Human Reproduction, Vol. 17, No. 2, 452-456,
February 2002
© 2002 European Society of Human Reproduction and Embryology
Collection of villous tissue under ultrasound guidance to improve the cytogenetic study of early pregnancy failure
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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BACKGROUND: The cytogenetic study of spontaneous miscarriage has been limited by poor karyotype success rates obtained from cell culture after surgical evacuation of retained products of conception. The aim of this study was to assess the effect of improving the method of collection of villous tissues at the time of surgery on the karyotype success rate of cell culture. METHODS: Villous samples were obtained prospectively from a cohort of 170 spontaneous miscarriages at the beginning of the surgical procedure using small biopsy forceps guided into the placenta by ultrasound imaging. This was compared with a retrospective series of 1191 spontaneous miscarriages, cultured in the same laboratory, following conventional collection of the sample from the aspiration recipient after surgery. RESULTS: In the prospective series, six (3.5%) of the original samples were classified by the laboratory as `decidua only' as compared with 162 (13.6%) in the retrospective series. The karyotype success rate was 94.5% in the prospective series compared with 83.7% in the retrospective series. The karyotype results revealed a chromosome abnormality rate of 65.8% in the prospective group and 64% in the retrospective group with a similar distribution in both groups. CONCLUSIONS: Our data show that a karyotype can be obtained from clean villous material collected at the time of surgical evacuation of miscarriage. Thus, it is not justified to subject women to transabdominal chorionic villus sampling to achieve a high karyotype success rate.
Key words: cytogenetic/karyotype/miscarriage/placenta/villous tissue
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Sporadic early pregnancy loss occurs in 10–20% of clinically recognized gestations (Alberman, 1992
) and accounts for 50 000 in-patient admissions to hospital in the UK annually (Bradley and Hamilton-Fairley, 1998). Feto-placental chromosome abnormalities account for ~50% of sporadic first trimester miscarriages; the remainder are usually classified as idiopathic (Hassold, 1986). Most of these abnormalities are numerical abnormalities and <10% are caused by structural abnormalities or other genetic mechanisms. The overall recurrence risk of numerical abnormalities is low and the risk of live born trisomy following an aneuploid early pregnancy failure is 1–2% (Alberman et al., 1992). In the case of recurrent miscarriage, a normal karyotype in a previous pregnancy is a predictor of subsequent miscarriage (Ogasawara et al., 2000)
The rate of miscarriage after assisted reproductive technology (ART) ranges between 12–40%, 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 31–35 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 48–59% 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 85–94%, 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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Study population
The study population consisted of a prospective cohort of 170 cases of spontaneous abortion presenting to University College Hospital (UCH), London, between October 1998 and September 2000. All women presenting to UCH with vaginal bleeding and a positive pregnancy test in the first trimester of pregnancy were offered a transvaginal ultrasound examination. Where an early pregnancy failure was diagnosed, with retained products of conception, management options, i.e. conservative versus surgical, were discussed with the couple and where surgical management was selected, the women were asked to participate in the study. The study was discussed in detail and consent was obtained prior to admission to the study. The study received approval from the University College London Hospitals Committee on the Ethics of Human Research. Results of karyotyping of placental tissue were offered at the time of admission to the study and were delivered 3–4 weeks later in writing with a follow-up telephone call. Karyotyping was performed by the laboratory (Cytogenetics Services); otherwise, the entire management of the cases was performed by a single operator.
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 10–20 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 1–7 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 1–7 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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The data of both study groups are summarized in Table I
. In the prospective group, six (3.5%, 95% CI: 1.3–7.5%) of the original samples were classified by the laboratory as `fetal or placental material not recognised, decidua only' as compared with 162 (13.6%, 95% CI: 11.7–15.5) in the retrospective group (2 = 13.95; P < 0.001). In the samples available for culture, successful karyotype was significantly (2 = 13.5; P < 0.001) higher in the prospective group (155 out of 164, 94.5%, 95% CI: 89.8–97.5) when compared with the retrospective group (861 out of 1029, 83.7%, 95% CI: 81.4–85.9). Thus the overall culture success rate (taking in to account errors of culture and collection) was significantly higher (2 = 28.03; P < 0.001) in the prospective (155 out of 170, 91.1%, 95% CI: 85.9–95.0) than in the retrospective group (861 out of 1191, 72.3%, 95% CI: 68.6–74.8). The samples in the prospective group that contained only decidua were collected during the initial phase, within the first 40 samples collected.
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The karyotype results revealed a chromosome abnormality rate of 65.8% (102 out of 155, 95% CI: 57.8–73.2) in the prospective group and 64% (551 out of 861, 95% CI: 60.8–67.2) in the retrospective group (OR: 1.08, 95% CI: 0.76–1.55). In the prospective group there were 69 autosomal trisomies (68%), 12 triploidies (11.8%) and five unbalanced translocations (4.9%). A similar distribution was found in the retrospective group (Figure 1
). The male to female ratio in the prospective series was 0.77.
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Discussion |
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The data of the present study indicate that the use of a transcervical placental biopsy method under ultrasound guidance before the surgical procedure of ERPC improves the chances of obtaining a successful karyotype compared with conventional techniques, with minimal morbidity incurred to the women. Collection of the samples under ultrasound guidance not only improves the quality of the sample, but also offers the clinical advantage of verifying complete emptying of the uterine cavity. In the study group, no patients returned with retained products of conception nor did they complain of persistent bleeding at their 4–6 week follow-up.
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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Direct preparation of villus material based on a method previously described (Simoni et al., 1983
) has been applied to the cytogenetic study of abortus material collected after ERPC (Guerneri et al., 1987; Eiben et al., 1990). Karyotype success rates were better than traditional cell culture studies previously quoted and were comparable with our prospective study in the former case. However, immediate access to sophisticated laboratory preparation renders this method less applicable to a local general hospital set-up. Our method presents a clinical advantage if such testing is to be incorporated in routine assessment of spontaneous miscarriage.
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 crown–rump 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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Recurrent miscarriage is a major issue for both fertile and infertile couples. Vidal et al. have shown that preimplantation genetic diagnosis may have a role in the management of recurrent miscarriage (Vidal et al., 2000
) and is being developed as an alternative to prenatal diagnosis in IVF (Delhanty and Harper, 2000). However, the epidemiology of genetic abnormality in early pregnancy failure after assisted reproduction remains to be investigated. We believe that our method could be adopted into clinical practice in some clinical settings if validated by a prospective study, although it is unlikely to be practicable for all IVF units. Within this context, it may help to identify which chromosomes may be important and may also establish what the likely contribution of genetic abnormality is to early pregnancy failure in this group of patients.
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Acknowledgements |
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The authors wish to thank Rodney Meredith and his team at Cytogenetic Services, Devonshire St. WC1, for performing all cell culture work in this study and Dr Gurmit Pahal, fellow researcher at University College Hospital for his help in carrying out this study.
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2 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Royal Free and University College Medical School, UCL campus, 86–96 Chenies Mews, London WC1 6HX, UK. E-mail: e.jauniaux{at}ucl.ac.uk
Submitted on December 29, 2000; resubmitted on May 21, 2001
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accepted on October 5, 2001.
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