Lee P. Shulman

Prenatal diagnosis with fetal cells isolated from maternal blood by multiparameter flow cytometry

A long-sought goal of medical genetics has been development of prenatal diagnostic procedures that do not endanger the conceptus. Reliable and universal screening for cytogenetic disorders would require analysis of fetal cells isolated from the maternal circulation. This would be applicable to all pregnant women, irrespective of their ages or histories. In the current study fetal nucleated erythrocytes were flow sorted on the basis of four parameters: cell size, cell granularity, transferrin receptor, and glycophorin-A cell surface molecule. By polymerase chain reaction with oligonucleotide primers flanking single-copy Y-specific deoxyribonucleic acid sequences, male fetuses were correctly identified among flow-sorted samples in 12 of 12 (100%) pregnancies; female fetuses were correctly identified in 5 of 6 (83%) pregnancies. We also achieved the prenatal diagnosis of fetal aneuploidies by use of flow-sorted nucleated fetal erythrocytes and in situ hybridization with chromosome-specific deoxyribonucleic acid probes: one case of trisomy 21 that was detected in maternal blood taken 1 week after chorionic villus sampling and one case of trisomy 18 that was detected in maternal blood taken immediately before chorionic villus sampling. Although our results are promising, additional data on the background sensitivity and specificity of in situ hybridization in flow-sorted fetal cells will be necessary to minimize subjective interpretation and permit clinical application.

Safety and efficacy of black cohosh and red clover for the management of vasomotor symptoms: a randomized controlled trial.

Objective: The aim of this study was to evaluate the safety and efficacy of black cohosh and red clover compared with placebo for the relief of menopausal vasomotor symptoms. Methods: This study was a randomized, four-arm, double-blind clinical trial of standardized black cohosh, red clover, placebo, and 0.625 mg conjugated equine estrogens plus 2.5 mg medroxyprogesterone acetate (CEE/MPA; n = 89). Primary outcome measures were reduction in vasomotor symptoms (hot flashes and night sweats) by black cohosh and red clover compared with placebo; secondary outcomes included safety evaluation, reduction of somatic symptoms, relief of sexual dysfunction, and overall improvement in quality of life. Results: Reductions in number of vasomotor symptoms after a 12-month intervention were as follows: black cohosh (34%), red clover (57%), placebo (63%), and CEE/MPA (94%), with only CEE/MPA differing significantly from placebo. Black cohosh and red clover did not significantly reduce the frequency of vasomotor symptoms as compared with placebo. Secondary measures indicated that both botanicals were safe as administered. In general, there were no improvements in other menopausal symptoms. Conclusions: Compared with placebo, black cohosh and red clover did not reduce the number of vasomotor symptoms. Safety monitoring indicated that chemically and biologically standardized extracts of black cohosh and red clover were safe during daily administration for 12 months.

Endometrial adenocarcinoma: Genetic analysis suggesting heritable site-specific uterine cancer

UNLABELLED Genetic factors are clearly integral to the etiology of neoplasia. A cancer family syndrome (Lynch syndrome II) consisting of uterine, colon, and ovarian cancer is recognized, but the heritability of isolated endometrial adenocarcinoma has not otherwise been thoroughly investigated. We have performed pedigree studies in index cases with endometrial adenocarcinoma, using spouses as controls. Preliminary results from 64 probands showed four families in which endometrial adenocarcinoma was diagnosed in at least one first-degree relative of the proband (mother, daughter, sister); none showed relatives with colon or ovarian cancer. In none of the 34 control pedigrees did either a mother or sister have endometrial adenocarcinoma. In four other families, multiple first- and second-degree relatives of probands had adenocarcinoma of the uterus, colon, or ovary, presumably representing a cancer family syndrome (Lynch syndrome II). CONCLUSION Our preliminary data not only show familial and probably heritable tendencies for endometrial adenocarcinoma, but further suggest that there are at least two distinct forms: (1) the previously described Lynch syndrome II (cancer family syndrome), and (2) a heretofore unemphasized entity characterized by a tendency to endometrial adenocarcinoma alone.

Objective hot flashes are negatively related to verbal memory performance in midlife women.

Objective: To test the hypothesis that hot flashes specifically relate to verbal memory performance by examining the relationship between objective hot flashes and cognitive test performance in women with moderate to severe vasomotor symptoms. Design: In an observational study, 29 midlife women (mean age, 53 y) with moderate to severe hot flashes provided measures of objective hot flashes with an ambulatory hot flash monitor, subjective hot flashes with a diary and questionnaire, and objective measures of verbal memory and other cognitive functions with standardized neuropsychological tests. Results: The mean number of objective hot flashes was 19.5 per day (range, 6 to 35), including 15.3 (range, 6 to 35) during waking hours and 4.2 (range, 0 to 9) during sleep. The mean sensitivity (ie, subjective detection of objectively measured hot flashes) was 60%. Regression analyses revealed that total number of objective hot flashes, sleep duration, and verbal knowledge were significant predictors of delayed verbal memory. Verbal fluency correlated positively with objective daytime hot flashes. Hot flashes did not predict performance on any of the other secondary cognitive measures (ie, attention, working memory, visual memory), although poor sleep predicted worse performance on several outcome measures. Conclusions: Highly symptomatic women underreport the number of objective hot flashes that they experience by 43%. Verbal memory performance relates significantly to the objective number of hot flashes women experience but not to the number of hot flashes that they report. These findings suggest that physiological factors related to hot flashes, rather than psychological factors, predict poorer verbal memory function.

Late First-Trimester Invasive Prenatal Diagnosis: Results of an International Randomized Trial

OBJECTIVE: To assess, in a randomized trial, the safety and accuracy of amniocentesis and transabdominal chorionic villus sampling (CVS) performed at 11–14 weeks of gestation, given that this time frame is increasingly relevant to early trisomy screening. METHODS: We compared amniocentesis with CVS from 77 to 104 days of gestation in a randomized trial in a predominantly advanced maternal age population. Before randomization, the feasibility of both procedures was confirmed by ultrasonography, and experienced operators performed sampling under ultrasound guidance; conventional cytogenetic analysis was employed. The primary outcome measure was a composite of fetal loss plus preterm delivery before 28 weeks of gestation in cytogenetically normal pregnancies. RESULTS: We randomized 3,775 women into 2 groups (1,914 to CVS; 1,861 to amniocentesis), which were comparable at baseline. More than 99.6% had the assigned procedure, and 99.9% were followed through delivery. In contrast to previous thinking, in the cytogenetically normal cohort (n = 3,698), no difference in primary study outcome was observed: 2.1% (95% confidence interval 1.5, 2.8) for CVS and 2.3% (95% confidence interval, 1.7, 3.1) for amniocentesis. However, spontaneous losses before 20 weeks and procedure-related, indicated terminations combined were increased in the amniocentesis group (P = .07, relative risk 1.74). We found a 4-fold increase in the rate of talipes equinovarus after amniocentesis (P = .02) overall and in week 13 (P = .03, relative risk = 4.65), but data were insufficient to determine this risk in week 14. CONCLUSION: Amniocentesis at 13 weeks carries a significantly increased risk of talipes equinovarus compared with CVS and also suggests an increase in early, unintended pregnancy loss. LEVEL OF EVIDENCE: I

Isolated fetal choroid plexus cysts and trisomy 18: A review and meta-analysis

OBJECTIVE Risk of trisomy 18 in a fetus with ultrasonographic diagnosis of choroid plexus cysts and no other anomalies is controversial. Using our data and current literature, we performed a meta-analysis and estimated the positive predictive value of isolated choroid plexus cysts for trisomy 18. STUDY DESIGN Between Jan. 1, 1989, and Dec. 31, 1992, all women undergoing ultrasonographic examination at our institution were prospectively evaluated for fetal choroid plexus cysts and cytogenetic outcome. In addition, all reports dealing with fetal choroid plexus cysts obtained from MEDLINE (1983 through 1992) were assessed. Only prospective studies with > 10 cases of choroid plexus cysts were further evaluated to determine the total number of fetuses with choroid plexus cysts and otherwise normal sonograms. Frequency of aneuploidy was determined by analysis of our data and the included studies. To estimate the positive predictive value of choroid plexus cysts from trisomy 18, a theoretic 2 x 2 table was constructed with values available from the literature. RESULTS Eighty fetuses with choroid plexus cysts were identified in our unit. Of 74 fetuses with isolated choroid plexus cysts, there were no cases of trisomy 18. Meta-analysis identified 2 cases of trisomy 18 among 748 fetuses with isolated cysts (1/374). To derive a positive predictive value of isolated choroid plexus cysts for trisomy 18, we reviewed the literature and found a total of 50 fetuses with trisomy 18 who underwent ultrasonographic examination in the midtrimester. There were 3 cases of isolated choroid plexus cysts, and 12 of 50 (24%) had otherwise normal ultrasonographic results. Using a midtrimester incidence of 1 in 2461 for trisomy 18 (Hsu LYF. In: Milunsky A, ed. Genetic disorders of the fetus. 3rd ed. Baltimore: Johns Hopkins University Press, 1992: 155-210; Hook et al. Am J Hum Genet 1989; 45:855-61) and a prenatal prevalence of 0.95% for choroid plexus cysts (based on a review of the literature), we obtained a positive predictive value of 1 in 390. CONCLUSION On the basis of the risk for trisomy 18 obtained from our meta-analysis (1/374) and its close approximation to the estimated positive predictive value (1/390), our data do not support the routine offering of invasive prenatal cytogenetic testing in cases of isolated choroid plexus cysts.

Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses.

Risk of fetal mosaicism when placental mosaicism is diagnosed by chorionic villus sampling

OBJECTIVE Our purpose was to determine the risk of fetal mosaicism when placental mosaicism is found on chorionic villus sampling. STUDY DESIGN We present a case of mosaic trisomy 22 detected on chorionic villus sampling and subsequently found in the fetus. A review of comprehensive chorionic villus sampling studies with emphasis on follow-up for fetal mosaicism was conducted. RESULTS Among 13 studies reviewed, 469 cases of placental mosaicism are presented; fetal mosaicism was found in 50 (10.7%). Factors associated with fetal mosaicism are (1) mosaicism on mesenchymal core culture and (2) type of chromosome abnormality involved--specifically, marker chromosomes (26.7%) and common autosomal trisomies (19.0%). Amniocentesis predicted fetal genotype in 93% to 100% of cases of placental mosaicism, depending on the cell type in which mosaicism was diagnosed. CONCLUSIONS Although mosaicism is usually confined to the placenta, the fetus is involved in about 10% cases. Patients should be counseled about this risk and the accuracy of follow-up amniocentesis.

Clinical studies of red clover (Trifolium pratense) dietary supplements in menopause: a literature review

Red clover (Trifolium pratense L., Fabaceae) botanical dietary supplements have received much attention recently for their potential use in the treatment of menopause symptoms, maintenance/improvement of bone and cardiovascular health, and reported benign effects on the breast and endometrium. Literature searches of four computerized databases were run to identify clinical studies of red clover botanical dietary supplements. The manufacturer of the red clover products used in the majority of the studies was contacted for unpublished information and/or clarification regarding the chemical content of their products. Red clover studies were reviewed that pertained to women’s health or menopause. Clinical evidence is presently lacking to support the efficacy of semipurified red clover isoflavone extracts for alleviation of climacteric vasomotor symptoms or reduction of low-density lipoprotein levels in the blood. Furthermore, the safety of use of red clover isoflavone supplements in patients with breast or endometrial cancer has not been established. Limited evidence suggests possible efficacy in maintenance of bone health and improvement of arterial compliance, a risk factor for atherosclerosis. This literature review covers red clover botanical dietary supplement clinical studies having a possible impact on the health care of mature and menopausal women, and provides historical perspective regarding the traditional uses of red clover.

Safety and efficacy of a continuous once-a-week 17β-estradiol/levonorgestrel transdermal system and its effects on vasomotor symptoms and endometrial safety in postmenopausal women: the results of two multicenter, double-blind, randomized, controlled trials

OBJECTIVE Two prospective multicenter, double-blind, randomized, controlled trials were conducted to examine the safety and efficacy of three once-a-week continuous combined 17beta-estradiol/levonorgestrel (E2/LNG) transdermal systems (E2 0.045 mg/day with 0.015, 0.030, and 0.040 mg/day LNG) for the treatment of vasomotor symptoms and prevention of estrogen-induced endometrial hyperplasia in healthy, postmenopausal women. DESIGN In study 1, performed in 293 hysterectomized and nonhysterectomized women with moderate to severe hot flushes, transdermal E2/LNG (E2 0.045 mg/day with 0.030 and 0.040 mg/day LNG) was compared with placebo for three 28-day treatment cycles. The frequency and severity of hot flushes were recorded daily. In study 2, performed in 845 women with intact uteri, transdermal E2/LNG (E2 0.045 mg/day with 0.015, 0.030, and 0.040 mg/day LNG) was compared with transdermal E2 0.045 mg/day monotherapy for thirteen 28-day treatment cycles. Women with endometrial tissue sufficient for evaluation underwent endometrial biopsy assessment at the end of cycle 13. Bleeding patterns were assessed throughout the study, and the Womens Health Questionnaire was used to assess well-being. RESULTS In study 1, transdermal E2/LNG (E2 0.045 mg/day with 0.030 and 0.040 mg/day LNG) significantly decreased the number and severity of hot flushes when compared with placebo. Symptom relief was seen as early as 2 weeks posttreatment. Similarly, in study 2, all three doses of transdermal E2/LNG and E2 controlled hot flushes with no differences between groups. In study 2, no women receiving transdermal E2/LNG developed endometrial hyperplasia compared with 19 (12.8%) who received transdermal E2 0.045 mg/day (p < 0.001 for each dose). Significant improvements from baseline in scores on the Womens Health Questionnaire for vasomotor symptoms, sleep problems, sexual function, cognitive difficulties, and total score were noted at all or most time points with both transdermal E2/LNG and E2. Application-site reactions, vaginal hemorrhage, and breast pain were the most common adverse events reported with transdermal E2/LNG. The proportion of women with amenorrhea increased over time in all treatment groups in study 2. CONCLUSIONS All three doses of this once-a-week combined E2/LNG transdermal system rapidly and effectively control vasomotor symptoms in postmenopausal women while protecting against endometrial hyperplasia. Amelioration of vasomotor symptoms also is accompanied by improvements in aspects of subjective well-being. Once-a-week transdermal E2/LNG, therefore, offers an effective and convenient formulation, the dosing of which can be individualized according to the needs of each patient.