Noonan syndrome and related disorders: Alterations in growth and puberty

Noonan syndrome is a relatively common multiple malformation syndrome with characteristic facies, short stature and congenital heart disease, most commonly pulmonary stenosis (Noonan, Clin Pediatr, 33:548–555, 1994). Recently, a mutation in the PTPN11 gene (Tartaglia, Mehler, Goldberg, Zampino, Brunner, Kremer et al., Nat Genet, 29:465–468, 2001) was found to be present in about 50% of individuals with Noonan syndrome. The phenotype noted in Noonan syndrome is also found in a number of other syndromes which include LEOPARD (Gorlin, Anderson, Blaw, Am J Dis Child, 17:652–662, 1969), Cardio-facio-cutaneous syndrome (Reynolds, Neri, Hermann, Blumberg, Coldwell, Miles et al., Am J Med Genet, 28:413–427, 1986) and Costello syndrome (Hennekam, Am J Med Genet, 117C(1):42–48, 2003). All three of these syndromes share similar cardiac defects and all have postnatal short stature. Very recently, HRAS mutations (Aoki, Niihori, Kawame, Kurosawa, Ohashi, Tanaka et al., Nat Genet, 37:1038–1040, 2005) have been found in the Costello syndrome and germline mutations in KRAS and BRAF genes (Rodriguez-Viciana, Tetsu, Tidyman, Estep, Conger, Santa Cruz et al., Nat Genet,2006; Niihori, Aoki, Narumi, Neri, Cave, Verloes et al., Nat Genet, 38:294–296, 2006) in the Cardio-facio-cutaneous syndrome. Phenotypic overlap between these genetic disorders can now be explained since each is caused by germline mutations that are major components of the RAS-MAPK pathway. This pathway plays an important role in growth factor and cytokine signaling as well as cancer pathogenesis.
Introduction
Noonan Syndrome (NS) has been recognized for about 40 years [1] but the genetic cause was not found until 2003 when mutations in the PTPN11 gene were reported [2]. Shortly afterwards LEOPARD syndrome (LS), a rare alleic variant of NS was found to have specific mutations in the PTPN11 gene [25]. Cardio-facio-cutaneous (CFC) syndrome and Costello syndrome (CS), both rare syndromes, share significant phenotypic overlap with NS especially in infancy. The common features of facial dysmorphism, short stature and similar cardiac findings in all four conditions suggest a similar underlying pathogenesis. In 2005 [6], mutations in the HRAS oncogene were found to be the cause of CS and in 2006 mutations in KRAS, BRAF, MEK1 and MEK2 genes [7, 8] were identified in CFC. All of these germline mutations are components of the RAS-MAPK pathway which plays an important role in growth factors and cytokine signaling. In this review, the clinical findings of the four syndromes will be reviewed. NS will be discussed in more detail since it is common and more clinical studies are available.
Noonan syndrome
NS is one of the more common non-chromosomal syndromes seen in children with congenital heart disease with an estimated incidence of 1 in 1,500 [1]. Although there is wide phenotypic variation in NS, distinctive facial features include hypertelorism, down-slanting palpebral fissures, a high arched palate, low set posteriorly rotated ears, malar hypoplasia, ptosis and often a short neck. The phenotype changes significantly with time. In the newborn, there is excessive nuchal skin which is the result of prenatal cystic hygroma. During infancy, the head is relatively large, the eyes are often prominent and round, there is a high nasal bridge which may be flat, and the neck appears short. At 3 to 4 years of age, the body becomes more stocky and the chest more prominent. The chest deformities often become significant. In later childhood, the facial appearance begins to show coarse features and becomes more triangular as the chin lengthens. The eyes become less prominent and the ptosis may be more apparent. In the teenager and young adult, as the neck lengthens webbing may become more apparent, the facial features are more triangular and become sharper, the nose has a pinched root and a thin high bridge. An older adult has prominent nasal labial folds, a high anterior hairline and the skin often appears rather transparent and wrinkled.
In most, the prenatal history is unremarkable but polyhydramnios is frequent. Height and weight are within normal limits at birth but height begins to drop off within a few months and over 70% of patients with NS have significantly short stature. Some patients with NS have significant feeding difficulties with resulting failure-to-thrive and require tube feedings. Although this may contribute to the poor gain in weight, short stature occurs equally in children who have no feeding problems in infancy. Other important findings include a chest deformity which may be in the form of a pectus carinatum or pectus excavatum, apparent widely spaced nipples and a relatively broad chest. Scoliosis and kyphosis occur in about 15% of patients. Muscle hypotonia is frequent and may account for some of the motor delay. Significant mental retardation is uncommon but some degree of learning disability is frequent and may require special help in school. Eye findings, especially strabismus and refractive errors are common and an occasional patient will have a coloboma. All children with NS should have a complete eye examination. Since conductive hearing loss is rather frequent, children should have a hearing evaluation. Over half of the males with NS have either one or both testes undescended and delay in puberty is common for both males and females.
Easy bruising is common in NS and a variety of bleeding problems have been reported [9]. These include deficiency of Factor XI, Von Willebrand’s disease, thrombocytopenia and platelet function defects. In addition, low levels of Factor VIII and XII have also been noted. Hepatosplenomegaly, usually unexplained, is present in about 25% of patients particularly in infancy. Lymphatic abnormalities occur in less than 20% of patients but may present serious problems. Over 80% of patients with NS have a cardiac finding. Pulmonary stenosis is, by far, the most common but nearly every cardiac lesion has been reported. In addition, hypertrophic cardiomyopathy may occur.
It was recognized early on that NS could be transmitted in an autosomal dominant manner. In 1994, Jamieson et al [10] was able to map the gene for NS to the distal part of chromosome 12q. Not all families with NS studied showed this linkage suggesting that more than one gene was likely to be involved in the etiology. In 2002, Tartaglia [2] found a mutation in the PTPN11 gene to be present in about 50% of patients with NS. This gene regulates the production of a protein called SHP-2 which is essential in several intracellular signal transduction pathways and controls a number of developmental processes including cardiac semilunar valvulogenesis. The protein is expressed throughout the body and it is an important player in cellular response to growth factors, hormones, cytokines and cell adhesion molecules. The PTPN11 mutations in NS are clustered in interacting portions of the N-SH2 in PTTP domains. This mutation results in a gain of function for SHP-2.
Children with NS often present to the endocrinologists because of the short stature, delayed puberty or undescended testes in males. Although height and weight are usually in the normal range at birth, height drops off within the first few months. In general, there is at least a 2-year delay between bone age and chronological age. Continued growth may occur until the early 20s. In both males and females, there is a delay in puberty. Females seem to possess normal fertility. Males, as expected due to undescended testes, appear to have decreased fertility but male transmission is well described and not uncommon.
The cause of short stature in NS is really not clear. After pharmacological stimuli, growth hormone secretion was usually normal in patients studied but a small number showed a subnormal response. Others have shown a neurosecretory growth hormone dysfunction to be present in some patients but this did not appear to have any effect on the response to growth hormone [11]. There have been previous studies looking at the IgF-1 levels which have been below normal for the majority reported. A considerable number of children have undergone treatment with human biosynthetic growth hormone. The majority of studies have shown similar results. There is a significant increase in growth velocity in the first and second year of growth hormone treatment [12–16]. The velocity in growth tends to diminish in succeeding years. Several authors claim that the predicted adult height has been increased in patients treated with growth hormone but there have been no real controlled studies to document the long-term effect of growth hormone on adult height. In those studies where bone age as well as actual height were measured, the increase in bone age was equal or slightly greater than the overall increase in height. If the bone age acceleration exceeds the acceleration in height, it is even possible that growth hormone therapy could actually decrease the adult height. Since there was little data available regarding adult height in NS, we recently [17] published a study of 73 adults who were all over 21 years of age at the time of their last measurement. Thirty-one percent of males and 32% of females achieved an adult height that was at the tenth percentile or greater which would be considered within a normal range. The remainder all fell below the tenth percentile but importantly 38% of males and 54.5% of females had an adult height below the third percentile. Adult height was unrelated to the presence or severity of cardiac disease and none of the adults achieving a normal height had been treated with growth hormone. Dr. Municchi et al [16] suggests that long-term serial height measurement over years comparing treated and untreated patients is needed to clearly show the benefit of growth hormone therapy on final adult height. There is no question that adult height can be achieved at an earlier age with the use of growth hormone.
With the discovery of the PTPN11 mutation, it is now possible to study patients with NS positive for a mutation and compare them with NS patients who do not carry a mutation of the PTPN11 gene. The large cohort reported by Ranke [18] suggested that weight and length were normal in 119 newborns with NS. With the availability of genetic testing, several recent studies have shown that mean birth length for NS patients with a mutation is slightly below normal and is less than that of the non-mutated NS newborns [19, 20]. In a study by Zenker [20], 88% of PTPN11 positive mutated children older than 3 years of age had a height less than two standard deviations and were significantly shorter than non-mutated children. All recent studies [21] suggest that there is a more severe mechanism acting on growth retardation in NS patients carrying a PTPN11 mutation. A recent study from France [19] evaluated 35 patients with NS, 20 of whom had a PTPN11 mutation. There was a trend to a shorter birth length in mutated versus non-mutated newborns and small for gestational age tended to be more frequent in mutated versus non-mutated patients. By 6 years of age, patients with mutations were significantly shorter than patients without mutations. The results of hormonal studies showed a normal growth hormone secretion after pharmacological stimuli and a low serum IgF-1 and ALS concentrations which is in contrast with a normal IgFBP-3 level. Since the PTPN11 gene has a negative effect on intracellular signaling downstream from several growth factor receptors, a growth hormone post receptor signaling resistance could represent the mechanism of stunted growth in NS. They felt that the lower growth response to growth hormone treatment observed in mutated versus non-mutated patients suggests some degree of resistance to growth hormone. Another recent study by Binder et al [22] showed a similar pattern. Data from these two studies would be in favor of growth hormone resistance by a late post receptor signaling defect specific for IgF-1 and ALS that does not effect IgFBP-3 stimulation. Changes in height during the first 2 years of growth hormone therapy in the pre-pubertal group show catch-up growth was less pronounced in patients with a mutation compared to those without a mutation. Fortunately, in all the studies using growth hormone, no adverse results have been observed. Many patients have undergone serial echocardiograms and none have shown an increase in their left ventricular mass index during growth hormone treatment [23]. These recent studies suggesting that there may be a primary IgF-1 deficiency in NS has stimulated and been incorporated into a phase II clinical study which will investigate the use of IPLEXtm (mecasermin rinfabate) (rDNA origin) which will be given by injection to treat growth failure due to insulin-like growth factor IgF-1 deficiency. Although we still do not understand how the mutation in the PTPN11 gene affects SHP-2, the result apparently is a disruption in the growth hormone IgF-1 axis and IgF-1 deficiency which could be the cause of growth failure.
LEOPARD syndrome (LS)
Gorlin [3], in 1969, introduced the acronym LEOPARD (LS) to describe a rare syndrome that shares many features similar to NS. These include autosomal dominant inheritance, similar facial dysmorphism and similar cardiac defects with an overabundance of hypertrophic cardiomyopathy compared to pulmonary stenosis. The characteristic cutaneous finding of lentigines is the main distinguishing characteristic. In addition, unilateral or bilateral hearing loss is frequent.
Sarkozy et al [24, 25] recently reported clinical and molecular studies in a consecutive study of 30 patients with LS and found mutations in the PTPN11 gene in 27 of the 30 patients studied. Mutations in LS have all occurred in exons 7, 12 and 13 while the more typical NS have the great majority of mutations occur in exons 3, 8 and 13. It is of interest that the mutations in patients with LS show a loss of function rather than gain a function as is found in the more typical NS patients.
Zenker [20] noted a specific mutation T468M in exon 12 which was also reported by Sarkozy in seven of their patients with LS to have less adverse effects on body growth. Only two of the ten patients with the T468M mutation have short stature.A cardiac abnormality was present in 71% of the patients with LS, with 80% of those showing hypertrophic cardiomyopathy. Pulmonary stenosis was present in two patients and a partial AV canal in another.
Cardio-facio-cutaneous syndrome (CFC)
It is often difficult to distinguish an infant with CFC from NS although, with time, the phenotype becomes more distinctive. Patients with CFC [4] have a high forehead, a relatively large head and bitemporal constriction. Like NS, they have a downward slant of the palpebral fissures, posteriorly rotated ears and a flat nasal bridge. The hair is usually sparse, curly and friable and absent eyebrows are frequent. The skin changes are variable but include keratosis pilaris with patchy hyperkeratosis [26]. In time, the phenotype for CFC becomes more distinctive and less Noonan-like. These patients are significantly delayed in both motor and mental skills and, like NS, they are hypotonic, often have failure-to-thrive with frequent gastrointestinal complaints and often require tube feedings. Cardiovascular abnormalities are similar to NS although hypertrophic cardiomyopathy is more common than in the typical NS patient. Although these children appear to be of normal height and weight at birth, they soon fail-to-thrive and short stature is found in 78% [27]. Bone age is significantly delayed and osteopenia is occasionally observed.
Very recently, mutations in four separate genes have been found to be associated with CFC [3, 4]. These four genes include BRAF, KRAS, MEK1 and MEK2. All of these genes belong to the same RAS-ERK pathway that regulates cell differentiation, proliferation and apoptosis. It is likely that the mechanism causing short stature in NS may be similar to that causing short stature in CFC. This is a very rare disease and there is little information regarding endocrine studies in patients with CFC.
Costello syndrome
Costello Syndrome (CS) [28] is a rare condition with a distinctive facial appearance which may be difficult to distinguish from NS and CFC in infancy. Although height and weight are normal or above normal at birth, severe growth retardation is the rule postnatally. Like NS, they have a large head, short wide nose and short neck. Unlike NS or CFC, these patients usually have thick and relatively prominent lips and tongue. They also have loose skin on the hands and feet and deep palmar and plantar creases. There is significant mental and motor delay. Cardiovascular abnormalities are found in about 60% and they are remarkably similar to that found in NS and CFC. Pulmonary stenosis, atrial septal defect and hypertrophic cardiomyopathy are the most common lesions [29]. Of particular interest is the high incidence of cardiac arrhythmias noted particularly in infancy [30] that is not characteristic of either CFC or NS.
Recently Aoki et al [4] reported mutations in HRAS a proto-oncogene to cause CS. Gripp et al [31] confirmed this finding and reported 33 of 40 patients with a clinical diagnosis of CS to have a HRAS mutation. All the mutations were de novo. Patients with CS are at a significantly increased risk for the development of malignancy, particularly rhabdomyosarcoma, neuroblastoma, ganglioneuroblastoma, and transitional cell carcinoma of the bladder. Although few studies of growth hormone have been carried out in Costello patients, the findings are very similar to that of NS. Response to growth hormone treatment has been variable. Kerr et al [32] have suggested that in CS, hormone treatment may be harmful because of the propensity to tumor formation and the presence of hypertrophic cardiomyopathy. He described two patients, one who had mild left ventricular hypertrophy with normal function before growth hormone was started. After 3 months of treatment, the cardiomyopathy progressed with significant left ventricular outflow tract obstruction with a gradient of 60 mmHg. He was treated with Propranolol. There was no further progression of the cardiomyopathy in spite of continuation of growth hormone. In the second case, growth hormone was started at 12 months and continued until age 26 months when a large pelvic mass was discovered which was proven to be an embryonal rhabdomyosarcoma. Growth hormone was stopped after the tumor was recognized. In spite of extensive chemotherapy, the patient died. Since CS is associated with both subaortic hypertrophic cardiomyopathy and tumors, the role of growth hormone in these two patients is unknown. At the present time, it is unclear whether growth hormone is beneficial or harmful in CS.
Conclusion
NS, LS, CFC and CS all have significant phenotypic overlap. Although they may be difficult to distinguish early on in life, in time they can usually be distinguished clinically. Recent studies show that each of these syndromes is caused by a germline mutation in a key component of the highly conserved RAS, RAF-ERK-MAP kinase cascade which is better known for its roles in growth factor and cytokines signaling in cancer pathogenesis. We still do not understand how these specific mutations cause the disease and why there are such distinct phenotypic differences in mutations within the same signaling pathway. Tartaglia et al [33] recently provided evidence that specificity in amino acid substitution is relevant to the functional deregulation of SHP-2 and disease pathogenesis. They showed NS mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated mutations and that Y279C and T468M amino acid substitutions noted in LS engender a loss of SHP-2 catalytic activity. It is not surprising that a mutation in the HRAS gene associated with CS has an increased incidence of tumors since these mutations are identical to the human tumor associated mutations. So far, CFC syndrome has not been associated with malignancy but the number of reported cases is still quite small with long-term follow-up limited. For the endocrinologist, it is interesting that all of the syndromes have a high incidence of short stature. LS, on the other hand, with loss of function appears to have a lower incidence of short stature but the higher incidence of hypertrophic cardiomyopathy. It will be of interest to see how effective IPLEXtm will be in treating the growth failure in NS and perhaps these related syndromes as well. Since the growth failure starts very soon after birth and may indeed start before birth, it is certainly possible that treatment may be necessary very early in life to achieve anywhere near a normal growth stature.

noonan syndrome
short stature
cardio-facio-cutaneous syndrome
costello syndrome
leopard syndrome
growth hormone


Objective Hypothermia is an adverse drug reaction (ADR) of antipsychotic drug (APD) use. Risk factors for hypothermia in ADP users are unknown. We studied which risk factors for hypothermia can be identified based on case reports.
Introduction
Antipsychotic drugs (APDs) can influence thermoregulation. Even before its psychotropic properties were clear in the early 1950s, the first manufactured APD, chloropromazine, was used to suppress compensatory responses to body cooling in surgery (artificial hibernation) [1].
The hypothermic effects of APDs seem less well known than the hyperthermic effects (e.g., malignant neuroleptic syndrome). Besides occasional case reports, little emphasis has been placed in scientific literature on hypothermia as adverse drug reaction (ADR). To our knowledge, a review of all reported and published cases of antipsychotic associated hypothermia has not yet been published.
Methods
The WHO international database for Adverse Drug Reactions was searched for reports of hypothermia and APD use (ATC-code N05A, with exclusion of lithium). The data in the database are collected from 77 countries participating in the WHO Program for International Drug Monitoring. This database comprises more than 3.5 million case reports, to which around 50,000 new reports are added quarterly. The relationship between the APD and hypothermia is evaluated by calculating the Reporting Odds Ratios (RORs) and 95% confidence intervals (95% CI) in a case/non-case design. The ROR compares the frequency of the reported ADR for a certain drug with the frequency of reports of that adverse drug reaction for all other drugs in the database. Reports concerning hypothermia were considered as cases, all other reports as non-cases. Index reports included all reports on an APD (ATC code beginning with N05A, with exclusion of lithium), all other reports were controls. When the number of reports of hypothermia in association with the APD is high and the number of reports of hypothermia in association with other drugs is low, ROR will be high. This also happens when the number of reports of other ADRs in association with the APD is low and the number of reports of other ADRs in association with other drugs is low. Since the vast majority of cases in the WHO database do not contain any details on indication of drug use, start and/or end dates and outcome, these factors cannot be analyzed with data from the WHO database. To get more information regarding characteristics of patients developing hypothermia during APD use, we performed a literature search in Medline and Embase for case reports with search terms “(antipsychotic OR neuroleptic) AND (hypothermia OR body temperature regulation), with no selection on date or language. From these articles, we searched the references for missing articles. Two reviewers judged all case reports. All relevant case reports were studied for patient, drug and environmental characteristics.
Results
In the WHO database, in January 2007, 480 reports were registered of patients developing hypothermia during the use of APDs. Characteristics of these reports are presented in Table 1. In the same period, 524 reports of hyperthermia associated with antipsychotic drug use were registered. Based on the reports, no specific pharmacological subgroup can be associated with an increased risk for hypothermia. Atypical antipsychotics are responsible for 55% of the reports, but this is mainly attributable to risperidone. Risperidone alone was responsible for 27% of all reports. A remarkable high association is found for pipamperone (ROR:24.62; 95% CI 13.16–46.07), an antipsychotic drug mainly used in Europe.
Table 1Antipsychotic drugs and hypothermia: reports from the WHO databaseAntipsychotic drugNumber of reports for hypothermiaNumber of reports for any ADR with this drugReported OR (CI)a for ADRs with ≥3 reportsTioxanthenes Zuclopenthixol13109415.88 (9.18–27.47) Flupenthixol616774.73 (2.12–10.55) Chlorprothixen450210.58 (3.95–28.31) Tiotixene39044.38 (1.41–13.62) Clopenthixol2293-Phenothiazines Thioridazine2344366.90 (4.57–10.41) Chlorpromazine1661823.43 (2.09–5.60) Levomepromazine1123486.21 (3.43–11.24) Cyamemazine911979.99 (5.18–19.27) Periciazine843025,00 (12.41–50.36) Pipothiazine319720.36 (6.50–63.71) Fluphenazine326561.49 (0.48–4.62) Trifluoperazine318952.09 (0.67–6.48) Perphenazine21522- Prochlorperazine24451- Promazine2307- Mesoridazine1215-Butyrofenones Haloperidol32105436.21 (3.43–11.24) Pipamperone1054624.62 (13.16–46.07) Droperidol21178- Benperidol1146-Benzamides Tiapride559611.14 (4.62–26.89) Sulpiride418282.89 (1.08–7.70) Amisulpiride11514- Sultopride172-Others Loxapine49285.70 (2.13–15.23) Pimozide36286.31 (2.03–19.65) Zotepine2260- Prothipendyl193- Penfluridol158-Atypical Risperidone129184319.65 (8.09–11.52) Clozapine68442552.05 (1.61–2.61) Olanzapine44160903.65 (2.71–4.91) Quetiapine2153745.19 (3.38–7.98) Aripiprazole1145663.18 (1.76–5.76) Ziprasidone829633.57 (1.78–7.15)aROR= (a/c) / (b/d); (a = no. of reports of adverse drug reaction with suspected drug; b = no. of reports of adverse drug reaction in total database; c = no. of reports regarding the suspected drug in database; d = total no. of reports in database). ROR is only calculated for APDs with three or more reports
The literature search resulted in 32 articles containing 43 case reports (December 2006) [3–34] from which characteristics are summarized in Table 2. Hypothermia following antipsychotic drug use is not associated with a specific age group. Reported ages vary from 0 to 90 years. In most cases, hypothermia is detected shortly following the start or dose increase of an antipsychotic drug. Most patients suffered from schizophrenia.
Table 2Characteristics of cases with hypothermia following antipsychotic drug use in literature (43 case reports, 46 episodes)CharacteristicsDataMale41%Age: mean (SD)49 (23.0)Range 0–90 yearsReported body temperature: mean (SD)32,6°C (2.7)Range 20.0–36.1°CDiagnosis known (n = 35)Schizophrenia51%Mental retardation11% Bipolar disorder11%Dementia11%Drug change Start or dose increase80% No change16%Interval drug change detection hypothermia <2 days57% 2–7 days16%Outcome death4% ICU admission24%Hospitalization (incl. prolonged)69%
Discussion
Hypothermia in patients using an APD is a serious, unpredictable, type B adverse event frequently leading to hospital and ICU admission and sometimes even to death. Some authors have even suggested that a substantial proportion of unexplained deaths should be attributed to antipsychotic-induced hypothermia [3, 35]. No single sufficient cause for hypothermia can be found in case reports. First, drug-receptor profile may play a role. Serotonin is associated with thermoregulation and APDs with a stronger affinity for the 5-HT2a receptor than for the D2-receptor (pipamperone, the atypical APDs) seem to be associated with hypothermia. The high association for relatively new drugs, like the atypical APDs, can partially be explained by reporting bias (reporting incidence for adverse drug effects is higher for new drugs and tends to decline in time; the so-called Weber-effect), but the high number of reports for risperidone should keep clinicians alert. Blocking alpha2-adrenergic receptors (e.g., chloropromazine, risperidone, clozapine, thioridazine) may also increase the hypothermic effect, by inhibiting peripheral responses to cooling (vasoconstriction and shivering). Next to this receptor profile, many patient-bound factors must be considered. Patients with pre-existing brain damage may be more susceptible to hypothermic effects. The pre-optic anterior hypothalamic region regulates body temperature. Animal studies show that lesions of this region give a hypothermic response following administration of an APD [36, 37]. In patients with multiple sclerosis, hypothermia is also associated with thalamic lesions [38]. Studies in schizophrenic patients show that core temperature decreases following the administration of APDs [39, 40]. Our search shows a predominance of case reports for schizophrenic patients and little for other frequent APD user groups like demented or delirious elderly. In schizophrenia, thermal regulation is altered. This may be explained by changes in neurotensine levels in schizophrenia. Neurotensine (NT) is one of the most important thermoregulatory peptides that also plays a role in the antipsychotic actions of APDs. In schizophrenic patients, NT concentration in cerebro-spinal fluid (CSF) is low and will be normalized following antipsychotic drug use [41]. The hypothermic reaction is also dependent on ambient temperature. In animal studies, APD administration at ambient temperatures below 22°C led to hypothermia, whereas APD administration in a room temperature of 29°C gave no thermal response and at 32°C an increase in rectal temperature [41, 42]. Normally, a cold environment will result in behavior aimed at protection against the cold (taking extra blankets or clothes). APDs, however, will induce apathy and indifference, resulting in unawareness of developing hypothermia. Since some case reports also mention the co-existence of infections at the time of development of hypothermia, this may also play a role in the dysregulation of thermal homeostasis.
There is an ongoing discussion concerning the value of case reports and spontaneous reports in the field of drug safety. There can be no discussion that pharmacodiligence requires prospective studies, access to regulatory filings for controlled and monitored use of drugs, and some sense of relative potency for comparison between different drug entities. However, large cohorts are needed for the detection and analysis of type B reactions. Since these data are lacking, case and spontaneous reports by alert clinicians must be analyzed [43]. The outcome of such an analysis cannot be seen as solid evidence, but can help to get more insight in the adverse drug reaction (ADR). The use of measures like ROR and disproportionality can help to detect drugs with an increased association with an ADR. A statistically significant ROR may be indicative of a higher risk for that particular event during the use of a specific medication, but is never conclusive for the actual existence of a causal relation [2].
The results of this study should alert physicians of the risk of hypothermia in psychiatric patients using APDs. There seems to be no direct relation between stable drug dose and the ADR; the period shortly after starting the APD or dose increase seems to be the high risk period. Often, drug changes are indicated by behavioral problems also leading to separation or isolation of the patient. In the case of separation, patients will be dressed lightly and, even at normal room temperature, can cool down easily. In these cases, the patient’s body temperature should be monitored daily (with a thermometer that can measure low body temperatures). Also, every change in behaviour or co-morbidity (e.g., infections) should be a warning sign to look for hypothermia.

hypothermia
thermoregulation
schizophrenia
adverse drug effect
antipsychotic agents


Frequency and diagnostic patterns of lymphomas in liver biopsies with respect to the WHO classification

The recent World Health Organization (WHO) classification of hematopoietic and lymphoid tissue tumors represents the first worldwide consensus classification of these malignancies. However, the applicability of this classification to a representative number of hepatic lymphomas in liver biopsy specimens has not yet been investigated. The frequency and infiltration pattern of a series of 205 liver biopsies with lymphoma manifestations was analyzed with the aid of immunohistochemical and molecular pathological analyses. Diffuse large B-cell lymphoma (DLBCL) was by far the most frequent entity, comprising 45% of the cases analyzed. Using a previously published immunohistochemical algorithm, 35% of 80 DLBCL were assigned to a germinal center B-cell-like (GCB) and 65% to a non-GCB group. Most B-cell lymphoma entities involving the liver revealed a characteristic infiltration pattern. Diagnostically challenging entities were T-cell-rich B-cell lymphomas, anaplastic large cell lymphomas and peripheral T-cell lymphomas, which frequently required additional molecular clonality assessment. Overall, the percentage of T-cell lymphomas in the liver (12%) was higher as compared to other extranodal sites except for the skin and the small intestine. This study provides relevant data on the distribution of hepatic lymphomas and demonstrates the applicability of the WHO classification proposing a diagnostic algorithm for liver biopsies.
Introduction
The liver is most commonly involved in non-Hodgkin lymphomas (NHL) next to lymph nodes, spleen, and bone marrow. In the vast majority of cases hepatic involvement reflects secondary dissemination in advanced disease [15, 18] rather than a primary site according to the definition of Caccamo and coworkers [5]. Histopathological analysis of liver biopsy may be required in patients with an established diagnosis of malignant lymphoma to differentiate lymphoma manifestation from other causes of hepatic dysfunction or to clarify elevated transaminases such as toxic damage because of chemotherapy or other medications. On the other hand, a biopsy may reveal a previously unknown lymphoma during the exploration of a solid hepatic mass or after measuring elevated liver enzyme serology [12]. Occasionally, a malignant lymphoma can be detectable in the setting of other liver diseases such as chronic hepatitis B [20, 22], chronic hepatitis C [2, 3, 7, 21, 25], or primary biliary cirrhosis (PBC) [24].
The goals of the present study were to test the feasibility of lymphoma subtyping according to the World Health Organisation (WHO) classification of tumors of hematopoietic and lymphoid tissues [16] in liver biopsy specimens and to describe the frequencies by which the different entities occur. For this purpose, a series of 205 liver biopsies with a diagnosis of malignant lymphoma were analyzed retrospectively with a specific focus on the histopathology, especially infiltration patterns, which may ultimately allow the use of diagnostic algorithms for subtyping of lymphomas in liver biopsies. This is an important addition in relation to previous studies, which have mainly described the frequency of liver involvement in autopsy material of patients with leukemia or lymphoma [8, 13, 26, 28, 29] differentiating only between low-grade and high-grade NHL [26] or having performed a categorization according to the outdated Kiel classification [29].
Overall, this study represents the largest series on hepatic lymphomas to date and due to its restriction to bioptic material reflects the primary diagnostic situation.
Materials and methods
All cases of hepatic lymphoma involvement diagnosed by liver biopsy during the years 1994–2003 were retrieved from the archives of the Institute of Pathology, Campus Benjamin Franklin, Charité-University Medicine Berlin and the Institute of Pathology, University of Cologne, Germany. The series comprised a total of 205 cases with 135 cases from Berlin and 70 cases from Cologne. All cases were reevaluated and reclassified independently by three pathologists (C.L., T.L., H.S.) according to the WHO classification [16]. For cases in which the diagnosis was not unanimous, a final consensus diagnosis was reached after further immunophenotyping, molecular analyses, and final consultation. In six cases initially considered “suspicious” for lymphoma, a final diagnosis of lymphoma was made after reevaluation and additional analyses. Nine liver biopsies with insufficient material for additional immunohistochemical or molecular analyses were excluded beforehand, as well as five cases where T-cell receptor (TCR) polymerase chain reaction (PCR) had revealed a polyclonal or oligoclonal rearrangement pattern without a reproducible dominant PCR product.
Overall, 32/205 (16%) cases were reclassified, including eight “low-grade” [5× B-CLL, 2× follicular lymphomas (FL), 1× marginal zone lymphoma (MZL)] and 20 “high-grade” [19× DLBCL, 1× Burkitt lymphoma (BL)] B-cell NHL that were assigned to a specific WHO lymphoma category as well as four cases with a change in the final diagnosis [BL, classical Hodgkin lymphoma (cHL), T-cell-rich B-cell lymphoma (TCRBCL), lymphoplasmacytic lymphoma (LPL) to DLBCL, anaplastic large cell lymphoma (ALCL), peripheral T-cell lymphoma (pTCL) and B-CLL, respectively].
The analyses of the infiltration pattern were based on the assessment of the hepatic architecture. Three main patterns were distinguished: Infiltrates for which an exclusive or predominant association to portal tracts was evident were recorded as portal infiltrates. The second pattern consisted of lymphoma infiltrates which showed a coherent growth pattern thus predominantly resulting in the replacement of the acinar structures. These infiltrates were designated as a nodular growth pattern. Finally, infiltrates that showed prominent intrasinusoidal dispersion of the lymphoma cells were recorded as a sinusoidal growth pattern. Additionally, the density of the infiltrate was semiquantitatively assessed. The presence of scattered neoplastic cells in a background rich in reactive bystander cells (e.g., nonneoplastic T-cells and/or macrophages in T-cell rich B-cell lymphoma) was defined as a loose infiltration pattern, whereas the appearance of coherently appearing neoplastic B- or T-cells was referred to as a dense infiltrate.
According to clinical information available, in 76/205 (37%) of the cases the diagnosis of a lymphoma had been previously established from extrahepatic biopsies or peripheral blood indicating secondary involvement of the liver. In four of these cases with known low-grade lymphoma (2× B-CLL, 2× FL) liver biopsy revealed transformation to a high-grade lymphoma (DLBCL). In the remaining cases, the biopsy obtained from the liver represented the site of primary diagnosis. In 26 (13%) patients, additional biopsies from extrahepatic sites were available (13 bone marrow biopsies, 10 lymph node biopsies, and three spleen biopsies). Furthermore, in a few cases clinical data regarding a potential predisposing condition for a primary hepatic lymphoma was available: chronic hepatitis C was reported in four patients (3× DLBCL, 1× B-CLL), HIV infection was present in three patients (2× DLBCL, 1× BL), and one marginal zone lymphoma occurred in a patient after liver transplantation.
The frequency of the various lymphoma entities diagnosed in liver biopsy specimen was compared to their frequency and distribution in other extranodal sites (cases from the Consultation and Reference Center for Lymph Node Pathology and Hematopathology, Berlin).
Immunohistochemistry
For immunostaining, 4 μm thick sections were cut, deparaffinized, and subjected to heat-induced epitope retrieval before incubation with antibodies. For this purpose, sections were immersed either in sodium citrate buffer at pH 6.0 or, alternatively, in ethylenediamintetraacetic acid (EDTA) at pH 8.0 and heated in a high-pressure cooker. After cooking, the slides were rinsed in running water, washed with Tris-buffered saline, pH 7.4, and incubated with the respective primary antibodies. All primary antibodies employed are listed in Table 1. With the exception of immunoglobulin detection, in which the streptavidin–biotin peroxidase method was applied [14], labeling was carried out using the alkaline–phosphatase/anti-alkaline–phosphatase complex method [6]. Alkaline phosphatase was developed using Fast Red as the chromogen, whereas peroxidase was visualized with diaminobenzidine chromogen as the substrate.
Table 1Antibodies used in this studyAntibodyCloneAntigen retrievalDilutionSourceALK1ALK1Citrate1:20DakoBCL2124Citrate1:25DakoBCL6594Citrate1:25DakoCD2AB75Citrate1:50NovocastraCD3F7.2.38Citrate1:100DakoCD41F6Citrate1:25NovocastraCD54C7Citrate1:25NovocastraCD7CD7-272EDTA1:50NovocastraCD8C8/144BCitrate1:100DakoCD1056C6Citrate1:25NovocastraCD15C3D1Citrate1:20DakoCD20L26Citrate1:50DakoCD211F8Protease1:50DakoCD231B12Citrate1:20NovocastraCD27137B4EDTA1:100NovocastraCD30BerH2Citrate1:50DakoCD43DF-T1Citrate1:50DakoCD68PG-M1Citrate1:50DakoCD79aJCB117Citrate1:100DakoCD138B-B4Citrate1:10SerotecCyclin D1P2D11F11Citrate1:50NovocastraEBV-LMPCS1-4Citrate1:100Dakoα-heavychainRabbit polyclonalCitrate1:40 000Dakoγ-heavy chainRabbit polyclonalCitrate1:30 000Dakoδ-heavy chainRabbit polyclonalCitrate1:2000Dakoμ-heavy chainRabbit polyclonalCitrate1:2000DakoKi-67MIB-1Citrate1:2000Dakoκ-light chainRabbit polyclonalCitrate1:100 000Dakoλ-light chainHP6054Citrate1:16 000DakoMUM1/IRF4MUM1pCitrate1:20Generously provided by Prof. B. Falini, Perugia, ItalyPax-524Citrate1:10Transduction LaboratoriesTIA-12G9Citrate1:500Coulter
Molecular pathology analyses
Additional molecular pathology analyses were selectively performed for those cases in which immunoprofiling alone was not sufficient to establish a definite diagnosis of a malignant lymphoma.
For this purpose, DNA was extracted after dewaxing 20-μm-thick paraffin sections with QIAEX (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. TCR-gamma rearrangements were analyzed using four different primer combinations (JGT1/2, JGT3, BioMed-2 Set A and Set B). The primer combinations JGT1/2 und JGT3 suitable to amplify the most frequent TCR-gamma rearrangements were used in a two-step seminested PCR. The BioMed-2 primer sets A and B able to detect all possible TCR-gamma rearrangements were performed as single round PCRs. The detailed cycling conditions (50 rounds of amplification for BioMed-2 set A and set B; two-time 35 cycles for primer combinations JGT1/2 and JGT3) for all PCRs have been described in detail elsewhere [9, 27].
Amplification of rearranged IgH genes was independently performed at least twice per case employing three different framework primer sets (BioMed-2 FR1, FR2, and FR3) separately in conjunction with a JH primer (JH22). PCR conditions consisted of 50 cycles of denaturation (95°C, 15 s), primer annealing (60°C, 40 s), and elongation (72°C, 45 s), and the reaction mixture contained 1.5 mM MgCl2, 0.8 mM deoxyribonucleotide triphosphates (dNTPs), 70 pmol VH primers, 30 pmol JH22 primer, and 2 U of AmpliTaq Gold polymerase (Applied Biosystems, Weiterstadt, Germany).
Results
Characteristics of hepatic lymphomas
Almost 90% (182/205) of the hepatic lymphomas belonged to the group of NHL, whereas only 23 of the 205 cases were cHL. Regarding NHL, 86% (157/182) were of B-cell and 14% (25/182) of T-cell origin. Overall, diffuse large B-cell lymphoma (DLBCL) was the most common type [51% (93/182) of all B-NHL analyzed] with TCRBCL—a morphologic variant of DLBCL—comprising 14% (13/93) of all DLBCL. According to the algorithm of Hans et al. [11] a predominance of DLBCL of non-GCB (germinal center B-cell-like) type (52/80; 65%) compared to the DLBCL of GCB type (28/80; 35%) was found (Fig. 1a–d,f). Only 4 out of 80 cases (5%) corresponded to the group of CD5-positive DLBCL (Fig. 1e).
Fig. 1Nodular pattern: DLBCL of GCB group positive for CD10 (a) and BCL6 (b) and negative for IRF4/MUM1 (few scattered plasma cells serve as positive intrinsic control; c). CD5 positive DLBCL of ABC type negative for CD10 (positive bile canaliculi serve as intrinsic control; d), positive for CD5 (e) and IRF4/MUM1 (f). TCRBCL with less than 10% large CD20 positive neoplastic B-cells (g) associated with numerous CD3 positive T-cells (h) and CD68 positive histiocytes (i)
In all lymphoma subtypes, men were affected more often, except for FL, which occurred with equal frequencies in both sexes. The male predominance was most striking in lymphomas of T-cell origin, in which ALCL and hepatosplenic T-cell lymphoma (HSTCL) were encountered exclusively in men. Additional molecular analyses were essential to confirm the suspected diagnosis of a lymphoma in 5 out of 19 T-cell lymphomas (26%) and 3 out of 116 (3%) B-NHLs (Fig. 2).
Fig. 2Size fragment analyses (GeneScan) of the amplificates after TCR-gamma PCR (primer combination JGT1/2). Fluorescence-labelled PCR products (blue lines) were separated on capillary electrophoresis system (Applied Biosystems, Model 310A) in parallel to a size standard (red line). The dominant PCR product (arrow) indicating the presence of a clonal T-cell population was reproducibly detectable in addition to a moderate oligo-/polyclonal T-cell background
The relative frequencies of the lymphoma subtypes, the available patient data and the infiltration patterns are summarized in Table 2.
Table 2Frequency, age distribution and pattern of infiltration in the different lymphoma entities presenting in the liver (Berlin/Cologne 1994–2003) BerlinCologneBerlin + Cologne, n (%)Age (range)Age (mean)Male/femalePatternDensity of infiltrateSinusoidalPortalNodularDenseLooseDiffuse large B-cell lymphoma (DLBCL)a522880(39)16–876450/3061972755T-cell rich B-cell lymphoma (TCRBCL)9413(6)35–85599/40115013B-cell chronic lymphocytic leukemia (B-CLL)131326(13)44–826616/107252251Classical Hodgkin lymphoma (cHL)101323(11)21–915414/912110320Follicular lymphoma (FL)11314(7)38–83627/70114140Marginal zone lymphoma (MZL)617(3)49–80706/127061Leukemic Plasmacytoma (PL)426(3)61–88704/241251Burkitt lymphoma (BL)5–5(2)30–70473/200550Mantle cell lymphoma (MCL)3–3(1)60–66632/113130B-lymphoblastic leukaemia (B-ALL)2–2(1)18–28221/120020Hairy cell leukaemia (HCL)1–142–0/110010Peripheral T-cell lymphoma, unspecified (pTCL)13518(9)38–846214/4668117Anaplastic large cell lymphoma (ALCL)b5–5(2)24–76585/033223Hepatosplenic T-cell lymphoma (HSTCL)112(1)38–51452/020020Total13570205aIncluding centroblastic (n = 69), immunoblastic (n = 3) and anaplastic variant (n = 8)bIncluding one anaplastic large cell lymphoma kinase (ALK) protein positive case
Infiltration patterns of the various lymphoma types
Most of the cases showed the predominance of a certain growth pattern, although some cases exhibited more than one growth pattern. Almost all DLBCL and BL showed a nodular infiltration pattern (91%) composed of a dense infiltrate (94%; Table 3), except for the TCRBCL variant, which was located predominantly within the portal tracts (85%) and exhibited the characteristic scattered infiltrate of fewer than 10% large neoplastic B-cells admixed with many reactive T-cells and histiocytes (Fig. 1g–i). Lymphomas showing a dense portal infiltration pattern were chronic lymphocytic leukemia/small lymphocytic lymphoma (96%; Fig. 3a–c), FL (79%; Fig. 3d–f), MZL (100%; Fig. 3g–i), and mantle cell lymphoma (100%). In contrast, in precursor B lymphoblastic leukemia/lymphoblastic lymphoma (B-ALL/B-LBL; Fig. 4a–c), HSTCL (Fig. 4d–f), leukemic plasmacytoma (Fig. 4g–i), and hairy cell leukemia, a sinusoidal infiltration pattern was observed in the majority of cases. The cHL displayed a predominantly portal infiltration pattern (Fig. 5a–c). Peripheral T-cell lymphoma and ALCL showed a spectrum of portal, sinusoidal, nodular, or mixed patterns with a varying density of infiltration, and thus did not allow for pattern-based selection of additional analyses (Fig. 5d–f). The infiltration patterns, differential diagnoses, and discriminative immunohistochemical markers are summarized in Table 3.
Table 3Infiltration pattern, differential diagnosis and characteristic immunohistochemical markersFig. 3Portal pattern: chronic lymphocytic leukemia (B-CLL) with a predominantly portal infiltrate of small lymphocytes (a) with expression of CD23 (b) and CD5 (c). FL (d) with expression of CD10 (e) and BCL2 (f). Marginal zone B-cell lymphoma with portal involvement (g), expression of memory B-cell marker CD27 (h) and a lymphoepithelial lesion of a CK7 positive bile duct (i)Fig. 4Sinusoidal pattern: precursor B lymphoblastic lymphoma (B-LBL) with a sinusoidal infiltrate of small blasts (a) with cytoplasmic expression of CD79a (b) and nuclear expression of terminal deoxynucleotidyl transferase (TdT; c). HSTCL with a sinusoidal infiltrate of monotonous neoplastic cells (d) with expression of CD3 (e) and the cytotoxic granule associated protein TIA-1 (f). Peripheral blood involvement (plasma cell leukemia) in a plasmacytoma with a sinusoidal infiltrate of plasma cells (g) with expression of CD138 (h) and IgG (i)Fig. 5Classical Hodgkin lymphoma vs. anaplastic large cell lymphoma: cHL with portal infiltrates of Hodgkin and Reed-Sternberg HRS cells (arrows) in a background rich in eosinophils (a). The HRS cells strongly express CD30 (b) and the EBV encoded latent membrane protein 1 (LMP1) (c). Anaplastic large cell lymphoma (ALCL) with portal infiltrates of pleomorphic large cells resembling HRS cells (arrows) (d) and strong positivity for CD30 (e) and the cytotoxic molecule perforin (f)
Comparison of hepatic lymphomas with other extranodal lymphomas
The percentage of T-cell lymphomas was considerably higher in the liver (12%, 25/205) when compared to other extranodal sites (5%, 165/3252) except for the skin (50%, mainly mycosis fungoides/Sézary syndrome) and small intestine (34%, mainly enteropathy-type T-cell lymphomas).
The high proportion of DLBCL in lymphomas of the liver (45%) was only surpassed by the small intestine (64%), the brain (83%), and the testis (97%). Of note, TCRBCL was extremely rare in other extranodal sites (0,3%) with only a few cases occurring in the bone marrow and spleen. Interestingly, one case fulfilled the criteria of TCRBCL in the liver, and an additional lymph node biopsy showed the picture of a typical DLBCL, whereas in another case, both liver and spleen biopsy showed the characteristic features of TCRBCL. The frequencies of other extranodal lymphomas compared to hepatic lymphomas are summarized in Table 4.
Table 4Frequency of extranodal lymphoma at different sites (Berlin 1994–2003) Brain (n = 31)Testis (n = 61)Lung (n = 79)Bone marrow (n = 1996)Stomach (n = 855)Small intestine (n = 71)Large intestine (n = 102)Spleen (n = 128)Skin (n = 427)Liver (n = 135)DLBCL255722662383032217852TCRBCL–––7–––3–9FL1–21681633209111B-CLL1–265612–410713PL3––473–––124MZL––401757741218*126MCL––310777411963LPL––3122––2135–HCL–––112–––6–1B-ALL–1–78–––1–2BL–1–4–32–25cHL––352–––31010B-NHL total (%)30/31 (97%)59/61 (97%)75/79 (95%)1862/1996 (93%)850/855 (99%)47/71 (66%)96/102 (94%)115/128 (90%)213/427 (50%)116/135 (86%)pTCL1–393466129413NK/T–1–––2––2–T-ALL–1–29––––9–ALCL––1816––265Enteropathy-type TCL–––––10––––HSTCL–––5–––1–1MF––––––––83–T-NHL total (%)1/31 (3%)2/61 (3%)4/79 (5%)134/1996 (7%)5/855 (1%)24/71 (34%)6/102 (6%)13/128 (10%)214/427 (50%)19/135 (14%)*Including nine cases of splenic marginal zone lymphoma (SMZL)
Discussion
To the best of our knowledge, this study represents the largest series focusing on the histopathology of hepatic lymphoma involvement and the first application of the WHO classification of tumors of hematopoietic and lymphoid tissues for this purpose [16]. Our data demonstrate the feasibility of in vivo lymphoma subtyping in liver biopsy with the aid of infiltration pattern analysis, the selection of differentiating immunophenotypic markers and additional molecular methods.
Many B-cell-derived lymphomas involving the liver revealed a characteristic infiltration pattern, which facilitated the use of a restricted panel of immunohistochemical markers to reach a final diagnosis. This approach is critical in those liver biopsies in which a lymphoma diagnosis has previously not been established, as accurate subtyping of lymphomas is fundamental for the initiation of adequate treatment.
Diffuse large B-cell lymphomas (DLBCL) accounted for 51% of all B-NHL cases. DLBCL was recently subdivided into different prognostic groups designated GCB, activated B-cell-like (ABC or non-GCB) and Type 3, according to their gene expression profiles [1]. The GCB group is described to have a better survival than the ABC group and the heterogeneous Type 3 group. Hans et al. [11] have proposed a simple immunohistochemical algorithm to assign cases to a GCB and a non-GCB group. In the present study, the frequency of either group in 80 liver biopsies with DLBCL was investigated, excluding the T-cell/histiocyte rich variant (n = 13). DLBCL of non-GCB type (65%) were more frequent in the liver than the GCB type (35%), whereas these two groups are described in other sites at about equal frequency [23]. As liver involvement with malignant lymphomas occurs secondary in advanced disease [15, 18], this observation may reflect the biological behavior of ABC-group DLBCLs through the selection of more aggressive cases by liver biopsy.
Approximately 10% of the de novo DLBCL express CD5 [4] and may have a poor outcome [31]. They tend to be associated with extranodal sites, especially bone marrow and spleen [19]. In the present series of hepatic lymphoma involvement, the number of CD5 positive DLBCL (4/80 or 5%) was not increased.
The TCRBCL, a variant of DLBCL in which the majority of cells are nonneoplastic T-cells and histiocytes, are often misdiagnosed as a reactive inflammatory condition or as a T-cell lymphoma infiltration of the liver [8, 17]. TCRBCL is relatively rare in lymph node biopsies [(23/976) or 2% of the DLBCL in the Berlin Reference Centre for Lymph Node Pathology from 1994–2003]. However, in the present study, 13/93 (14%) of the DLBCL in the liver were found to fulfill the WHO criteria for TCRBCL thus confirming previous data from Dargent and coworkers in their series of 62 liver specimens [8]. Additionally, one case with TCRBCL characteristics in the liver showed the typical morphology of a conventional DLBCL in an additional lymph node biopsy. This finding indicates that the composition of the reactive coinfiltrate may be influenced by the tissue-specific microenvironment. Therefore, the specific environment of a certain organ may influence the intensity of the T-cell response and subsequently explain the higher frequency of the TCRBCL variant of DLBLC presenting in the liver [10]. Limitations were encountered in the grading of follicular lymphoma and the subtyping of the cHL, which is generally not possible with sufficient certainty in needle biopsies.
In contrast to B-cell lymphomas, T-cell lymphomas infiltrating the liver generally lack a typical infiltration pattern, making their diagnosis more challenging. In particular, ALCL may mimic cHL due to the possible resemblance of Hodgkin and Reed-Sternberg cells and expression of CD30. Thus, additional markers against pan-T and pan-B cell antigens (including pax-5), CD15, ALK-1, cytotoxic molecules, e.g., TIA-1, granzyme B, and perforin) are required when confronted with this differential diagnosis (Fig. 5).
Similarly, the distinction between a T-cell lymphoma and a drug-induced or viral hepatitis (e.g., due to Epstein-Barr virus) can be very difficult in biopsies containing an increased number of sinusoidal T-cells. Therefore, additional clonality analyses by TCR or IgH PCR were necessary in 5 out of 19 (26%) T-cell lymphomas compared to only 3 out of 116 (3%) B-cell lymphomas to establish a diagnosis.
The reason for the relatively high frequency of liver involvement by T-cell lymphomas is not known. As T-cells play an important role in the regulation of the hepatic immune responses, especially in chronic viral and autoimmune hepatitis [30], it is tempting to speculate whether this may influence the relatively high incidence of T-cell lymphomas in the liver compared to other extranodal sites. In the present study, 25 of the 205 (12%) cases were found to represent lymphomas derived from T-cells, whereas lymphomas of the testis, brain, lung, and stomach were B-cell lymphomas in nearly all instances (Table 4).
In summary, the present study demonstrates the feasibility of subtyping lymphoma infiltrates in liver biopsies according to the WHO classification. The large number of 205 cases provides reliable information regarding the relative frequencies of the different lymphoma entities encountered in the liver and demonstrates the usefulness of infiltration pattern analysis for diagnostic purposes.

lymphoma
liver
pcr
immunohistochemistry
differential diagnosis


Is there an association between depressive and urinary symptoms during and after pregnancy?

Depressive symptoms and urinary symptoms are both highly prevalent in pregnancy. In the general population, an association is reported between urinary symptoms and depressive symptoms. The association of depressive and urinary symptoms has not yet been assessed in pregnancy. In this study, we assessed (1) the prevalence of depressive symptoms, over-active bladder (OAB) syndrome, urge urinary incontinence (UUI) and stress urinary incontinence (SUI) during and after pregnancy using the Center for Epidemiologic Studies Depression Scale (CES-D) and the Urogenital Distress Inventory (UDI) and (2) the association of depressive symptoms with urinary incontinence and over-active bladder syndrome during and after pregnancy, controlling for confounding socioeconomic, psychosocial, behavioural and biomedical factors in a cohort of healthy nulliparous women. Our data show a significant increase in prevalence of depressive symptoms, UUI, SUI and OAB during pregnancy and a significant reduction in prevalence of depressive symptoms, SUI and OAB after childbirth. UUI prevalence did not significantly decrease after childbirth. In univariate analysis, urinary incontinence and the OAB syndrome were significantly associated with a CES-D score indicative of a possible clinical depression at 36 weeks gestation. However, after adjusting for possible confounding factors, only the OAB syndrome remained significantly associated (OR 4.4 [1.8–10.5]). No association was found between depressive and urinary symptoms at 1 year post-partum. Only OAB was independently associated with depressive symptoms during pregnancy. Possible explanations for this association are discussed.
Introduction
The lifetime risk of depressive symptoms in women (5.9–21.3%) is about twice that in men and symptoms often start in the childbearing years [1–3]. Prevalence rates of 10–25% during pregnancy [4–9] and of 6–16% post-partum are reported [5, 6, 8, 10–12]. Prevalence rates vary because of the diversity of populations and diagnostic methodology. Most researchers found higher rates during pregnancy than after childbirth [5, 6, 8]. Urogenital symptoms are also more prevalent in pregnancy than in the general population. Prevalence rates of 9–50% have been reported for urinary incontinence (UI) during first pregnancy and 34–95% for frequency and urgency symptoms (over-active bladder [OAB] syndrome) [13, 14]. Strong associations are found between depressive symptoms, urinary incontinence and over-active bladder syndrome in non-pregnant women [15–19]. Whilst the relationship of depressive symptoms and urinary symptoms has not yet been explored in pregnancy, several factors have been found to be associated with depressive symptoms in a general, pregnant and post-partum population. These factors include biomedical factors such as obesity, age, chronic pain (like back pain), previous depressive symptoms [12, 20, 21], behavioural factors like excessive use of alcohol, smoking, lack of leisure time physical exercise and socioeconomic factors like unemployment and low job satisfaction [5, 7, 22]. Psychosocial factors, such as poorer social support, stressful life events and personality features have also been found related to depressive symptoms [11, 22–25].
The aim of this study was to (1) analyse the prevalence of depressive symptoms and urinary symptoms during and after pregnancy and (2) assess the association of urinary symptoms with depressive symptoms, controlling for psychosocial, behavioural, socioeconomic and biomedical factors during and after pregnancy.
Materials and methods
Study population
Between January 2002 and July 2003, 1,366 nulliparous pregnant women from 10 urban midwifery practices in the center of The Netherlands were approached to take part in a prospective longitudinal cohort study assessing pelvic floor problems, sexuality and back pain during first pregnancy until 1 year after delivery. All nulliparous pregnant women received information about the study from the midwives. After 1 week, the women were approached by phone and asked if they wanted to participate in the study. Inclusion criteria were a singleton low risk pregnancy and sufficient knowledge of the Dutch language.
One hundred and twenty-two women were excluded due to having a twin pregnancy (n = 2), miscarriage (n = 13) or insufficient knowledge of the Dutch language (107). Thus, 1,244 women met the inclusion criteria. Of these, 672 (54%) decided to participate in the study. The most common reasons for refusal were lack of time and the intensity and intrusiveness of the questions. The present study is a separate analysis of data collected from the larger study. The Medical Ethics Committee of the University Medical Center Utrecht approved the study. All participants signed an informed consent form.
Data collection
To assess the prevalence rates at different points in time of depressive symptoms, stress urinary incontinence (SUI), urge urinary incontinence (UUI) and over-active bladder (OAB) syndrome, we used data obtained from all respondents of questionnaires sent at 12 and 36 weeks gestation and 3 and 12 months post-partum. In addition, we analysed data obtained from the questionnaires sent at 36 weeks gestation and 12 months after delivery to assess the possible association between urinary symptoms and depression. We did so because the prevalence of urinary symptoms peak in the third trimester and because pelvic floor symptoms, occurring in pregnancy and persisting 1 year after delivery, may be associated with depression at this time [26].
Depressive symptoms were investigated using the Center for Epidemiologic Studies Depression Scale (CES-D). This scale is developed for use in non-psychiatric populations and gives an impression of depressive symptoms [27, 28]. The total score ranges from 0 to 60; a higher score corresponds with more depressive symptoms. A cut-off score of 16 is frequently used as an indication of a possible clinical depression. We refer in this study to women who scored B16 on the CES-D as having depressive symptoms.
Urinary symptoms were assessed with the Urogenital Distress Inventory (UDI) [29, 30]. The UDI is a validated, standardised questionnaire, translated in Dutch. This questionnaire consists of 19 questions about urogenital symptoms and the experienced discomfort of these symptoms. We looked specifically at self-reported urge and stress urinary incontinence and over-active bladder syndrome. Although the UDI also consists of questions on prolapse, obstructive micturition and pain and heaviness in the pelvic area, these symptoms have not been linked to depression in current literature. Therefore, these questions were not used in the present study. We followed the definitions of the International Continence Society (ICS) [31]. Urge incontinence was determined by a positive answer to the question: “Do you experience urine leakage related to the feeling of urgency?” Stress incontinence was determined by a positive answer to the question: “Do you experience urine leakage related to physical activity, coughing or sneezing?” Over-active bladder syndrome was determined when both of the following questions were answered positively: “Do you experience frequent urination?” and “Do you experience a strong feeling of urgency to empty your bladder?”
Validated questionnaires were used to assess potential confounding variables. The Dutch Personality Questionnaire (DPQ) contains 133 statements, which are divided into 7 domains: inadequacy, social inadequacy, rigidity, hostility, egoism, dominance and self-esteem [32]. The higher the score, the more these characteristics are part of the subject’s personality. Personality traits are stable and this questionnaire was completed at 24 weeks gestation for logistic reasons. The Maudsley Marital Questionnaire (MMQ) was used to measure the subjective emotional and sexual relationship of the woman with her partner [33]. The MMQ consists of 15 questions, of which 10 concern emotional aspects (range 0–80) and 5 concern sexual aspects (range 0–40) of the relationship. The higher the score, the worse this specific aspect of the relationship is perceived. In addition, the questionnaires addressed biomedical and socioeconomic variables.
Biomedical factors included length and weight at 36 weeks gestation and 1 year after delivery, age at time of delivery, the presence of a chronic illness or back pain and the use of medication. Data on mode of delivery and pregnancy-related complications (hypertensive disorders, premature delivery, growth retardation and vaginal blood loss) were obtained from the case files of obstetricians and midwifes. Data on length and weight were transformed into a body mass index (BMI = weight/(height)2). Socioeconomic factors consisted of level of education, marital state, employment and job satisfaction. For practical reasons, the education level was dichotomised into high school or less and more than high school. Marital state was dichotomised into married and unmarried/divorced. Behavioural factors we studied were whether participants smoked, used alcohol or were involved in leisure time physical activity in early pregnancy. In addition, women were able to add comments to the standard questions.
Statistical analyses
Changes in prevalence of depressive symptoms, UUI, SUI and OAB between 6 and 12 weeks gestation and between 36 weeks gestation and 3 and 12 months after delivery was determined using the McNemar tests. Possible variables associated with depressive symptoms, including urinary symptoms, were analysed in women that responded to the questionnaire at 36 weeks gestation and 1 year after delivery. Women with and without CES-D B16 were compared in univariate analyses using the independent samples t-test for continuous variables and chi-squared test for categorical variables. The relationship between depressive symptoms (CES-D _16) (dependent variable), urinary symptoms and potential confounders was assessed using multivariate logistic regression analysis (stepwise forward method) to assess whether urinary symptoms remained associated with depressive symptoms.
Only significantly associated variables in univariate analyses (p < 0.05) were put in the model. Odds ratios (Exp (B)) for continuous variables were calculated when appropriate. The Hosmer and Lemeshow test was used to establish the goodness of fit of the model. A p value of >0.05 indicates that the model provides a valid representation of data. All analyses were performed with the SPSS for Windows 11.5.
Results
Of the 672 women who started in the study at 12 weeks gestation, 642 (95%) women responded to the personality questionnaire sent at 24 weeks gestation. The questionnaires at 36 weeks gestation and 3 and 12 months after delivery were answered by 511 (76%), 503 (75%) and 509 (76%) women, respectively. The average age at delivery was 30.0 years (standard deviation (SD) 3.6 years). Average BMI at 36 weeks gestation was 27.9 kg/m2 (SD 4.1 kg/m2). The percentage of women whose education stopped after high school was 52.7%. None of the women were breast-feeding 1 year after delivery. The percentage of women who did not have a spontaneous vaginal delivery was 33.9%. Pregnancy-related complications were reported in 18.3%. In our series, urinary tract infection was not mentioned by any of the women.
The prevalence rates of depressive symptoms, UUI, SUI and OAB at different points in time during and after pregnancy are shown in Table 1. The increase in prevalence during pregnancy (from 12 to 36 weeks gestation) and decrease in prevalence after pregnancy (from 36 weeks gestation to 12 months after delivery) of all symptoms, except urge urinary incontinence, was statistically significant in the McNemar tests (p < 0.05). The increase in prevalence of urge incontinence during pregnancy was significant; however, the seemingly decrease after childbirth was not significant. Factors significantly associated with depressive symptoms at 36 weeks gestation in univariate analyses and in multivariate logistic regression model are shown in Table 2. Many factors were significantly associated in univariate analysis; however, in logistic regression only (social) inadequacy (personality traits), worse sexual relationship with the partner, not being involved in leisure time physical activity, pregnancy-related complications and OAB syndrome remained independently associated with depressive symptoms. The Hosmer and Lemeshow test provided a p value of 0.214, which indicates a proper goodness of fit for this model. Explained variance in this model (R2) is 40.9%. At 1 year post-partum, no significant association was found between depressive and urinary symptoms in the chi-squared tests; therefore, no logistic regression analysis was performed.
Table 1Incidence of depressive symptoms (CES-D ≥ 16), incontinence and over-active bladder syndrome 12 weeks gestation (%)36 weeks gestation (%)3 months post-partum (%)12 months post-partum (%)Depressive symptoms18.220.716.712.2Urge incontinence7.319.116.115.6Stress incontinence20.142.226.534.3Over-active bladder syndrome54.260.17.814.4Table 2Univariate and multivariate associated factors with depressive symptoms at 36 weeks gestation CES-D < 16 (n = 405)CES-D ≥ 16 (n = 106)Crude OR (95% CI)Adjusted OR (95% CI)UDIUrge incontinence17.4%26.7%1.73 (1.05–2.86)Stress incontinence39.2%52.4%1.71 (1.11–2.63)Over-active bladder syndrome57.0%73.3%2.08 (1.29–3.34)4.40 (1.84–10.48)MMQEmotionality6.62 (6.21)13.74 (11.33)1.10 (1.07–1.13)Sexuality11.52 (6.95)16.53 (8.12)1.09 (1.06–1.13)1.12 (1.06–1.17)NPVInadequacy8.08 (5.09)14.50 (7.79)1.17 (1.13–1.22)1.21 (1.13–1.30)Social inadequacy6.89 (6.19)9.84 (6.96)1.07 (1.04–1.10)1.08 (1.01–1.16)Rigidity24.65 (6.81)26.47 (5.91)1.04 (1.01–1.08)Hostility12.36 (5.36)17.12 (6.71)1.14 (1.10–1.19)Egoism9.03 (4.23)10.56 (4.90)1.08 (1.03–1.13)Self-esteem30.30 (4.83)27.09 (4.56)0.88 (0.84–0.92)BMI (kg/m2)27.59 (3.65)29.22 (5.24)1.10 (1.04–1.15)Age (years)30.50 (3.67)29.66 (4.02)0.94 (0.89–1.00)Education high school/below46.9%63.2%1.95 (1.25–3.02)Unemployed4.5%12.3%3.00 (1.42–6.34)Low job satisfaction5.2%15.1%3.23 (1.57–6.68)Smoking7.1%16.2%2.53 (1.33–4.83)Use of alcohol16.0%6.7%0.38 (0.17–0.85)No leisure time physical activity43.2%65.1%2.46 (1.57–3.83)2.83 (1.35–5.92)Back pain51.9%67.0%1.90 (1.21–2.98)Pregnancy related complications8.8%20.8%2.71 (1.38–5.34)3.22 (1.12–8.87)Shown are significantly associated factors at the p < 0.05 level with depressive symptoms. Non-significantly associated factors are left out in this table.CES-D: Center for Epidemiologic Studies Depression Scale, UDI: Urogenital Distress Inventory, MMQ: Maudsley Marital Questionnaire, DPQ: Dutch Personality Questionnaire, BMI: body mass index, OR: odds ratio, 95% CI: 95% confidence interval.
Discussion
In our study, we set out to analyse the prevalence of depressive and urinary symptoms during and after pregnancy and to assess the possible association of urinary symptoms with depression, whilst controlling for psychosocial, behavioural, socioeconomic and biomedical factors. We showed a significant increase in prevalence of depressive symptoms, UUI, SUI and OAB during pregnancy and a significant reduction in the prevalence of depressive symptoms, SUI and OAB after childbirth (p < 0.05). The prevalence of UUI did not significantly decrease after childbirth. In univariate analysis, UUI, SUI and the OAB syndrome was significantly associated with a CES-D score of B16 at 36 weeks gestation. However, after adjusting for possible confounding factors, only the OAB syndrome remained significantly associated. At 1 year after delivery, no association was found between urinary incontinence, OAB syndrome and depressive symptoms.
In non-pregnant women, an association is reported between depression and urinary incontinence, especially urge incontinence, and over-active bladder syndrome with and without urge incontinence [15–19]. These studies mainly used univariate statistics and did not account for potential confounding factors. Our univariate analysis also showed that urinary incontinence and over-active bladder syndrome were significantly associated with depressive symptoms. However, in multivariate analysis, we did not find an association between depressive symptoms and stress or urge urinary incontinence during or after pregnancy. On the other hand, we did find an independent significant relation between depressive symptoms and over-active bladder syndrome at 36 weeks gestation, though not 1 year after delivery. In a research in a non-pregnant population, several explanations are suggested. Firstly, urinary incontinence as a chronic disorder may lead to depressive symptoms. The finding that depression at 1 year after delivery was not associated with urinary symptoms does not support this explanation. Secondly, it is suggested that psychological factors might influence urgency and detrusor instability [34]. Thirdly, a mutual pathologic origin of urinary incontinence and depression is proposed: both are suggested to be caused by the reduction of serotonin [15, 35]. This suggestion is at the least dubious because serotonergic depletion as the suggested aetiology in depression has been seriously questioned [36]. Furthermore, the therapeutic effect of selective serotonin re-uptake inhibitors (SSRIs) in the treatment of depression is only slightly better than placebo [37].
In a pregnant population, several other explanations can be considered. First, during the third trimester of pregnancy, the increase in cardiac output, increasing size of the uterus with compression of the bladder and sleep disturbances may all contribute to an increased voiding frequency. Secondly, the definition used for over-active bladder syndrome (combination of urgency and frequency) has been developed for use in a non-pregnant population. Because the majority of women will have frequency and urgency symptoms as part of their normal third trimester pregnancy, it is questionable if the diagnosis of OAB in pregnancy has the same meaning and impact as OAB in non-pregnant women. The strength of this study is that we used a prospective, longitudinal design with validated questionnaires in a large number of healthy nulliparous women. By measuring not only urogenital symptoms and depressive symptoms, but also psychosocial, behavioural, socioeconomic and biomedical factors, we were able to perform solid multivariate analysis techniques on the associations between bladder symptoms and depression.
Our study has also some potential drawbacks that need to be discussed. First, because of the observational, epidemiological design of the study, all information gathered through the questionnaires is subjective. We used a symptom-based definition of urinary symptoms recommended by the ICS [31]. Although we could rule out, to a certain extend, urinary tract infection as potential confounding factor for urinary symptoms from the records of the midwife or gynecologist, we were unable to confirm the reported symptoms by means of a clinical diagnosis. However, the sensitivity of clinical tests like the stress test and also urodynamic investigation in relation to urogenital symptoms is still under debate [38, 39]. Our data have to be interpreted from a symptom-based point of view, which is most often the best there is to get out of epidemiological studies. It would be worthwhile to test our findings in a clinical setting, although the use of urodynamics during pregnancy in healthy women may turn out to be a difficult ethical question. Secondly, we had a moderate participation rate. Due to the quantity and intimate nature of the questions on sexuality and pelvic floor discomfort, the response rate was 54%. However, the prevalence rates of pelvic floor problems and back pain found in this cohort are in concordance with prevalence rates in other studies and the obstetrical outcome (mode of delivery, birth weight, etc.) of the study population was similar to that of comparable women registered in The Netherlands Perinatal Registry 2001 [13, 40, 41]. In addition, the percentage of women scoring above the cut-off score of 16 on the CES-D is similar to that found in a large American study among pregnant women [7]. Finally, the associations we found between psychosocial, behavioural, socioeconomic and biomedical factors at 36 weeks gestation have all been reported before in ante-natal and post-natal study populations [5, 7, 21, 22, 42–45]. So, the chance that our results are biased by the moderate participation rate is low. A third limitation is that we did not have information on urogenital and depressive symptoms before pregnancy. We decided not to ask about previous symptoms because of the risk of recall bias. Longstanding pre-pregnancy depressive disorders or urogenital symptoms may affect the prevalence rate recorded in pregnancy, but it is questionable if it would affect the associations we studied. Our main conclusion that there is no association between UUI, SUI and depressive symptoms during and after pregnancy was reached after multivariate adjustment in which many factors were accounted for. Finally, the prevalence of depressive symptoms was measured with the CES-D and diagnosis of depression could therefore differ from diagnosis obtained through interview following the DSM-IV criteria. However, the CES-D is a widely used questionnaire with adequate sensitivity and specificity [46], gives a good indication of depressive symptoms and is very suitable for large cohort studies.
Conclusion
We found significantly higher rates of depressive symptoms, SUI and OAB syndrome during pregnancy than after childbirth. After controlling for other associated factors, we found an independent association between depressive symptoms and the OAB syndrome in pregnancy but not with urinary incontinence. This association between OAB syndrome and depressive symptoms is lost after pregnancy, so it is likely that pregnancy-related factors have confounded this association. In general practice, this information on the natural course of the OAB syndrome, and its lack in causing major depressive symptoms after childbirth, can be used to counsel women who are confronted with these problems during pregnancy.

depression
pregnancy
urge urinary incontinence
stress urinary incontinence
ces-d
over-active bladder syndrome


Preconception Brief: Occupational/Environmental Exposures

In the last decade, more than half of U.S. children were born to working mothers and 65% of working men and women were of reproductive age. In 2004 more than 28 million women age 18–44 were employed full time. This implies the need for clinicians to possess an awareness about the impact of work on the health of their patients and their future offspring. Most chemicals in the workplace have not been evaluated for reproductive toxicity, and where exposure limits do exist, they were generally not designed to mitigate reproductive risk. Therefore, many toxicants with unambiguous reproductive and developmental effects are still in regular commercial or therapeutic use and thus present exposure potential to workers. Examples of these include heavy metals, (lead, cadmium), organic solvents (glycol ethers, percholoroethylene), pesticides and herbicides (ethylene dibromide) and sterilants, anesthetic gases and anti-cancer drugs used in healthcare. Surprisingly, many of these reproductive toxicants are well represented in traditional employment sectors of women, such as healthcare and cosmetology. Environmental exposures also figure prominently in evaluating a woman’s health risk and that to a pregnancy. Food and water quality and pesticide and solvent usage are increasingly topics raised by women and men contemplating pregnancy. The microenvironment of a woman, such as her choices of hobbies and leisure time activities also come into play. Caregivers must be aware of their patients’ potential environmental and workplace exposures and weigh any risk of exposure in the context of the time-dependent window of reproductive susceptibility. This will allow informed decision-making about the need for changes in behavior, diet, hobbies or the need for added protections on the job or alternative duty assignment. Examples of such environmental and occupational history elements will be presented together with counseling strategies for the clinician.
Introduction
The influence of environmental exposures on the general status of health has been increasingly acknowledged for numerous disease outcomes. The connection between air pollution and acute respiratory disease, for example and, more recently, the observation linking poor indoor air quality to increases in the incidence of childhood asthma has been widely publicized. Such epidemiologic observations are often reported in the media and, when combined with the growing public interest in a clean and healthy environment, have translated into an increasingly sophisticated patient population that expects healthcare providers to be conversant with the environmental contribution to disease risk. Indeed, the lay public often has shown more interest in– and occasionally knowledge of– the relationship between health and the environment than has the medical community.
Medical educators and the Institute of Medicine (IOM) both have addressed this dilemma by promoting enhanced medical student and physician training in environmental medicine in order to develop ‘environmentally literate’ physicians. In this brief, the term ‘environmental medicine’ will refer to “diagnosing and caring for people exposed to chemical and physical hazards in their homes, communities and workplaces through such media as contaminated soil, water and air” [1, 2]. This definition was taken from a 1992
IOM Committee report on Curriculum Development in Environmental Medicine. The preconception clinical visit already includes environmental history queries regarding smoking and alcohol use, [3] but must be enlarged to address the broader concept of environmental exposures occurring in the woman’s home, community and workplace.
Sources of environmental exposure
Organizing the environmental exposure history can be facilitated by using the definition referred to above and by eliciting information on aspects of the patient’s larger environment; this includes the specific community locale (workplace and her home environment) and infrastructure which relates to air quality and soil or water pollution, all of which the patient deals with on a regular basis.
Community
Surprisingly, a clinician would likely not know if a given patient in his/her practice was living in an area near by an environmentally polluted location, such as a National Priority Listed (NPL) waste site or a local toxic waste dump. In large metropolitan areas, many such sites are present and unknown to most citizens. Although clinically significant exposure to a toxic hazard present on the site would be unlikely to threaten the wider community, a risk may exist for some residents living very close to the site, possibly allowing for soil or drinking water contamination. In specific instances often covered by the news media, patients would likely be able to report knowledge of living in the vicinity of such a place. Such a ‘self-report’ response could be elicited by simply asking the question, “Do you live near or have contact with a waste site?” Probably the most potentially important exposure opportunity is via contaminated drinking water. Here the biggest risk is usually from a small private water source such as a well, not subject to municipal water treatment standards and testing. According to the EPA approximately 15% of the U.S. population has a private drinking water source.
Workplace
An intimate relationship exists between occupational and environmental health, because often the source of environmental contamination is a former (or present day) work site. The workplace is an important ‘special case’ in environmental history taking, because populations exposed at work tend to be exposed at higher concentrations than the larger community, regardless whether contamination is via air, water or soil. Thus, though the working population is smaller than the public at large, workers are exposed at higher ‘doses’. It is thus sometimes observed that workers are the ‘canaries’ or ‘sentinels’, exhibiting first the health effects which might be expected in a wider community exposure from an environmental pathway.
It is fair to say that the principal exposure opportunity to an environmental reproductive or developmental toxicant in patients will be from their work place. Many toxicants with unambiguous reproductive and developmental effects are still in regular commercial or therapeutic use, and thus pose a continued potential risk in the occupational environment. Several employment sectors where such toxicants commonly are found employ women workers primarily and include: laboratory and clinical medicine, printing and dry cleaning [4] though this is clearly not always the case and the gender mix varies by sector. (See Table 1). Whereas some of these chemical toxicants are regulated by public health agencies, the majority of chemicals considered for regulation are not evaluated for reproductive endpoints [5] but rather, for other toxic effects. This gap in the regulatory safety net allows reproductive toxicants to be encountered in both environmental and work settings by men and women.Table 1Employment sectors and associated reproductive/develop-mental toxicantsSectorToxicantExamplesAgriculturePesticides/HerbicidesEthylene DibromideManufacturingOrganic SolventsGlycol ethers, lead,Heavy MetalsCadmiumDry CleaningSolventsPerchloroethylenePrintingSolvents/inksPharmaceutical Compounding/ManufactureHazardous DrugsAntineoplastics, hormones, immunologic modifiersHealth CareBiologicsRubella, CMV, Hepatitis virusPhysical AgentsIonizing Radiation/HeatChemicalsAntineoplastics/Hazardous Drugs Anesthetic Gases SterilantsPhysical ExertionLifting/Prolonged Standing Shift WorkNote. Ref: Summarized from GAO, 1988; Stellman, 1994.
Home
Here, one must consider the exposure opportunities posed by the woman’s residential environment, such as those involved with household tasks, those related to her pursuit of hobbies and those related to her ‘micro-environment,’ including diet.
Diet
Beyond the current recommendations related to healthy eating, the uses of non-prescription herbal or alternative medicines or supplements are crucial parts of the medical history for women planning a pregnancy. Also, some ethnic home remedies must be queried for, as some contain hazardous contaminants such as lead and mercury [6]. There are also several dietary cautions, which should be reviewed with the woman contemplating pregnancy. One is not new and relates to the warning regarding exposure to food borne listeria infections. Although listeriosis may cause only mild flu-like symptoms in the pregnant mother, serious outcomes in the fetus may result. These include premature delivery, stillbirth or neonatal infection [7–8]. General advice relies upon avoidance of soft cheese such as feta, Brie, Camembert, blue-veined and Mexican-style cheeses and unpasteurized milk or milk products [9]. Strict adherence to food preparation safety, such as vigilant washing of raw fruits and vegetables, avoidance of undercooked and raw meat and careful separation of stored raw food from uncooked meats serves to enhance safety for the entire family as most food borne illness is preventable.
Consumer advisory on methylmercury in fish
In 2004, the Food and Drug Administration (FDA) and the Environmental Protection Agency (EPA) issued a joint consumer advisory on methylmercury in fish and shellfish. The warning specifically targets women who may become pregnant, are pregnant or are nursing mothers, and also includes young children. The document emphasizes the health benefits of fish in the diet generally as a good source of protein, and also warns of several types of fish which contain comparatively higher concentrations of mercury and should be avoided. These include: shark, swordfish, King mackerel and Tilefish. Other types of fish may be consumed in up to two meals (6 oz each) per week, including: shrimp, canned light tuna (but not albacore, which has more mercury), salmon, pollock and catfish. The Advisory also warns about local fish advisories, which are generally posted for specific populations which supplement their diets with locally, caught fish. Appropriate discussion of relevant advisories should take place in the preconception visit and is especially important for non-meat eating patients whose total dietary intake of fish may be relatively greater than is the case for meat consumers. Also of note, the U.S. Department of Health’s Women’s Infants, and Children Program (WIC) sometimes gives canned tuna as a diet supplement. This practice should be weighed in light of the above concerns, however, at least in terms of the relative amount of tuna in a weekly diet. The complete consumer advisory can be found at: www.epa.gov/ost/fish.
Hobbies and home-based work
The hobbies of concern generally would include those involving similar types of chemical toxicants discussed in the occupational section above, including heavy metals (lead, cadmium, arsenic) and solvents (paints (other than latex based), furniture stripper, metal cleaners etc.) Hobbies to be discouraged include: painting, ceramics, stained-glass window making (lead solder), furniture re-finishing and the like. Leisure activities to be avoided include use of saunas and hot tubs.
A number of home-based work activities, which resulted in contamination of the home and family members, have been documented in the literature. Although uncommon, they deserve mention here. At one end of the spectrum most involve the use of metals, either in metal parts reclamation (lead batteries being melted down) or jewelry making (heating and soldering) [10]. At the other, the home environment may be unwittingly contaminated by family members who bring work contaminants home on their shoes or dirty work clothes. If the work clothes are laundered at home, there not only is opportunity for exposure to the launderer but also other household members. For this reason, some OSHA standards require changing rooms so work clothes will not secondarily expose family members, in the case of lead and asbestos work, for example [11].
Household exposures
Pesticides, herbicides and rodenticides are likely the most common chemical toxicants in the average home. Certainly, a pregnant woman or one actively attempting to conceive, should not apply any of these [12]. Because the concept of ‘second hand’ exposure as described above applies here also, care needs be taken so that the chemicals are not ‘tracked’ into or throughout the house, no matter who performs the chemical applications. Of far greater importance on a population basis is second hand smoke in the home and/or workplace. Numerous polycyclic aromatic hydrocarbons present in cigarette smoke are carcinogenic and have been shown to affect reproductive outcomes in animal studies. They cross and affect the placenta, and enter breast milk [13]. Laundering of contaminated work clothes is also a concern, as discussed above. A hiatus from caring for the family cat and litter box should be mentioned due to the toxoplasmosis risk.
Fig. 1Example checklist for initial qualitative evaluation of reproductive hazards, (modified from Grajewski, 2005)Fig. 2Example of checklist for initial quantitative evaluation of reproductive hazards, (modified from Grajewski, 2005)
Many non-latex paints are solvent based and contain small amounts of metals to enhance wear, and as preservatives. This is especially true of exterior paint, such as that used on porches or building exteriors or even in the interiors of older buildings. Rehabbing older homes, which often involve paint stripping, either with a heat gun or a chemical stripper is particularly hazardous. Inhalation is a very efficient means of producing a clinically significant exposure to lead which was commonly used years ago in interiors of homes. Many commercial paint strippers contain methylene chloride (dichloromethane), which metabolizes to carbon monoxide, and is particularly toxic to the fetus.
Guidance for clinician
Certainly, awareness that occupational and environmental hazards encountered by patients may play a clinically significant role in a pregnancy outcome is the first step in effective patient management. Therefore, enlarging the standard health history form, completed by the patient on or before the first visit to obtain a more detailed environmental history is a necessary first step and is also often very informative. Whereas a number of preconception checklists exist, only some of which address fish consumption or residence near a waste site, most poorly capture, or even fail to query about occupation, despite this being the likely greatest ‘environmental’ risk the patient faces.
A ‘qualitative’ evaluation of a patient’s potential exposure to reproductive and developmental toxicants can be obtained with a screening questionnaire as seen in Fig. 1. This involves examining occupation by industry sector of employment and then by chemical, physical and biological agents of concern. If a reproductive hazard is present in the patient’s work environment, an initial quantitative assessment can be made regarding the exposure intensity (Fig. 2). However, at this point, the clinician may want to consult an occupational medicine colleague to elicit a more detailed history and assist in the preconception recommendations.
Preconception management strategies, should be based on the occupational history and include a decision on the safety of continued employment during the preconception period and pregnancy. If the patient works with bona fide reproductive and development toxicants, continued employment and under what circumstances must be determined. This may involve identifying additional protections, such as a respirator, gloves or other personal protective apparel or equipment that may make the job safer for the patient. There are some jobs, however, where it is recommended by professional organizations as well as governmental safety and health agencies, that pregnant persons, or those actively trying to conceive be provided with alternative duty [14]. A good example here is nurses who handle cancer chemotherapy [15]. However, a related issue involves crafting elements of an existing job which the pregnant patient, such as the oncology nurse may safely continue, such as patient education, telephone triage and the like. This arrangement is termed ‘alternative duty’. Also involved in preconception counseling is the potential impact on the quality of breast milk if the mother is planning to breastfeed. The majority of organic solvents and pharmaceuticals absorbed by the mother in the workplace invariably make their way into milk. Thus, both work during pregnancy and return to work while breastfeeding need to be considered.
We have thus far focused on the working patient’s current job, which is generally considered the most influential on pregnancy outcome, as most reproductive toxicants are thought to act via an acute toxicity mechanism during the three to four months prior to conception and pregnancy [16–18]. However, there are also several examples of remote past exposure that are important to elicit, though likely less commonly encountered. An example includes the patient who has high bone lead stores from remote past (childhood) exposure that can be mobilized during pregnancy and expose the fetus [19–20]. Medical management at the preconception visit would include a blood lead level so this can be tracked during pregnancy. Also, maternal stores of fat-soluble organic chemicals, such as dioxins and PCBs may also expose the fetus [21, 22]. Positive responses to exposure to occupational or environmental agents by the preconception patient may require consultation. The Association of Occupational and Environmental Clinics can supply referrals to clinicians in various locales nationwide at (www.aoec.org).
Conclusion
The preconception office clinic visit presents a strategic opportunity to minimize the environmental and occupational sources of reproductive risk facing the preconception patient. This requires the two-way exchange of information between the patient and her clinician, clarifying misunderstandings and implementing reasonable strategies to minimize exposures from the wider community, in the workplace and at home. Taking an environmental and occupational history and tailoring recommendations based on that, enlarges the likelihood that preventable, adverse pregnancy outcomes can be avoided.

environmental exposure
reproductive
reproductive toxicants