A postpartum haemorrhage package with condom uterine balloon tamponade: a prospective multi-centre case series in Kenya, Sierra Leone, Senegal, and Nepal.

OBJECTIVE: To evaluate the effectiveness and safety of an ultra-low-cost uterine balloon tamponade package (ESM-UBT™) for facility-based management of uncontrolled postpartum haemorrhage (PPH) in Kenya, Sierra Leone, Senegal, and Nepal. DESIGN: Prospective multi-centre case series. SETTING: Facilities in resource-scarce areas of Kenya, Sierra Leone, Nepal, and Senegal. POPULATION: Women with uncontrolled postpartum haemorrhage in 307 facilities across the four countries. METHODS: A standardised ESM-UBT package was implemented in 307 facilities over 29 months (1 September 2012 to 1 February 2015). Data were collected via a multi-pronged approach including data card completion, chart reviews, and provider interviews. Beginning in August 2014, women who had previously undergone UBT placement were sought and queried regarding potential complications associated with UBT use. MAIN OUTCOME MEASURES: All-cause survival, survival from PPH, and post-UBT use complications (surgery, hospitalisation, antibiotics for pelvic infection) associated with UBT use. RESULTS: 201 UBTs were placed for uncontrolled vaginal haemorrhage refractory to all other interventions. In all, 38% (71/188) of women were either unconscious or confused at the time of UBT insertion. All-cause survival was 95% (190/201). However, 98% (160/163) of women survived uncontrolled PPH if delivery occurred at an ESM-UBT online facility. One (1/151) potential UBT-associated complication (postpartum endometritis) was identified and two improvised UBTs were placed in women with a ruptured uterus. CONCLUSIONS: These pilot data suggest that the ESM-UBT package is a clinically promising and safe method to arrest uncontrolled postpartum haemorrhage and save women's lives. The UBT was successfully placed by all levels of facility-based providers. Future studies are needed to further evaluate the effectiveness of ESM-UBT in low-resource settings. TWEETABLE ABSTRACT: Evidence for ESM-UBT as a clinically promising and safe method to arrest uncontrolled PPH and save women's lives.

Association of marital status and colorectal cancer screening participation in the USA.

AIM: In the USA, for both men and women, colorectal cancer (CRC) ranks third in incidence and second in mortality. Despite evidence that it decreases mortality, CRC screening in the USA remains under-utilized. Some European studies have suggested that marital status affects participation in CRC screening, but the effect of marital status on CRC screening participation in the USA is unknown. In this study, the aim was to compare CRC screening participation rates among married and unmarried couples, separated, widowed, never married and divorced adults living in the USA. METHOD: This was a retrospective data analysis of the 2010 Behavioural Risk Factor Surveillance System survey. The population studied included 239,300 participants, aged 50-75 years, who completed the 2010 survey. Logistic regression analysis was conducted to assess the association between adherence with CRC screening guidelines and marital status while accounting for survey stratum/weight and covariates. RESULTS: Individuals who were divorced or separated, never married or widowed had decreased odds of adherence with CRC screening guidelines compared with individuals who were married and unmarried couples. CONCLUSION: In this study, individuals living in the USA who were married and unmarried couples had increased odds of undergoing CRC screening compared to individuals in other marital status groups. Public health interventions are needed to promote CRC screening participation in these other groups.

Perinatal health care in a conflict-affected setting: evaluation of health-care services and newborn outcomes at a regional medical centre in Iraq.

A field-based assessment was conducted to assess maternal and newborn health-care services, perinatal and newborn outcomes and associated risk factors at Bint Al-Huda Maternal and Newborn Teaching Hospital, a large referral hospital in southern Iraq. The multi-method approach used interviews, discussions, observation and review of perinatal and newborn outcome data. There is limited assessment of maternal vital signs, labour pattern, fetal response, and complications during pregnancy and labour. Perinatal and neonatal mortality rates are 27.4/1000 births and 30.9/1000 live births respectively. Associated neonatal mortality factors were gestational age < 37 weeks, male sex, birth weight < 2.5 kg, maternal age > 35 years, rural maternal residence and vaginal delivery. Improving birth outcomes in southern Iraq requires evidence-based clinical guidelines, additional supplies and equipment, quality improvement initiatives and in-service training.

First Report of Soybean Rust Caused by Phakopsora pachyrhizi on Kudzu (Pueraria montana var. lobata) in Illinois.

Soybean rust, caused by Phakopsora pachyrhizi Syd., first was observed in the continental United States during 2004 on soybean (Glycine max (L.) Merr.) in Louisiana (4), and on kudzu (Pueraria montana (Lour.) Merr. var. lobata (Willd.) Maesen & Almeida) in Florida (2). Kudzu is a leguminous weed that is prevalent in the southern United States with its range extending northward into other states including Illinois. In October 2009, a kudzu patch located in Pulaski County in southern Illinois was investigated for the presence of soybean rust. Twenty-five leaflets were collected, and the abaxial sides of leaflets were evaluated visually for the presence of uredinia with a dissecting microscope. Uredinia and urediniospores were found on two leaflets. When viewed with a compound microscope, urediniospores were hyaline, echinulate, and measured 20 × 25 µm. On the basis of uredinia and urediniospores, the disease tentatively was identified as soybean rust caused by P. pachyrhizi. To confirm the identification, one leaflet with pustules was assayed with a Soybean Rust QuickStix Diagnostic Kit (Envirologix, Portland, ME). For the other leaflet, the area of the pustule was excised (approximately 28 mm2) and an area of the leaflet at the margin on the opposite half of the leaflet with no visible pustule (approximately 54 mm2) was excised. DNA was extracted from the excised areas of the leaflet for confirmation by quantitative PCR (Q-PCR) using primers and probe specific to P. pachyrhizi and P. meibomiae (Arthur) Arthur (1). Both the QuickStix Diagnostic Kit and the Q-PCR confirmed the diagnosis as soybean rust caused by P. pachyrhizi. Q-PCR also suggested the presence of a nonsporulating latent rust infection on the same kudzu leaflet at the margin on the opposite side of the midrib. Soybean rust first was confirmed on soybean in Illinois in 2006 (3), but to our knowledge, this is the first observation of the disease on kudzu in the state. This report confirms that at least some kudzu plants in Illinois are susceptible to soybean rust and that latent kudzu infection may exist without outward signs of the fungus. Currently, this is the most northern observation of soybean rust on kudzu in North America. It is unknown what role, if any, Illinois kudzu will play in the epidemiology of soybean rust in the state. Since kudzu tops die after the first frost, there is no expectation of P. pachyrhizi to overwinter in Illinois on kudzu as it does in some states adjacent to the Gulf of Mexico. References: (1) R. D. Frederick et al. Phytopathology 92:217, 2002. (2) P. F. Harmon et al. Online publication. doi:10.1094/PHP-2005-0613-01-RS. Plant Health Progress, 2005. (3) G. L. Hartman et al. Plant Dis. 91:466, 2007. (4) R. W. Schneider et al. Plant Dis. 89:774, 2005.

Partial characterization of the human CYP1A1 negatively acting transcription factor and mutational analysis of its cognate DNA recognition sequence.

Previous studies in our laboratory identified a negative regulatory domain in the 5'-flanking region of the human CYP1A1 gene containing two negative regulatory elements (NRE). Characterization of one of these elements revealed three nuclear protein binding regions: a 21-bp palindrome with a point of symmetry at -784 and two guanine- and cytosine-rich elements that flank the palindrome. Functional studies suggested the palindrome is critical for transcriptional repression, whereas the guanine- and cytosine-rich sequences play a secondary role. In this study, the interaction between nuclear proteins and the CYP1A1 NRE was further defined. Electrophoretic mobility shift assays (EMSA) indicated that the NRE -784 palindrome alone, but not the guanine- and cytosine-rich sequences minus the palindrome, was capable of specific nuclear protein binding. Competitive cotransfection experiments confirmed this observation in intact cells. Specific residues important for DNA-protein interactions were identified by site-directed mutagenesis and competitive EMSA. The loss of specific protein binding was also correlated with the loss of negative regulatory activity in a transient-expression assay. Finally, competitive EMSA was performed with consensus oligonucleotides for known transcription factors. An NF-Y consensus sequence efficiently competed with the NRE probe for specific nuclear protein binding. EMSA supershift analyses indicate that a protein immunologically related to NF-YB is part of the specific nuclear protein complex binding the human CYP1A1 NRE. These studies have refined our understanding of the sequences critical for the transcriptional repression of human CYP1A1. To our knowledge, this is also the first report implicating a member of the NF-Y transcription factor family in negative gene regulation.

Late Season Occurrence of Soybean Rust Caused by Phakopsora pachyrhizi on Soybean in Illinois.

Soybean rust, first reported in the continental United States in Louisiana in 2004 (2), is one of the most important foliar diseases of soybean worldwide. On 10 October 2006, 20 soybean leaflets from 20 plants at physiological maturity were arbitrarily collected in research plots near Glendale, IL at the University of Illinois Dixon Springs Agricultural Center in Pope County and sent by overnight courier. On 11 October, leaflets were examined with a dissecting microscope at the Soybean Disease Laboratory at the National Soybean Research Center, and then at the Plant Disease Clinic, University of Illinois. Tan, angular lesions that were 2 to 4 mm in diameter were observed on the lower leaf surfaces of two of the 20 leaflets. Within these lesions, there was one uredinum on one leaflet and four on the other leaflet exuding hyaline, echinulate urediniospores (20 × 25 µm). On 11 October 2006, these leaflets were sent by overnight courier to the USDA/APHIS/PPQ/NIS Laboratory, Beltsville, MD Plant Disease Clinic for identification by morphological examination and by PCR using primers specific to Phakopsora pachyrhizi (1). Both tests confirmed the presence of P. pachyrhizi. The 18 leaflets that did not have sporulating pustules on 11 October were incubated in the laboratory for 5 days at near 100% relative humidity. Following incubation, nine leaflets were observed to have uredinia exuding urediniospores with a range of 1 to 43 uredinia per leaflet. These results indicate that incubation may be necessary to maximize the potential to observe uredinia exuding urediniospores. To our knowledge, this is the first report of P. pachyrhizi infecting plants in Illinois. References: (1) R. D. Frederick et al. Phytopathology 92:217, 2002. (2) R. W. Schneider et al. Plant Dis. 89:774, 2005.

Altered regulation of the cytochrome P4501A1 gene: novel inducer-independent gene expression in pulmonary carcinoma cell lines.

The cytochrome P450 (CYP) systems catalyze the metabolic transformation of a wide variety of xenobiotics including procarcinogens present in cigarette smoke condensate as well as atmospheric pollutants. The CYP1A1 isoenzyme is of particular interest because it has been implicated as a risk factor in the etiology of lung cancer in heavy cigarette smokers. The identification and expression of the structural CYP1A1 gene in either normal human lung or lung cancer cells has not been reported. Because of its potential significance in human lung cancer, we investigated the expression of the CYP1A1 structural gene in 24 established human lung cancer cell lines including 15 non-small cell (eight adenocarcinomas, three large cell undifferentiated carcinomas, two bronchioloalveolar cell carcinomas, and two squamous cell carcinomas) and nine small cell lung carcinomas. CYP1A1 mRNA was detected in 14 of 15 (93%) of the non-small cell lung carcinoma cell lines examined following 24-hour treatment with benz[a]anthracene (BA) and in nine of 15 (60%) of the non-small cell lines cultured without an inducer in the medium. When the small cell lung cancer lines were evaluated for CYP1A1 gene expression, two of nine (22%) expressed detectable CYP1A1 mRNA in both BA-induced cell cultures and constitutive (control) cultures. A positive correlation was noted between BA-induced CYP1A1 mRNA levels and the corresponding aryl hydrocarbon hydroxylase activity expressed as absolute BA-induced enzyme activity (r = 0.74; P less than .01; n = 24), which further demonstrated that CYP1A1 mRNA expression reflects CYP1A1 enzyme activity in the individual cell lines. These observations represent the first known demonstration of constitutive (non-induced) CYP1A1 gene expression in human cells and suggest altered regulation of the CYP1A1 gene in selected lung cancer cell lines. These human pulmonary carcinoma cell lines, which have documented regulatory defects, could be useful for further identification of the mechanisms associated with CYP1A1 gene regulation.

The role of P(i) in glycolytic inhibition of calcium ion uptake by ELD ascites tumor cells.

In contrast to previous investigations at 25 degrees C, glucose was shown to support 45Ca2+ uptake at 37 degrees C in intact ELD ascites tumor cells. Intact ascites tumor cells in vitro accumulated up to 5.0 micromol of 45Ca2+ per g cells dry wt. within 20 min. In the presence of 10.0 mM glucose, intracellular P(i) levels fell from 40.0 micromol x g(-1) cells dry wt. to 20.0 micromol x g(-1) cells dry wt. in 5 min. Intracellular P(i) levels were maintained by 20.0 mM extracellular Tris-P(i). 45Ca2+ uptake was inhibited in P(i)-depleted cells, even though the metabolic rate (as measured by Q(lactate)) and energy state (as measured by ATP levels) were at acceptable levels. Evidence has been presented suggesting that previous reports of glucose inhibition of calcium uptake can be attributed to a competition for available intracellular P(i) between glycolytic processes and the mitochondrial calcium uptake mechanism.

Oligonucleotide design and optimized protocol for site-directed mutagenesis.

We have optimized conditions for the successful execution of site-directed mutagenesis (SDM) in systems that utilize mutagenic oligonucleotide primers to direct the synthesis of mutant plasmids. In this report, we describe strategies for the design of single-strand DNA templates for SDM, mutagenic oligonucleotide primers, as well as conditions for the annealing, synthesis and propagation of mutant plasmids. The primary focus of the report details the technical aspects of computer-assisted mutagenic oligonucleotide design. Important features include oligonucleotide length, number of matched bases flanking the point(s) of mutation(s), melting temperature, internal stability of the 5' and 3' ends, hairpin and dimer formation, and potential false-priming sites. Largely through a retrospective analysis of our successes and failures, we describe features of the mutagenic oligonucleotide primer that appear critical in this mutagenesis system. Specific examples of efficient and inefficient oligonucleotides are discussed. These characteristics and guidelines should be applicable for SDM of a broad range of target sequences of varying composition, complexity and secondary structure.

Large-scale purification of plasmid DNA by anion-exchange high-performance liquid chromatography.

Numerous methods have previously been reported for the final steps in the large-scale purification of plasmid DNA. Although gel permeation and reverse-phase high-performance liquid chromatography have been utilized for this procedure in the past, the limited capacity of these systems often necessitated multiple rounds of chromatography, especially with the high copy number plasmids commonly in use today. In this paper, the use of the high-capacity, high-resolution Protein-Pak DEAE 8HR column is presented for the large-scale isolation of highly purified plasmid DNA from crude E. coli cell lysates. Up to 5 mg of plasmid DNA have been purified in a single 50-minute chromatography run. The purified DNA demonstrated excellent biological activity as demonstrated by restriction endonuclease digestion, E. coli transformation and DNA-mediated gene transfection of eukaryotic cells.

Induction of cytochrome P450 1A1 gene expression, oxidative stress, and genotoxicity by carbaryl and thiabendazole in transfected human HepG2 and lymphoblastoid cells.

Carbaryl and thiabendazole, two widely used pesticides, have been shown to induce cytochrome P450 1A1 (CYP1A1) expression, but neither compound is capable of displacing [3H] 2,3,7,8-tetrachlorodibenzo-P-dioxin from its aryl hydrocarbon receptor binding site. In the present study, we investigated the transcriptional regulation of CYP1A1 as well as other genes in various human hepatoma HepG2 cell lines stably transfected with the chloramphenicol acetyl transferase (CAT) reporter gene and cloned under the control of each of 14 promoters or response elements from relevant stress genes. Carbaryl and thiabendazole were found to activate CYP1A1 at the level of transcription, as demonstrated by the dose-dependent increase in reporter CAT and CYP1A1 mRNAs. Moreover, this effect appeared to be mediated via the xenobiotic responsive element (XRE), because both pesticides specifically activated various fusion constructs containing XRE sequences (CYP1A, glutathione S-transferase, and XRE). Carbaryl and to a lesser extent thiabendazole also activated other stress genes such as c-fos and NF-kappaBRE, HSP70 and GRP78, and GADD153 at a transcriptional level. These data suggest that these molecules induce early alert genes, including those known to be sensitive to oxidative stress. This led us to examine the genotoxic effect of carbaryl and thiabendazole by an in vitro DNA repair solid-phase assay. Both compounds provoked a strong DNA-damaging activity in the human lymphoblastoid cell line that constitutively expresses human CYP1A1 cDNA, but not in the parental line, indicating that CYP1A1 is chiefly implicated in carbaryl and thiabendazole genotoxicity. This effect was confirmed on HepG2 cells. These observations support the notion that intracellular signals leading to CYP1A1 induction, oxidative stress, and genotoxicity are intimately related.

The effect of calcium on pulmonary vascular resistance and right ventricular function.

Calcium chloride is frequently administered to patients immediately after separation from cardiopulmonary bypass to improve the contractile state of the myocardium. Animal studies suggest that calcium chloride may produce increases in pulmonary vascular resistance, which can precipitate right ventricular failure. In an attempt to determine the effect of calcium chloride administration after cardiopulmonary bypass on right ventricular function, this study was designed to evaluate patients with normal and elevated pulmonary vascular resistance. Fifty patients scheduled for elective cardiac surgery were prospectively studied for changes in ionized calcium levels before and after bypass. The impact of calcium administration on right ventricular function was assessed by a pulmonary artery catheter modified for the measurement of right ventricular ejection fraction. In all patients the level of ionized calcium decreased during bypass from a mean of 4.91 to 4.29 mg.dl-1. However, the infusion of calcium chloride (10 mg.kg-1) after bypass resulted in increasing the ionized calcium levels to prebypass levels. Administration of calcium chloride after bypass to patients with normal right ventricular function resulted in a transient increase in both cardiac output and right ventricular ejection fraction without any change in pulmonary vascular resistance. Eight patients with both elevated pulmonary vascular resistance and depressed right ventricular function were evaluated to determine the effect of calcium chloride after bypass on pulmonary vascular resistance and right ventricular ejection fraction. Administration of calcium chloride (10 mg.dl-1) to these patients did not result in any significant increase in pulmonary vascular resistance or depression of right ventricular performance. More important, in these patients, right ventricular ejection fraction and cardiac output were significantly increased after calcium chloride administration. In summary, the results of this study fail to demonstrate any increase in pulmonary vascular resistance or deterioration of right ventricular function with the administration of calcium chloride (10 mg.kg-1) after bypass in patients with elevated pulmonary vascular resistance.

Experimental murine acid aspiration injury is mediated by neutrophils and the alternative complement pathway.

Acid aspiration may result in the development of the acute respiratory distress syndrome, an event associated with significant morbidity and mortality. Although once attributed to direct distal airway injury, the pulmonary failure after acid aspiration is more complex and involves an inflammatory injury mediated by complement (C) and polymorphonuclear leukocytes. This study examines the injurious inflammatory cascades that are activated after acid aspiration. The role of neutrophils was defined by immunodepletion before aspiration, which reduced injury by 59%. The injury was not modified in either P- or E-selectin-knockout mice, indicating that these adhesion molecules were not operative. C activation after aspiration was documented with immunochemistry by C3 deposition on injured alveolar pneumocytes. Animals in which C activation was inhibited with soluble C receptor type 1 (sCR1) had a 54% reduction in injury, similar to the level of protection seen in C3-knockout mice (58%). However C4-knockout mice were not protected from injury, indicating that C activation is mediated by the alternative pathway. Finally, an additive effect of neutrophils and C was demonstrated whereby neutropenic animals that were treated with sCR1 showed an 85% reduction in injury. Thus acid aspiration injury is mediated by neutrophils and the alternative C pathway.

Molecular analysis of implantation.

The great frontier in reproductive medicine is implantation biology. The scientific understanding of normal implantation lags behind what is known in areas such as fertilization. For obvious reasons this process is difficult to analyze. Recent investigations utilizing molecular biology approaches have scratched the surface of the interaction between the developing trophoblast and the receptive endometrium. This article describes these approaches with techniques such as serial analysis of gene expression (SAGE) and complementary DNA array hybridization, which will soon reveal the key mediators of normal implantation and result in an understanding of failed implantation.

DH82 cells: a macrophage cell line for the replication and study of equine infectious anemia virus.

In vivo, tissue macrophages have been implicated as an important cell for the replication of equine infectious anemia virus (EIAV). Laboratory investigations of EIAV/macrophage interactions, however, have been hampered by the laborious blood monocyte isolation procedures. In addition, adherent equine macrophage cultures generally have poor long-term viability and are resistant to transfection. This report describes an adherent canine macrophage-like cell line, DH82, that supports the replication of EIAV. This cell line was easily transfectable and supported EIAV Tat transactivation of the LTR. Electrophoretic mobility shift assays were carried out to determine which transcription factor binding sites within the LTR enhancer region were bound by DH82 nuclear extracts. It was found that five different motifs were occupied. The ets motifs that are bound by PU.1 in primary macrophage nuclear extracts specifically interacted with DH82 nuclear extracts. In addition, the PEA-2, Lvb and Oct motifs that are occupied by fibroblast nuclear extracts were also bound by DH82 nuclear extracts. Finally, the methylation-dependent binding protein (MDBP) site that is bound by all nuclear extracts investigated to date demonstrated specific interactions with DH82 nuclear extracts. The observation that both macrophage-specific and fibroblast-specific motifs were utilized by DH82 nuclear extracts suggested that both macrophage-adapted and fibroblast-adapted EIAV could replicate in DH82 cells. Indeed, infectivity studies demonstrated that strains of virus that exclusively replicate in macrophages can replicate in DH82 cells and fibroblast-adapted strains of virus can also replicate in these cells. Finally, these cells could be transfected readily with the EIAV molecular clone, pSPeiav19-2, and virus spread was detected within the culture. In conclusion, this study has identified a useful cell line that should facilitate the study of EIAV expression and replication.

Intentional and unintentional injury in the State of Nevada: 1989-1992.

INTRODUCTION: Data from a large population-based trauma registry were used to assess risk factors and outcomes associated with injuries that were either caused unintentionally, were self-inflicted, or resulted from an assault. DESIGN: A retrospective analysis was conducted of all cases of serious trauma (N = 19,505) occurring in the State of Nevada during the 4-year period from January 1, 1989, through December 31, 1992. The outcome measures of interest were Injury Severity Score, hospitalization, and mortality. RESULTS: Of all patients with unintentional injuries, 9.9% died compared with 44.8% of those whose injuries were intentionally caused. Nearly half (49.0%) of all deaths occurred in persons who were intentionally injured. Of the intentional injuries, 70% were from assaults. Self-inflicted injuries accounted for 5.2% of all injuries but 28.2% of the deaths in the registry. Firearms were most often used in both assaults (38.8%) and self-inflicted injuries (87.4%). Logistic regressions showed that, compared with unintentional injuries, assaults were more likely to occur in urban counties, among males, African Americans, and young adults. Also, compared with unintentional injuries, self-inflicted injuries were more likely to occur in urban counties, among Caucasian, and the 65+ age group. CONCLUSION: Although the overwhelming number of injuries in the registry were unintentionally caused, deaths from intentionally caused injuries accounted for almost half of all deaths. The data from Nevada's registry provided the ability to identify who is at risk for trauma-related injury and death. Prevention programs should be designed to target these populations. As prevention programs are implemented, trauma registries such as Nevada's will provide a sound source of data for assessing long-term trends in injury patterns.

Ovarian cholelithiasis after laparoscopic cholecystectomy associated with chronic pelvic pain.

OBJECTIVE: To report a case of chronic pelvic pain associated with ovarian cholelithiasis and discuss prevention and management of this condition. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 39-year-old woman who presented with right lower abdominal pain after a laparoscopic cholecystectomy. INTERVENTION(S): Diagnostic laparoscopy followed by laparotomy with lysis of adhesions and removal of three to four dozen gallstones. MAIN OUTCOME MEASURE(S): Patient's subjective report of pain. RESULT(S): Resolution of patient's pain. CONCLUSION(S): Gallstones spilled into the peritoneal cavity may migrate and adhere to the dependent portions of the pelvis, potentially resulting in pelvic pain or infertility. This suggests the importance of removing inadvertently spilled gallstones at the time of surgery or using nonsurgical methods of gallstone management in reproductive-aged females.

Ovulation induction in clomiphene-resistant anovulatory women with normal dehydroepiandrosterone sulfate levels: beneficial effects of the addition of dexamethasone during the follicular phase.

OBJECTIVE: To evaluate the effect on ovulation of a 10-day course of dexamethasone (DEX) initiated concurrently with a 5-day course of clomiphene citrate (CC) in CC-resistant patients with normal DHEAS levels. DESIGN: Retrospective review. SETTINGS: Patients from the clinical practice of the authors at the Medical College of Georgia, Augusta, Georgia. PATIENTS: Thirteen oligomenorrheic women with normal DHEAS levels who failed to ovulate on a graduated regimen of CC up to a dose of 150 mg for 5 days. INTERVENTIONS: Ten-day course of DEX initiated concurrently with a 5-day course of CC; ovulation and pregnancy outcomes recorded. MAIN OUTCOME MEASURE: Pregnancy. RESULTS: Eleven of 13 women had evidence of ovulation. Five clinical pregnancies were achieved. CONCLUSION: These initial data support improvements in follicular development with an overlapping follicular phase regimen of CC and DEX in patients with normal DHEAS levels and a previous poor response.

Paternal somatic and germ-line mosaicism for a sex-determining region on Y (SRY) missense mutation leading to recurrent 46,XY sex reversal.

OBJECTIVE: To determine the etiology for recurrent 46,XY sex reversal in a family with two Swyer siblings. DESIGN: Deoxyribonucleic acid (DNA) from peripheral lymphocytes and sperm were analyzed for duplication of the dosage sensitive sex locus (DSS) and for mutations in sex-determining region on Y (SRY). SETTING: An academic teaching hospital. PATIENTS: A family consisting of mother, father, and five phenotypic daughters, of which two were 46,XY sex-reversed females. INTERVENTION: Deoxyribonucleic acid (DNA) extraction, polymerase chain reaction (PCR), Southern blotting, dosage densitometry, single-strand conformation polymorphism (SSCP), and sequencing. MAIN OUTCOME MEASURE: Comparison of control and subject DNA. RESULTS: Deoxyribonucleic acid (DNA) analysis of SRY in genomic DNA from the 46,XY sex-reversed siblings revealed identical missense mutations (T-->G) in both sisters. Analysis of the SRY gene in paternal lymphocyte and sperm DNA revealed mosaicism for wild and mutant (T-->G) SRY sequences. SRY analysis of sperm DNA also demonstrated the same mosaicism for the T-->G missense mutation. CONCLUSION: A postembryonic SRY mutation gave rise to paternal mosaicism for two distinct cell populations (SRY+/SRY-). The presence of a wild type SRY in the somatic cell line may account for a normal pattern of male sexual differentiation, whereas the presence of a mutated SRY in the germ line resulted in two 46,XY sex-reversed offspring. These results confirm a proposed mechanism for the condition of recurrent 46,XY sex-reversed females.