1 Early Pregnancy Unit, Department of Obstetrics and Gynaecology, Obstetric Hospital, University College Hospital, London, UK
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Abstract |
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Key words: cytogenetic/karyotype/miscarriage/placenta/villous tissue
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Introduction |
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The rate of miscarriage after assisted reproductive technology (ART) ranges between 1240%, depending on the technique used (Wennerholm et al., 1997; Aytoz et al., 1999
; Palermo et al., 2000; Westergaard et al., 2000
), the number of embryos transferred (Balen et al., 1993
) and maternal age (Lass et al., 1998
; Pantos et al., 1999
; Nikolettos et al., 2000
). In both spontaneous gestations and pregnancies resulting from ART, the latter factor, i.e. age, is the most important parameter in the evaluation of the risk of pregnancy complications, and in women aged
45 years, the risk of miscarriage is ~75% (Nybo et al., 2000
). Overall, in infertile women there is a five-fold increase in early pregnancy loss from the age of 40 years compared with ages 3135 years, which is independent of the infertility diagnosis, mode of insemination and ovulation induction protocol (Smith and Buyalos, 1996
). Although, new ARTs such as ICSI may be associated with a higher incidence of aneuploidy and in particular, of sex-chromosomal de novo aberrations (Tarlatzis and Grimbizis, 1999
), there is little information on the epidemiology of chromosome abnormalities in miscarriage after ART.
Historically, the cytogenetic study of miscarriage material has been flawed by low karyotype success rates of 4859% from material obtained at the time of evacuation of retained products of conception (ERPC) (Boue et al., 1975; Kajii et al., 1980
; Hassold, 1986
). More recently, with the development of prenatal methods of sampling such as chorionic villous sampling (CVS), several groups have advocated the use of this invasive method to obtain placental samples in women presenting with early pregnancy failure and requiring karyotyping. Although these authors presented improved karyotypic success rates of 8594%, their patients were subjected to additional invasive procedures at the time of ultrasound diagnosis of miscarriage. Our study aims to illustrate that if placental tissue collection is optimized at the time of ERPC then cytogenetic analysis performs very well and that pre-operative invasive testing may not be justified.
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Materials and methods |
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Control population
The control population consisted of a retrospective series of 1191 cases of spontaneous abortion held in the records of the laboratory (Cytogenetic Services). The populations were matched for gestational and maternal age. Products of conception had been sent from 25 different gynaecologists whose patients requested karyotyping after spontaneous abortion and ERPC. All samples were processed by the same laboratory between January 1998 and December 1999.
Tissue samples
Tissue samples were obtained at the time of the surgical ERPC, which was performed within 48 h as an elective procedure under light general anaesthesia. All surgical procedures were performed under transabdominal ultrasound guidance enabling clear identification of the placenta and directed retrieval of this tissue using small biopsy forceps. The sample was thus collected intact on many occasions. Where suction was used to remove remaining villi and decidua, tissues were retrieved from the end of the plastic curette prior to the products passing through the suction tubing. In this way, damage to the sample was minimized. The tissues were then examined and cleaned by blotting on an absorbent tissue pad. Maternal blood was removed from the placenta, the decidua was kept separately and the 1020 mg of placental villi were inserted into culture medium. In all cases, a clean, villous-rich, fresh sample was delivered to the cytogenetics laboratory within 17 h. All samples were collected by N.G., who prior to undertaking the research project worked as a clinician in the same London hospital. The technique was developed by E.J. over the period of collection and could easily be taught to other junior clinical staff.
In the retrospective group, samples had been collected by a method which is representative of current clinical practice in the UK. No ultrasound is used and the retained products of conception are removed from the uterus using a suction curette which is connected to a collection vessel. The contents of this vessel are delivered, prior to any cleaning or dissection, to the laboratory over an average interval of 17 days. Dissection and cleaning was undertaken by the laboratory on receipt of the sample.
Laboratory method of karyotyping
Placental tissues were set up in the cytogenetics laboratory after examination under a microscope to confirm identification of villous material. A culture medium composed as follows was used and will be referred to as `POC medium': complete amniochrome 500 ml + supplement (75% of total mix). Composition: 0.5 ml gentamycin; 10 ml kanamycin; 10 ml; 5 ml L-glutamine; 5 ml HEPES. Complete chang D 500 ml (25% of total mix). Composition: 5 ml amphoterecin; 0.5 ml gentamycin; 10 ml kanamycin; 10 ml; 5 ml L-glutamine; 0.3 ml HCl; 5 ml HEPES. Villous material was then added to the POC medium and finely chopped using a sterile scalpel blade. The sample was then applied and spread finely to a flat tissue culture surface and allowed to dry for 2 h at room temperature to allow the tissue to adhere. Following the addition of POC medium to the culture tubes, each tube was placed flat surface down in an incubator and left undisturbed for 5 days.
Analysis of results
The data are expressed as proportions, 95% confidence intervals (CI) and odds ratios (OR) and were compared using the 2 test. Results were considered significant at P < 0.05.
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Results |
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Discussion |
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The culture success rate of 94.5% in our prospective group was much higher than those reported previously in cytogenetic studies employing cell culture (Boue et al., 1975; Kajii et al., 1980
; Hassold et al., 1986); in our retrospective group (Table I
) and in a recent study (Carp et al., 2001
) (Table II
). In the early studies, the samples were collected at the end of the surgical procedure and were most probably contaminated with maternal tissues. The fact that the success rate was higher in our retrospective group than in earlier studies suggests that changes in cytogenetic techniques have also had an influence on the success of karyotyping villous tissue from early pregnancy failure. However, the more recent study also displays limitations to karyotypic success. This indicates that refined collection of the sample not only reduces the number of samples rejected for culture, but also improves the quality of the sample, i.e. low risk of maternal cell and bacterial contamination.
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The management of spontaneous miscarriage has changed very little over the past 30 years and epidemiological studies now need to be updated (Alberman, 1992). Research into the cytogenetic aspects of miscarriage has been limited by the poor performance of karyotype testing. Furthermore, inaccurate dating of miscarried pregnancies, which were classified according to time of expulsion of the pregnancy and not according to time of fetal demise, flawed the interpretation of cytogenetic findings at different gestations. The timing of fetal demise can now be assessed more accurately by transvaginal ultrasound measurement of the crownrump length at the time of diagnosis. It is of epidemiological interest, that the distribution of karyotypic abnormalities between the prospective and retrospective groups was very similar (Figure 1
). This indicates that, in the case of miscarriage, culture failure is not directly linked to a specific chromosomal abnormality that may impair placental villous growth in culture.
Several authors have reported on the use of modern prenatal diagnosis techniques for the evaluation of karyotypic abnormalities in early pregnancy failure. Transabdominal chorionic villus sampling (CVS) was offered to patients pre-operatively (Johnson et al., 1990; Strom et al., 1992
; Sanchez et al., 1999). Karyotypic success was similar to our prospective study; however, patients had to undergo a separate invasive procedure, which our data show to be unnecessary. Furthermore, CVS is only performed in specialized units which could not accommodate the large numbers needed for an epidemiological study.
Cytogenetic study remains important as a method of classifying miscarriage into chromosomally normal and abnormal groups. The normal group represents the group of major interest, since it is this group that seems to repeatedly miscarry (Warburton et al., 1987; Ogasawara et al., 2000
). In our study, this amounts to 32.8% of the population. Patient request is also a very significant reason to offer karyotypic information. With the increase in pregnancies from ART, one can anticipate that this demand from patients will only increase. The epidemiology of miscarriage after ART is not well defined. Studies have described the outcome of ART pregnancies following prenatal diagnosis, but there appear to be no cytogenetic data regarding pregnancy loss at <11 weeks gestation. The rate of miscarriage in IVF quoted in the literature is widely variable and displays the same increase in incidence with maternal age as in spontaneous miscarriage (Table III
). Cryopreserved IVF and ICSI do not appear to alter the rate of miscarriage relative to fresh procedures, but ICSI is associated with a higher incidence of sex-chromosomal de novo aberrations (Tarlatzis and Grimbizis, 1999
). However, there is very little information available regarding karyotype results in cases of miscarriage. In a study on the pregnancy outcome after ICSI (Palermo, 2000) karyotypic information was available but limited. A study on oocyte donation (Westergaard et al., 2000
) reported a high rate of miscarriage. Since oocyte donors are required to be <35 years old, it would be of value to assess the contribution of genetic abnormality to this group of miscarriages.
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Acknowledgements |
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Notes |
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Submitted on December 29, 2000; resubmitted on May 21, 2001
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References |
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accepted on October 5, 2001.