The cause of death remains unexplained in about two-thirds of stillbirths.1–5 It is useful to identify a cause of death: it helps parents in their mourning process, it may determine the recurrence risk, aid counseling for future pregnancies, siblings and families, enables comparison of national and international health care, and aids prevention.6–8
One of the causes of stillbirth is a chromosomal anomaly. The incidence of these anomalies, which are detectable through karyotyping, has been reported to be 6–12% in a random selection of stillborns.3,9,10 There are no international uniform protocols for cytogenetic analysis available based on evidence of stillbirths.2,10 Some studies recommend cytogenetic analysis for all fetal deaths,9,10 whereas others advise testing for a selected population, mainly due to the substantial costs. This selection would cover deaths with, eg, congenital anomalies, fetal growth restriction, nonmacerated fetuses, advanced maternal age, or recurrent pregnancy loss.5,10–15 In several European countries it is common practice that cytogenetic analysis is only indicated (funded by insurance companies) if congenital anomalies are present either at ultrasound or at birth.16 Gynecologists (both qualified and in training) or midwives are often responsible for evaluating these morphologic abnormalities at birth and also, therefore, for the decision to perform cytogenetic analysis.
Results of tissue cultures after intrauterine fetal death are often disappointing. Overall, successful tissue culture followed by karyotyping has been reported in 41% of intrauterine fetal deaths but depends on the type of tissue examined. Karyotyping in this group was successful in 18% of intrauterine fetal deaths for skin biopsies, in 7% of other fetal tissues, and in up to 71% of extrafetal tissues such as placenta.17 To increase the rate of successful tests, amniocentesis or chorionic villus sampling (CVS) before induction of labor is advised.18–20 However, good alternatives are needed if this is not feasible due to objection by parents, inexperience of the gynecologist, diminished amniotic fluid, or logistic problems.2 There is no consensus on which tissue type is best for cytogenetic testing postpartum. The aim of our study was to estimate success rates of cytogenetic analysis in different tissue types after intrauterine fetal death, to study selection criteria for cytogenetic analysis, and the value of cytogenetic testing in determining the cause of death. Our goal was to design a flow chart to help determine which intrauterine fetal deaths should have cytogenetic analysis performed and to guide counseling of the parents through the decision on whether to allow this test.
MATERIALS AND METHODS
In 2002 we initiated a national Dutch study on intrauterine fetal death at the University Medical Centre Groningen, with 50 participating hospitals located throughout the Netherlands. Inclusion criteria for the study were singleton intrauterine fetal deaths diagnosed antepartum (heart beat ceased before start of labor) after 20 weeks of gestation and confirmed by ultrasonography. Pregnancy terminations were excluded. The University Medical Centre Groningen Institutional Review Board Committee determined that this study is exempt. The Institutional Review Board of each participating hospital reviewed local feasibility. Clinical data were collected after informed consent was obtained. For each intrauterine fetal death, a case record form was filled in and a standard diagnostic workup protocol was followed. There is no national diagnostic work-up guideline for intrauterine fetal death. Our diagnostic study protocol was therefore based on local protocols currently used in different Dutch hospitals.
Patient data included medical and obstetric history, maternal characteristics, fetal characteristics, pregnancy details, and obstetric discharge letters. The diagnostic test results included maternal and fetal blood tests; maternal and fetal viral serology; microbiologic cultures from mother, fetus, and placenta; autopsy; placental examination; and cytogenetic analysis. Parental consent was obtained for all the diagnostic work.
In the Dutch genetic guidelines, cytogenetic analysis is indicated in the event of stillbirth after 16 weeks of gestation if fetal congenital anomalies are present at ultrasound or at birth. There is no national consensus on which tissue is best to use. Our study protocol prescribed cytogenetic evaluation of all intrauterine fetal deaths either by invasive testing (amniocentesis or CVS) before induction of labor or by postpartum tissue testing of fetus (fetal blood, fascia lata, pericardium, cartilage, or skin), umbilical cord, or placenta. In some cases, amniocentesis, CVS, or preimplantation diagnostics had already been performed during the ongoing pregnancy and before fetal death for various reasons. In most cases, cytogenetic analysis was performed after intrauterine fetal death was diagnosed, but in a small group of cases both types of cytogenetic analysis were performed. Multiple tissues were analyzed in some cases.
The biopsies were collected in a sterile pot containing saline solution or culture medium and transported to the laboratory. Cytogenetic analysis was performed in local, specialized genetic laboratories following standard laboratory procedures consistent with the guidelines of the Dutch Association of Clinical Cytogeneticists. Mostly, chromosomal analysis was performed in fibroblast cultures. In some cases, additional molecular cytogenetic analyses, such as fluorescent in situ hybridization, multiplex ligation-dependent probe amplification, or comparative genomic hybridization were done if fibroblast cultures failed.
Autopsy and placental examination were performed by local pathologists in the participating hospitals. Pathologists were urged to follow the study guidelines for autopsy and placental examination based on those published by the Royal College of Obstetricians and Gynecologists, the Royal College of Pathologists,21 and the College of American Pathologists.22,23
Morphologic abnormalities determined at birth, small for gestational age (SGA), maternal age 35 or more years, and maceration were studied as possible selection criteria for cytogenetic analysis. Morphologic abnormalities of the fetus at birth, as determined by the gynecologist, gynecologist-in-training, or midwife, were all adjudicated by an independent perinatal clinical geneticist as common variant, minor or major abnormalities, based on the phenotypic abnormality classification by Merks et al.24,25 The geneticist did not have access to the cytogenetic or autopsy results. We added hydrops as a major anomaly and single umbilical artery as a minor anomaly. Fetal growth percentiles for birth weight by gestational age at time of diagnosis of intrauterine fetal death were calculated according to the Kloosterman growth charts,26 which start at 25 weeks of gestation. Small for gestational age was defined as a birth weight at less than the 10th percentile. The stage of maceration was classified according to Wigglesworth.27
Panel classification sessions were set up for determining cause of death and the value of cytogenetic analysis in this determination. Procedures were agreed upon in advance and the Tulip classification for cause of death was used.28 The panel consisted of two obstetricians, an obstetric resident, and a pediatric pathologist. All panel members first prepared each case individually using the patient information records, then panel discussions were held and a consensus reached on cause of death and the value of the diagnostic test. No other information sources were consulted. The value of cytogenetic analysis in determining cause of death was assessed under four headings as “establishing cause of death” (abnormal results of cytogenetic analysis established a cause), “excluding cause of death” (the results of cytogenetic analysis excluded this cause in an intrauterine fetal death with a suspected chromosomal abnormality), “missing for determination of cause of death” (in an intrauterine fetal death with a suspected chromosomal abnormality, the results of cytogenetic analysis were missing), or “not valuable in determining cause of death” (cytogenetic analysis was not regarded as valuable in this process).
Continuous variables were expressed as median values and ranges and categorical data were expressed as counts and percentages. Differences between groups for categorical data were evaluated by using the Fisher exact test or χ2. In addition, exact 95% confidence intervals were presented when applicable. A two-tailed P<.05 was considered to indicate statistical significance. Statistical analyses were performed using SAS 9 software (SAS Institute, Inc., Cary, NC).
A total of 750 intrauterine fetal deaths were studied during a 4-year period from 2002 to 2006. The participating hospitals started including intrauterine fetal deaths at different points in time and the inclusion rates per hospital differed. An investigation into these rates yielded an average inclusion rate of 75% of intrauterine fetal deaths that met our selection criteria. The reasons for exclusion were informed consent denied, a language barrier, logistic problems, and a doctor’s decision to exclude an intrauterine fetal death in the case of a “known” cause of death at birth (placental abruption, known chromosomal abnormalities, and major congenital anomalies).
The median age of the mothers was 31 years (range 17–46 years), and the median gestational age at determination of intrauterine fetal death was 31 weeks and 4 days (range 20–42 weeks and 1 day). The median fetal weight was 1,470 g (range 12–4,630 g). Oligohydramnions or anhydramnions was present at diagnosis of intrauterine fetal death in 188 cases (25.1%). Significantly more intrauterine fetal deaths were males (n=408) than females (n=339; P<.01). In three cases, the fetal sex could not be determined by physical, cytogenetic, or pathologic examination. Autopsy was performed on 525 fetuses (70.0%).
Cytogenetic analysis was performed for 508 intrauterine fetal deaths (67.7%), and a successful result was obtained in 246 deaths (48.4%). Amniocentesis and/or CVS or preimplantation diagnostics were performed during 74 (14.6%) ongoing pregnancies before intrauterine fetal death. The overall success rate during ongoing pregnancy was 100%. Cytogenetic analysis was performed after death in 453 intrauterine fetal deaths (cultures and molecular cytogenetic analysis) and was successful in 39.7%. In 19 intrauterine fetal deaths, tissue samples were taken during ongoing pregnancy as well as after death. The success rates of cytogenetic analyses in relation to tissue type are presented in Table 1. The highest success rates after death were seen for invasive samples (84.6%), CVS (two cases—both successful) or amniocentesis (84.0%), and for intrauterine fetal deaths for which invasive sampling was performed in combination with fetal, placental, or umbilical cord biopsies (85.7%). Invasive cytogenetic testing after death yielded significantly more successful cytogenetic results than postpartum analyses (P<.001). Cytogenetic success rates on tissues taken postpartum varied between 32.1% for umbilical cord and 0% for pericardium. After excluding the pericardium from this group, umbilical cord samples (32.1%, 95% confidence interval [CI] 25.6–39.2) were no more successful than fetal blood and “other tissue types” (20.0%, 95% CI 8.4–36.9; P=.17). The group “other tissue types” included a placenta biopsy (n=1), skin biopsies (n=15), and unknown tissue type (n=10). Additional molecular cytogenetic analysis after failure of fibroblast cultures was performed in six cases during ongoing pregnancy and in 39 cases postpartum. These comprised 36 fluorescent in situ hybridization (4 abnormal), 8 multiplex ligation-dependent probe amplification (1 abnormal), and 1 comparative genomic hybridization.
The prevalence (prior probability) of a chromosomal abnormality in the 246 intrauterine fetal deaths for which a successful cytogenetic result was obtained was 13.0% (95% CI 9.1–17.9). The 32 chromosomal abnormalities in relation to the moment cytogenetic analysis was performed, tissue type tested, and cause of death are shown in Table 2. Trisomy 21 was established in 10 of 32 (31%), trisomy 18 in 7 of 32 (22%), monosomy (45, X) in 7 of 32 (22%), trisomy 13 in 2 of 32 (6%) and other chromosomal abnormalities in 6 of 32 (19%). Fewer than one half of the chromosomal abnormalities (40.6%) were determined in second trimester intrauterine fetal deaths.
The outcome of cytogenetic analysis in relation to the morphologic abnormalities seen at birth by the physician attending the delivery and classified afterward by a perinatal geneticist are presented in Table 3. Of the 246 chromosomal results, 180 were determined after death. In this latter group of 180 intrauterine fetal deaths with morphologic abnormalities seen at birth, there were significantly more chromosomal abnormalities (38.0%) than in the group without morphologic abnormalities (4.6%, P<.001). In 19 of the 25 (76.0%, 95% CI 54.9–90.6) intrauterine fetal deaths with a chromosomal abnormality (in this group of 180 deaths), morphologic abnormalities were seen at birth (sensitivity), whereas in the group with normal chromosomal results (n=155), there were no morphologic abnormalities in 124 intrauterine fetal deaths (80.0%, 95% CI 72.8–86.0; specificity). Overall, in 143 of 180 cases (79.4%, 95% CI 72.8–85.1), morphology matched with the chromosomal results. The absence of morphologic abnormalities for a fetus at birth had a negative predictive value of normal chromosomes of 95.4%, whereas the posterior probability of a chromosomal abnormality was 4.6% (95% CI 1.7–9.8). In contrast, the prior probability of a chromosomal abnormality in our cohort was 13%. The presence of morphologic abnormalities had a 38.0% (95% CI 24.7–52.8) positive predictive value of a chromosomal abnormality.
The outcome of chromosomal analysis in relation to fetal birth weight is presented in Table 4. In the group of SGA fetuses with successful chromosomal analysis, we did not observe a statistically significant difference in deaths with a chromosomal abnormality (15.5%) compared with non-SGA (9.2%; P=.22). Nine of 21 intrauterine fetal deaths, (42.9%, 95% CI 21.8–6.0; sensitivity) with a chromosomal abnormality were SGA. Whereas in the group with normal chromosomes (n=167), 118 intrauterine fetal deaths were non-SGA (70.7%, 95% CI 63.1–77.4; specificity). The negative predictive value of non-SGA for normal chromosomes at birth was 90.8%. The posterior probability of a chromosomal abnormality in non-SGA was therefore 9.2% (95% CI 4.9–15.6). The positive predictive value of SGA for a chromosomal abnormality was 15.5% (95% CI 7.4–27.4). Small for gestational age was not associated with a higher failure of cytogenetic analysis (58.9% 95% CI 50.3–76.1 compared with 50.4% 95% CI 44.2–56.6; P=.12). None of the fetuses with a chromosomal abnormality were reported to be hydropic. For 58 intrauterine fetal deaths, a birth weight percentile could not be calculated. Fifty-seven of these intrauterine fetal deaths were less than 25 weeks of gestation.
The outcome of chromosomal analysis in relation to maternal age is presented in Table 5. Women older than 35 years of age with intrauterine fetal deaths with successful chromosomal analysis did not have significantly more deaths with a chromosomal abnormality (16.7%) than women of 35 years or younger (12.0%, P=.37). Nine of 32 (28.1%, 95% CI 13.8–46.8; sensitivity) women with an intrauterine fetal death with a chromosomal abnormality were older than 35 years of age, whereas in the group with normal chromosomes (n=214), 169 women (79.0%, 95% CI 72.9–84.2; specificity) were 35 years of age or younger. The negative predictive value of maternal age 35 years or younger for normal chromosomes was 88.0%. The posterior probability of a chromosomal abnormality for these women was therefore 12.0% (95% CI 7.8–17.4). The positive predictive value of maternal age older than 35 years for a chromosomal abnormality was 16.7% (95% CI 7.9–29.3).
The outcome of successful cytogenetic analysis after death (normal and abnormal chromosomes) compared with the group in which this was unsuccessful was studied in relation to maceration stage. Maceration stage was unknown for 16 intrauterine fetal deaths. Success rates in intrauterine fetal deaths without maceration was significantly higher 58.5% (95% CI 45.6–70.6) than in intrauterine fetal deaths with maceration (36.6, 95% CI 31.7–41.7; P=.001). There was no difference in success rates between mildly (43.9%, 95% CI 34.6–53.0), moderately (32.2%, 95% CI 24.9–40.3), or severely macerated fetuses (34.9, 95% CI 25.9–44.8; P=.14).
Overall, in our cohort of 750 intrauterine fetal deaths, cytogenetic analysis was valuable for determining the cause of death in 18.7% (n=140) of cases. In 2.8% (n=21) of cases the cytogenetic analysis established cause of death, in 7.2% (n=54) the cause of death could be excluded, and in 8.7% (n=65) the panel missed the results of this test for determining cause of death. In 81.3% (n=610) cases, the cytogenetic analysis was not valuable for determining the cause of death.
Invasive cytogenetic testing is recommended for all intrauterine fetal deaths before induction of labor because this had the highest success rates. Fetal deaths with morphologic abnormalities determined after birth accounted for most of the chromosomal abnormalities. However, the posterior probability of a chromosomal abnormality in the absence of morphologic abnormalities was still 4.6%. Small for gestational age, advanced maternal age, and maceration stage were not adequate selection criteria for cytogenetic analysis. Cytogenetic analysis postpartum in a selected group of intrauterine fetal deaths was not effective, because almost 70% of these tests failed. If invasive testing was not feasible, samples from the umbilical cord showed the highest success rates. Cytogenetic analysis was valuable in determining the cause of death in one fifth of intrauterine fetal deaths (18.7%).
Chromosomal abnormalities were established in 13% of intrauterine fetal deaths for which cytogenetic analysis was successful. This is higher than the 6–12% reported previously.3,9,10,29 Furthermore, the 13% we found is in fact an underestimation, because investigation of missing inclusions indicated a selection bias for deaths with known chromosomal abnormalities and congenital anomalies. However, the prevalence of the different chromosomal abnormalities we found compared with those published earlier30 was comparable. Parents with an intrauterine fetal death should be counseled about the prior probability of chromosomal abnormalities, which is at least 15-fold more than in livebirths.
Cytogenetic analysis was performed for 67.7% of the 750 intrauterine fetal deaths included in this study and was successful in 48.4%. The major reason this test was not performed in all the intrauterine fetal deaths was due to noncompliance with the study protocol. Others have reported overall successful chromosomal analysis in 41% of intrauterine fetal deaths17 and 43% of stillborns.9 Our highest success rates after death were for amniocentesis, CVS, and invasive testing in combination with fetal, placental, or umbilical cord biopsies. Amniocentesis and CVS were also the most successful in the study by Khare et al (90% and 100%, respectively)19 and in other studies.18,20 Identification of cytogenetic abnormalities during ongoing pregnancy compromised the completeness of the data in the postpartum evaluation, although this has no consequences for our conclusions. A uniform approach to obtaining karyotypes in one center and culturing fibroblasts in a central laboratory could have raised our low culture success rate. Amniocenteses and CVS rates were not high in our study. It is not common practice in the Netherlands to perform these tests after intrauterine fetal death, probably due to ignorance, inexperience with the procedure, or missing logistics. A similar situation has also been reported for the United States.2 Diminished or absent amniotic fluid (25%) may have also negatively influenced the decision to perform amniocentesis. In our experience, and in that of others, invasive testing after intrauterine fetal death is accepted well by parents.19,29 In contrast to earlier published clinical guidelines,11,13 we therefore recommend invasive cytogenetic analysis after intrauterine fetal death.
Alternatives to invasive cytogenetic analysis are fetal, placental, or umbilical cord biopsies postpartum. However, these tissue cultures may fail to grow due to nonvital tissue or bacterial or fungal overgrowth, contamination during passage through the birth canal, inappropriate collection of biopsies, and transport problems. In our study, overall cytogenetic analysis of tissues solely postpartum was unsuccessful in 71.5% of cases. Success rates varied between 32.1% for umbilical cord and 0% for pericardium. Pericardium was, however, often represented in the “multiple” tissue group, which had a success rate of 25.4%. Success rates of skin and placenta biopsies (19.2%, combined in one group) were comparable to earlier published skin biopsy success rates of 18%17 and 14%.19 Much higher success rates in skin biopsies (76%) were established in pregnancy terminations for fetal abnormalities.31 However, as success rates decrease with the duration of time that the fetus has been dead, skin biopsies for intrauterine fetal death should be avoided in our opinion. Our success rates for cartilage were comparable to the 27.3% published for stillbirths from 16 weeks of gestation,32 whereas placenta success rates (65.6%) in this study were much higher. Others have also advised placenta biopsies because they are often a successful source of viable fetal cells surviving for days after fetal death.14,19,33,34 The few placenta biopsies included in our study might have led to a lower yield of successful cytogenetic analysis and our postpartum failure rate might therefore be an overestimate. We only included a few placenta biopsies because earlier studies indicated that maternal cell contamination was a problem and placental mosaicism.17 Other fetal tissue types in our cohort showed comparable success rates with other studies, although some reported even lower success rates of 7%.17 We recommend cytogenetic analysis of umbilical cord postpartum as an alternative to invasive analysis, because this seems to have the highest culture success rate. Besides, sampling of umbilical cord is not as mutilating as sampling other tissue types. This is in contrast with earlier published advice that samples of fetal blood, skin, or fascia lata are good tissue sources.2,10,11,13
Additional molecular cytogenetic analyses, such as fluorescent in situ hybridization, multiplex ligation-dependent probe amplification, comparative genomic hybridization and quantitative fluorescent polymerase chain reaction, have been advised if cells do not grow in culture. Although the numbers in our study were small, these techniques seem promising; the majority of analyses were fluorescent in situ hybridization or multiplex ligation-dependent probe amplification because comparative genomic hybridization is more expensive. The feasibility of these techniques in common practice needs further evaluation.
Some authors recommend cytogenetic analysis for all fetal deaths,9,10,29 while others have suggested that it be attempted in a selected group.5,12,15,34,35 Prevalence of chromosomal abnormalities has been reported to be substantially higher in the presence of congenital anomalies.5,35–38 Some morphologic abnormalities will have been diagnosed at ultrasonography. However, interpretation of ultrasonography after intrauterine fetal death is not always conclusive. Prevalence of morphologic abnormalities determined at birth (27.8%) in our cohort was higher than the 20% reported earlier.9,39 Determination of these abnormalities is indeed significant for predicting abnormal chromosomes. However, whereas most chromosomally aberrant fetuses show some morphologic abnormalities, in our study 24% did not, and previously a figure of 10% was reported.9 In morphologically normal fetuses, the posterior probability of a chromosomal abnormality in our study was 4.6%, one third of our prior probability (13%). Others expected this to be less than 2%.10 In the presence of morphologic abnormalities, the posterior probability of a chromosomal abnormality was 38%. Others reported 25–50% fetal aneuploidy in the context of fetal abnormalities noted at postmortem examination.10,40 Our perinatal clinical geneticist observed some common variants diagnosed as morphologic abnormalities by the physician, illustrating an interobserver variation. A new prediction of the missed chromosomal abnormalities in morphologically normal fetuses reduced the missed chromosomal abnormalities to 4.4%. First-hand evaluation of morphologic abnormalities by trained pediatricians, geneticists, or pathologists would probably have given better posterior probability results, but this was not feasible in our study. However, if morphologic abnormalities are determined after birth there only remains the option of postpartum cytogenetic analysis, and much effort is required for little result. Parents who object to invasive cytogenetic analysis need to be counseled about these consequences, whereas after birth, the presence of morphologic abnormalities may influence the parents’ decision to allow cytogenetic analysis. Some may not find the level of 4.6% chromosomal abnormalities seen in morphologically normal fetuses acceptable.
In our study the chances of fetal aneuploidy were not increased for SGA fetuses, advanced maternal age (older than 35 years) or decreased gestational age, in contrast to results reported by others.10,14,19 Neither did we find SGA or advanced maternal age to be an adequate selection criterion for performing cytogenetic analysis. However, because we excluded intrauterine fetal deaths at 20–24 weeks from our SGA analysis, we could have introduced some bias. Khare et al19 found most chromosomal abnormalities in the second trimester. If resources are limited, they advised cytogenetic analysis for this group and for the third trimester if the intrauterine fetal death is associated with fetal anomalies. We disagree here, because we found more than one half of our chromosomal abnormalities in third-trimester intrauterine fetal deaths.
Some argue that cytogenetic analysis after intrauterine fetal death should only be attempted in nonmacerated or mildly macerated fetuses;41,42 again, we disagree. Similar to other reports, we found no significant differences in success rates between mildly, moderately, or severely macerated fetuses.9,33 Furthermore, we would have missed 20 chromosomal abnormalities if no effort had been made to assess macerated fetuses using cytogenetic analysis.
The decision to perform any test should be based on the value of the test, its cost, the resources involved, the risk of harm by the test, the ease with which the test can be performed, the effect of missing a positive result, and the diagnostic discrimination of the particular test.29 On most points, cytogenetic analysis for all intrauterine fetal deaths seems justified. Cost aspects were not evaluated in our study but others have done this and concluded that if nonselective stillbirth assessment is performed, new information relevant to a recurrence risk estimation, prenatal diagnosis recommendation, or preconceptual and prenatal treatment will be gained from 51% of stillborns.43
On the basis of our results we have drawn up a flowchart (Fig. 1) illustrating the clinical implications of our findings. We recommend invasive cytogenetic analysis as standard diagnostic workup after intrauterine fetal death. The flowchart also addresses the issues which should be presented to parents during counseling and some alternative options with their consequences. The risk of a chromosomal abnormality in an intrauterine fetal death cohort is substantially higher than for most cases of prenatal testing. Cytogenetic analysis after intrauterine fetal death, regardless of whether morphologic abnormalities are present, should be funded. Waiting to take samples for cytogenetic testing until after the birth means results are missed unnecessarily and money is wasted. Gaining skills and experience in invasive cytogenetic analysis should be part of the training program for all gynecologists.
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