Maternal origin of monosomy 21 derived from ICSI: Case report

Sai Ma1,3, Wendy Robinson2, Ryan Lam1 and Basil Ho Yuen1

1 Department of Obstetrics and Gynecology and 2 Department of Medical Genetics, University of British Columbia, Vancouver, Canada


    Abstract
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
With the worldwide diffusion of the intracytoplasmic sperm injection (ICSI) procedure in recent years, the issue of possible genetic risks of this new and powerful technique has attracted considerable attention. An important concern is whether ICSI facilitated the passage of genetic defects from spermatozoa to offspring. ICSI was performed with spermatozoa from a frozen–thawed sperm sample from a testicular sperm extraction (TESE) of a 38 year old man who suffered from azoospermia. His wife was 36 years old. The resulting pregnancy spontaneously aborted at 8 weeks gestation after embryo replacement. Cytogenetic investigation displayed monosomy 21. The paternal origin of the single chromosome 21 was determined by molecular analysis. The segregation error leading to loss of one chromosome 21 is likely to have occurred during oogenesis rather than as a direct consequence of ICSI. Nonetheless, monosomy 21 is extremely rare and it cannot be excluded that ICSI assisted the fertilization of an abnormal oocyte.

Key words: intracytoplasmic sperm injection (ICSI)/monosomy 21/oogenesis/origin of chromosomal abnormality/polymorphic microsatellite markers


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
Intracytoplasmic sperm injection (ICSI) has markedly improved the chances for successful treatment of male factor infertility. However, the issue of possible genetic risk of this new and powerful procedure has attracted considerable attention. One important aspect in the debate about the genetic implications is the possible increased rate of chromosomal abnormalities in the resulting pregnancies (Johnson, 1998Go).

An increase in de-novo chromosomal abnormalities and the higher frequency of transmitted chromosomal aberrations were found in a large cohort of 1082 conceptions through prenatal genetic diagnosis from a single IVF centre (Bonduelle et al., 1999Go). Limited studies from the literature have indicated that most of these chromosomal abnormalities are of paternal origin (Van Opstal et al., 1997Go; Bartels et al., 1998Go).

It has been suggested that men with abnormal sperm parameters may have a higher incidence of aneuploidy in their spermatozoa as compared with fertile men. The selection of spermatozoa for ICSI from a male factor infertility patient may unwittingly result in the transmission of chromosomal abnormalities. Studies using fluorescence in-situ hybridization (FISH) for the analysis of aneuploidy in human spermatozoa suggest that oligozoospermic men are especially at risk for sperm chromosomal abnormalities (Moosani et al., 1995Go; Bernardini et al., 1997Go; Ushijimal et al., 2000Go).

Our previous study and those of others, using cytogenetic methods for the analysis of aneuploidy in human unfertilized oocytes, revealed a high incidence of aneuploidy (Ma et al., 1989Go; Pellestor, 1991Go). Whether ICSI could cause increases in chromosomal abnormalities of both paternal and maternal origin in the resulting embryos is unknown. Examination of spontaneous abortuses derived from ICSI is one feasible area for study because ~15% of pregnancies from ICSI are spontaneously lost.

Recently, Bartels et al. reported a paternal origin of trisomy 21 from a spontaneous miscarriage after ICSI (Bartels et al., 1998Go). We are, to the best of our knowledge, reporting the first case of a maternally derived monosomy 21 in an abortus conceived by ICSI.


    Case report
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
The couple (female age 36 years, male age 38 years) presented with a 3 year history of primary infertility. The female partner had no evidence of tubal, ovulatory or pelvic infertility factors. The male partner, however, was found to have obstructive azoospermia. Testicular sperm extraction (TESE) was performed. Spermatozoa from the TESE showed no motility. These spermatozoa were then cryopreserved. One year later, IVF, combined with ICSI, was undertaken. A standard luteal phase `long protocol' of controlled ovarian hyperstimulation using a gonadotrophin-releasing hormone (GnRH) agonist and recombinant FSH with intravaginal progesterone as luteal support was undertaken in the female partner. Of the 24 oocytes retrieved, 19 metaphase II oocytes were identified by the presence of a single polar body. After the spermatozoa from the frozen TESE specimen were thawed, all thawed spermatozoa were found to be immotile. Viable spermatozoa were selected based on the phenomenon of sperm tail curling (Ma et al., 2000Go).

Of the 19 oocytes injected, 18 survived the procedure (94.7%) and 17 of them fertilized normally (94.4%), as shown by the presence of two pronuclei 18 h after injection. Four good quality embryos, three at the 6-cell stage and one at the 5-cell stage, were transferred on day 3 after oocyte retrieval. Ten good quality embryos (<20% fragmentation) were cryopreserved. A positive pregnancy test was obtained 14 days after embryo transfer. At 6 weeks of pregnancy, the first ultrasound showed a single gestational sac without heartbeat. At 8 weeks gestation after embryo replacement the patient had a spontaneous miscarriage. Cytogenetic analysis of cultured chorion from placental tissue revealed a male karyotype with monosomy of chromosome 21 (all five karyotypes showed 45, XY, –21). To assess the parental origin of the single chromosome 21 in the fetus, highly polymorphic microsatellite markers on chromosome 21 were analysed using the DNA of both parents and DNA isolated from the cryopreserved fetal tissue sample after patient consent. This study was conducted with the approval of the Clinical Ethics Board at the University of British Columbia.

DNA was extracted from maternal and paternal blood samples as well as sample of chorion from the aborted material using standard protocols. DNA typing of parents and chorion was done using highly polymorphic microsatellite markers listed in Table IGo. Primers were obtained from Research Genetics Inc. (Huntsville, Alabama, USA). PCR amplification was performed by standard methods, samples were run on a 0.4 mm thick 6% polyacrylamide/50% ureal gel and visualized by silver staining. A summary of results is given in Table IGo.


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Table I. Polymorphic microsatellite analysis
 

    Discussion
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 Discussion
 References
 
Although aneuploidy accounts for ~50% of karyotyped spontaneous abortions, most of these are non-mosaic trisomics (Jacobs, 1990Go). 45,X is the only common monosomy that occurs in clinically detectable pregnancies and 99% will result in spontaneous loss. Only eight cases of monosomy 21 have been described (Dziuba et al., 1976Go; Kuliev et al., 1977Go; Fryns et al., 1977Go; Houston and Chudley, 1981Go; Herva et al., 1983Go; Pellissier et al., 1987Go; Garzicic et al., 1988Go; Joosten et al., 1996Go) previously. Six of them were live-born and two were from spontaneous abortions (Kuliev et al., 1977Go; Joosten et al., 1996Go). However, only the case of Joosten et al. was confirmed molecularly. In that case, monosomy 21 was prenatally diagnosed in chorionic villi using FISH and DNA investigations revealed a paternal origin of the single chromosome 21.

Although FISH was not used in the present case, all seven microsatellite markers mapping to chromosome 21 that were tested yielded only a single allele, consistent with full monosomy 21. Furthermore, the lack of a maternal contribution at several markers indicated that there was a loss of the maternal chromosome 21.

The loss of a chromosome 21 could occur as a result of non-disjunction or anaphase lag at either a meiotic division during gametogenesis, or an early division of a normal zygote. However, an early post-zygotic loss of one chromosome 21 in a diploid conceptus seems quite unlikely as the diploid cell line would be expected to have a selective advantage over the monosomy cell line and would be unlikely to be lost (Peterson et al., 1992). Thus, the monosomy 21 most likely originated through a meiotic error during oogenesis. By using ICSI, a nullisomic oocyte for chromosome 21 was fertilized with a normal haploid spermatozoon.

It is well established that most chromosomal abnormalities originate from female meiosis, contributing considerably to pregnancy failures particularly in women of advanced maternal age. Recent evidence from a study of the rates of aneuploidy from first and second polar bodies showed that non-disjunction could occur in both meiotic divisions (MI and MII) (Verlinsky et al., 1999Go). In some cases non-disjunction could result in a disomic second polar body and a nullisomic oocyte. It is not clear whether the monosomy 21 in this study is derived from the first meiotic division or the second meiotic division due to the limitation of the technique used to determine the origin of the abnormalities.

Chromosome abnormalities prenatally detected in ICSI pregnancies are frequently found to involve the sex chromosomes. Those reported were predominantly of paternal origin (Van Opstal et al., 1997Go; Bartels et al., 1998Go). Especially, two cases of 47,XXY and four cases of 45,X evaluated were of paternal origin (Van Opstal et al., 1997Go), as is the case for 80% of 45,X, and ~50% of 47,XXY in non-ICSI pregnancies (Hassold et al., 1988Go; Abruzzo and Hassold, 1995Go). However, maternally-derived autosomal trisomies 18 and 21 and one paternally-derived trisomy 21 were also reported (Van Opstal et al., 1997Go; Bartels et al., 1998Go). Our report, to our knowledge, is the first report of monosomy 21 derived from ICSI.

It has been argued that bypassing natural sperm selection, which occurs when spermatozoa penetrate into the oocyte, may promote fertilization by genetically abnormal spermatozoa. Studies of spermatozoa from infertile men with normal somatic karyotypes using FISH showed increased numerical sex chromosome abnormalities (Moosani et al., 1995Go; Bernardini et al., 1997Go). The slightly increased sex chromosome abnormalities among newborn after ICSI may relate to the genetically abnormal spermatozoa among infertile men.

In standard IVF, the fertilization rate remains at 60–70%, while in ICSI, the fertilization rate could reach as high as 90% (Ma and Ho Yuen, 2000Go). This high fertilization rate achieved by ICSI raises the possibility that ICSI could fertilize an oocyte with an abnormal chromosome complement, which may not be fertilized in standard IVF procedures. It is well known that the high incidence of aneuploidy is one of the causes for the failed fertilization in IVF. Our results and other data in the literature demonstrated that about 25% and 30% of human unfertilized oocytes were aneuploid (hyperhaploid and hypohaploid) and diploid (Pellestor, 1991Go; Ma et al., 1994Go). These findings suggest that natural selection against chromosome abnormalities may occur even prior to fertilization (Ma et al., 1994Go). 45,X is the only monosomy discovered among spontaneous abortions (Jacobs, 1990Go), except for the rare cases of monosomy 21 (Joosten et al., 1996Go). Perhaps most of the hypohaploidy oocytes could not be fertilized or die shortly after fertilization. Our hypothesis is that ICSI, bypassing the fertilization barrier (zona pellucida), may promote the fertilization of genetically abnormal oocytes.

Under normal circumstances, the resulting monosomy 21 in this case could not have occurred because the partner did not have spermatozoa in his semen. The high fertilization was only achieved by using ICSI with spermatozoa retrieved from testicular tissues. A hypohaploid oocyte may be fertilized by means of the ICSI procedure. It is also possible that the ICSI procedure itself may cause the chromosomal abnormality by disturbing the second meiotic division. It has been demonstrated that the presumptive localization of the spindle together with metaphase chromosomes in oocytes cannot be predicted very accurately by the position of the first polar body (Silva et al., 1999Go). Thus, in some cases, the ICSI procedure may damage the spindle. At the present time, whether this disturbance does or does not cause an error in chromosomal segregation during the second meiotic division cannot be determined from the data present in the literature.

It seems that ICSI may have the potential to generate abnormal embryos by introducing genetically abnormal spermatozoa from infertile men, and also produce abnormal embryos by fertilizing genetically abnormal oocytes, events which may not occur in IVF or natural conception. However, most genetically abnormal embryos may not implant or would die at an early stage of pregnancy due to the lethal nature of these abnormalities. This may be one of the important factors for the low implantation rate of ICSI embryos (10–15%) despite high fertilization rates after ICSI (Ma et al., 2000Go).

The cytogenetic investigation of fetuses and newborns from ICSI and the origin of their chromosomal abnormalities are of great importance to clinical practice. It may provide two pieces of important information: (i) whether paternally derived chromosomal abnormalities are increased after ICSI when compared with those from the normal population; (ii) whether maternally derived chromosomal abnormalities are also increased after ICSI when compared with those from normal population.

Another important area that can also be taken into consideration is the study of spontaneous abortions. Chromosomal aberrations are known to be the major reason for pre- and post-implantation human embryo wastage. The cytogenetic study of spontaneous abortuses and the origin of chromosomal abnormality may provide more information because the spontaneous abortuses contain high incidences of chromosomal abnormalities (Poland and Ho Yuen, 1978Go; Jacobs, 1990Go). This approach combined with prenatal diagnosis and follow-up of newborns will provide a better estimate of the paternally and maternally derived risk of aneuploidy after ICSI. As compared with spontaneously conceived pregnancy, conception after assisted reproductive technologies tends to be closely monitored. Thus, abnormalities in pregnancies conceived after assisted reproduction techniques are more likely to be detected. Hence, conclusions concerning the safety of ICSI can only be made from large multicentre trials.


    Notes
 
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Room 313, Willow Pavilion, Vancouver Hospital and Health Sciences Center, 855 West 12th Avenue, Vancouver BC V5Z 1M9, Canada.E-mail: sai{at}unixg.ubc.ca Back


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Submitted on October 17, 2000; accepted on February 23, 2001.