chromosomal abnormalitiesamong pregnancies from the ICSI program.

Overall, the results have shown a total incidence of an-euploidy of 2% (95% CI: 0.4%–11.8%) in children bornthrough this clinic. Data in the literature suggest a 0.6%

incidence of chromosomal abnormality (including sex chro-mosome aneuploidy) expected in the general populationusing prenatal diagnosis (2). Although our sample size wastoo small to detect any difference, the rate we observed wassimilar to that found in other studies (1, 3). When the resultsfrom umbilical cord blood analysis and prenatal diagnosisare combined, the incidence of abnormality in our clinic isapproximately 1.9% (95% CI: 0.4%–10.7%), with the totalnumber of umbilical cord blood and prenatal diagnosis casesrepresenting 87% of all babies derived from our clinic.

The abnormalities found in these infants can be accountedfor by the nature of ICSI itself. It may also be due to anadvanced maternal age. It estimated that a 40-year-oldwoman has at least three times the rate of chromosomalabnormalities as a 30-year-old woman (4). There has been nostudy to date that confirms or denies an association betweennormal-looking sperm (according to the WHO criteria) and anormal cytogenetic constitution. Selection of a spermato-zoon for use in ICSI therefore could conceivably result inchoosing an abnormal sperm, thus accounting for the in-crease in the incidence of children born with a chromosomalabnormality (1). The lack of sex chromosome abnormalitiesin our population was likely only a consequence of our smallsample size as compared with the other studies (3).

The one abnormality found among the karyotypes ofinfants was a 47, XX,�21. The origin of this abnormalitycannot be certain because of the parents declining furtherinvestigation. Although it is possible that an abnormal spermselected for ICSI resulted in a trisomic embryo, trisomy 21 isrelatively common in natural conceptuses. Like other auto-somal trisomies, the maternally derived trisomy 21 may berelated to maternal age. Livebirths with trisomy 21 occur in�1% of women age 40 but in only 0.15% for a 30-year-old(4). In addition, there has been evidence from our group as

T A B L E 1

Obstetrical outcome of pregnancies derived from ICSI from 1997–1999.

Parameter Total no. Total analyzed Umbilical cord blood (n) Prenatal diagnosis (n)

Couples who conceivedin ICSI program

46 — — —

Pregnancies 50 — — —Miscarriages 9 5 — —

Live births 55 52b 43b 9b

Singletons 29 29 22 7Twins 10 (20 infants) 10 9 1Triplets 2 (6 infants) 1 1 0Male 29a 29 23 5Female 25a 25 20 4Major malformations 2 1 1 0

a One couple could not be contacted—as a result, the sex of the infant is unknown, but umbilical cord blood was not collected an therefore did not affectthe results of the study.b Four of the nine couples opted for both forms of analysis—prenatal diagnosis and cytogenetic study of postnatal umbilical cord blood.

Lam. Cytogenetic analysis of ICSI infants. Fertil Steril 2001.

1274 Lam et al. Cytogenetic analysis of ICSI infants Vol. 76, No. 6, December 2001

well as from other investigators to suggest that many of theabnormalities could be due to maternal error at meiosis, asthe majority of aneuploidies generally are (5, 6).

Among the five spontaneous losses analyzed, two werefound to harbor abnormalities. One of them was a 45,XY,�21 (7). Only eight other cases of monosomy 21 havebeen reported in the literature, which indicates the rareoccurrence of this type of abnormality among spontaneousabortuses as well as infants (8). Results on the 45, X casealso reveal a maternal origin. Although these surprisingresults are the first cases in ICSI to be found, the concept ofthe oocyte being the carrier of the abnormality is understand-able. Studies from our group as well as others have beenperformed that have detected a 15%–20% incidence of an-euploidy in morphologically normal human unfertilized oo-cytes (5, 6). These studies not only indicate that oocytes cancontain chromosomal abnormalities but also that ICSI canfertilize these aneuploid oocytes, thus possibly contributingto the overall incidence of aneuploidy seen in the ICSIpopulation.

The findings of this study have significant implicationsfor the use of ICSI. They add to the current body of knowl-edge and provide new information on maternal origins ofabnormalities in pregnancies derived from ICSI. Althoughsignificance above the general population’s rate of abnor-mality cannot be determined in our study, the rate we founddoes not seem to differ from the incidence suggested byother studies in the literature. When quoted to the patients,this risk estimate is usually acceptable for these couples, whohave no alternative to having their own biological children.

More important, it stresses the importance of combining bothresults from umbilical cord blood samples and prenatal di-agnosis samples for a more accurate representation of theICSI population when determining rates of abnormality.

Acknowledgments: The authors thank the British Columbia Cancer AgencyCytogenetics Laboratory, the nurses and physicians at the UBC IVF clinic,the physicians and nurses who aided in the collection of umbilical cordblood, and the clinicians and staff of the clinical cytogenetic laboratory atthe British Columbia Research Institute for Children’s and Women’sHealth.

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Assche E, et al. Seven years of intracytoplasmic sperm injection andfollow-up of 1987 subsequent children. Hum Reprod 1999;14(Suppl1):243–64.

2. Ferguson-Smith M. Prenatal chromosomal analysis and its impact on thebirth incidence of chromosomal disorders. Br Med Bull 1983;39(4):355–64.

3. Tarlatzis BC, Bili H. Intracytoplasmic sperm injection. Survey of worldresults. Ann NY Acad Sci 2000;900:336–44.

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5. Ma S, Kalousek DK, Ho Yuen B, Gomel V, Katagiri S, Moon YS.Chromosome investigation in in vitro fertilization failure. J Assist Re-prod Genet 1994;11:445–51.

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