Benjamin D. Hamar

Proteomic Profiling of the Amniotic Fluid to Detect Inflammation, Infection, and Neonatal Sepsis

Background Proteomic analysis of amniotic fluid shows the presence of biomarkers characteristic of intrauterine inflammation. We sought to validate prospectively the clinical utility of one such proteomic profile, the Mass Restricted (MR) score. Methods and Findings We enrolled 169 consecutive women with singleton pregnancies admitted with preterm labor or preterm premature rupture of membranes. All women had a clinically indicated amniocentesis to rule out intra-amniotic infection. A proteomic fingerprint (MR score) was generated from fresh samples of amniotic fluid using surface-enhanced laser desorption ionization (SELDI) mass spectrometry. Presence or absence of the biomarkers of the MR score was interpreted in relationship to the amniocentesis-to-delivery interval, placental inflammation, and early-onset neonatal sepsis for all neonates admitted to the Newborn Special Care Unit (n = 104). Women with “severe” amniotic fluid inflammation (MR score of 3 or 4) had shorter amniocentesis-to-delivery intervals than women with “no” (MR score of 0) inflammation or even “minimal” (MR score of 1 or 2) inflammation (median [range] MR 3–4: 0.4 d [0.0–49.6 d] versus MR 1–2: 3.8 d [0.0–151.2 d] versus MR 0: 17.0 d [0.1–94.3 d], p < 0.001). Nonetheless, a “minimal” degree of inflammation was also associated with preterm birth regardless of membrane status. There was a significant association between the MR score and severity of histological chorioamnionitis (r = 0.599, p < 0.001). Furthermore, neonatal hematological indices and early-onset sepsis significantly correlated with the MR score even after adjusting for gestational age at birth (OR for MR 3–4: 3.3 [95% CI, 1.1 to 9.2], p = 0.03). When compared with other laboratory tests routinely used to diagnose amniotic fluid inflammation and infection, the MR score had the highest accuracy to detect inflammation (white blood cell count > 100 cells/mm3), whereas the combination of Gram stain and MR score was best for rapid prediction of intra-amniotic infection (positive amniotic fluid culture). Conclusions High MR scores are associated with preterm delivery, histological chorioamnionitis, and early-onset neonatal sepsis. In this study, proteomic analysis of amniotic fluid was shown to be the most accurate test for diagnosis of intra-amniotic inflammation, whereas addition of the MR score to the Gram stain provides the best combination of tests to rapidly predict infection.

Value of placental microbial evaluation in diagnosing intra-amniotic infection

OBJECTIVE: To evaluate the ability of microbiologic and pathologic examination of the placenta to accurately diagnose intraamniotic infection and inflammation. METHODS: One hundred eighty-three women with a clinically indicated amniocentesis were enrolled prospectively. We applied our analysis to 56 women with evidence of preterm labor or preterm premature rupture of membranes who delivered within 48 hours of amniotic fluid testing results. Twenty-three patients, assessed for fetal lung maturity in the third trimester, served as controls. Amniotic fluid was cultured for aerobic, anaerobic, Ureaplasma, and Mycoplasma species. We used mass spectrometry to assess the degree of intraamniotic inflammation (Mass Restricted scoring). After delivery, microbiologic and histologic studies of the placenta were performed. These results were interpreted in comparison with the direct microbiologic and inflammatory analysis of the amniotic fluid. A sample size of 45 patients was required to show a test accuracy of 80% or more. RESULTS: Ninety-two percent of women with positive amniotic fluid cultures tested with at least one positive placenta culture. Eighty percent of women who had negative amniotic fluid cultures also tested with a positive placenta culture. The accuracy of placental cultures in predicting amniotic fluid infection varied from 44% to 57%. Placental pathology showed an accuracy of only 58% in diagnosing intraamniotic inflammation. CONCLUSION: Placental microbiologic and histologic studies poorly reflect the infectious and inflammatory status of the amniotic fluid. Results of such studies should be interpreted with caution in the management and future counseling of women with preterm labor or preterm premature rupture of membranes. LEVEL OF EVIDENCE: II

Proteomic but Not Enzyme-Linked Immunosorbent Assay Technology Detects Amniotic Fluid Monomeric Calgranulins from Their Complexed Calprotectin Form

ABSTRACT Four proteomic biomarkers (human neutrophil peptide 1 [HNP1], HNP2 [defensins], calgranulin C [Cal-C], and Cal-A) characterize the fingerprint of intra-amniotic inflammation (IAI). We compared proteomic technology using surfaced-enhanced laser desorption-ionization-time of flight (SELDI-TOF) mass spectrometry to enzyme-linked immunosorbent assay (ELISA) for detection of these biomarkers. Amniocentesis was performed on 48 women enrolled in two groups: those with intact membranes (n = 27; gestational age [GA], 26.0 ± 0.8 weeks) and those with preterm premature rupture of the membranes (PPROM; n = 21; GA, 28.4 ± 0.9 weeks). Paired abdominal amniotic fluids (aAFs)-vaginal AFs (vAFs) were analyzed in PPROM women. Quantitative aspects of HNP1-3, Cal-C, Cal-A, and calprotectin (a complex of Cal-A with Cal-B) were assessed by ELISA. SELDI-TOF mass spectrometry tracings from 16/48 (33.3%) aAFs and 13/17 (88.2%) vAFs were consistent with IAI (three or four biomarkers present). IAI (by SELDI-TOF mass spectrometry) was associated with increased HNP1-3 and Cal-C measured by ELISA. However, immunoassays detected Cal-A in only 4 of the AFs even though its specific SELDI-TOF mass spectrometry peak was identified in 19/48 AFs. Calprotectin immunoreactivity was decreased in AFs retrieved from women with IAI (P = 0.01). In conclusion, IAI is associated with increased HNP1-3 levels. In the absence of isoform-specific ELISAs, mass spectrometry remains the only way to discriminate the HNP biomarker isoforms. Monomeric Cal-A is not reliably estimated by specific ELISA as it binds to Cal-B to form the calprotectin complex. Cal-C was reliably measured by SELDI-TOF mass spectrometry or specific ELISA.

Ultrasound evaluation of the uterine scar after cesarean delivery: a randomized controlled trial of one- and two-layer closure.

OBJECTIVE: To survey the uterine scar thickness by ultrasonography in women randomly assigned to one- or two-layer hysterotomy closure after primary cesarean delivery. METHODS: This was a randomized, blinded trial of uterine scar closure with ultrasonographic follow-up. Thirty consecutive patients undergoing primary cesarean delivery were enrolled and randomly assigned to one- or two-layer closure of the hysterotomy. Ultrasound surveillance of the uterine scar thickness was performed at baseline (before surgery) and 48 hours, 2 weeks, and 6 weeks post partum. RESULTS: Patient compliance with the postpartum surveillance protocol was 90%, and the uterine scar was visualized in 99% of attempted ultrasonographic examinations. There were no differences between groups at baseline or at any of the follow-up evaluations. An initial 5- to 6-fold increase in uterine scar thickness was observed, followed by a gradual decrease with the 6-week measurements still thicker than baseline. Repeated measures analysis of variance showed significant variation across time points starting either at baseline (P<.001) or at 48 hour postoperatively (P<.001), but this variation did not depend on closure type (P=.79 for all visits and P=.81 beginning with 48-hour postoperative time point). CONCLUSION: The process of uterine scar remodeling can be successfully monitored by ultrasonography. Uterine scar thickness diminishes progressively after both one- or two-layer closure but does not vary with mode of hysterotomy closure. The uterine scar thickness remains increased even at 6 weeks post partum, suggesting that the process of uterine scar remodeling extends beyond the traditional postpartum period. CLINCAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00224250 LEVEL OF EVIDENCE: I

Premature rupture of membranes, placenta increta, and hysterectomy in a pregnancy following endometrial ablation

Endometrial ablation has become a popular method of managing menorrhagia. Pregnancy after endometrial ablation has a high rate of complications. We present the case of a parous woman with a history of endometrial ablation with preterm premature rupture of membranes. Despite the absence of established sonographic markers for abnormal placentation, placenta accreta was noted at the time of cesarean delivery. In women with history of endometrial ablation, the endometrium is not normal and may allow for more aggressive placental invasion or adherence. Consequently, the sonographic indices described for evaluating placenta accreta may not be present. We believe that placentation in women with prior endometrial ablations should be considered extremely high risk for placenta accreta or increta and managed accordingly when preparing for delivery.

Expectant management of uterine dehiscence in the second trimester of pregnancy

BACKGROUND Uterine dehiscence and rupture are serious complications of pregnancy after a cesarean delivery. Management of uterine dehiscence diagnosed in second trimester can be controversial. CASE A woman with a previous cesarean delivery was diagnosed with a uterine dehiscence at 20 weeks in the area of her prior cesarean incision. Although she was counseled regarding risks to herself and the fetus, she decided to continue the pregnancy. She was, therefore, managed expectantly until 31 weeks and delivered by cesarean because of fetal heart rate decelerations. The infant did well and was discharged home at 3 weeks of age. The patient remained asymptomatic after delivery. CONCLUSION With close monitoring, expectant management of uterine dehiscence diagnosed in the second trimester is possible.

Management of a cervical heterotopic pregnancy presenting with first-trimester bleeding: case report and review of the literature

OBJECTIVE To report a rare case of a cervical heterotopic pregnancy resulting from intrauterine insemination (IUI) that presented with first-trimester bleeding. DESIGN Case report and literature review. SETTING Large university-affiliated infertility practice. PATIENT(S) A 40-year-old gravida 2 para 1 Asian woman at 7-3/7 weeks gestational age following clomiphene citrate/IUI for the treatment of secondary infertility presented with heavy vaginal bleeding for several days. INTERVENTION(S) Transvaginal ultrasound on admission revealed a single live intrauterine pregnancy and a cervical gestational sac containing a nonviable embryo. The patient continued to have vaginal bleeding and 2 days later underwent removal of the cervical ectopic pregnancy tissue with ring forceps, as well as an ultrasound-guided intracervical Foley balloon and cerclage placement. The bleeding subsided, and 48 hours later the Foley and cerclage were removed. MAIN OUTCOME MEASURE(S) Pregnancy outcome. RESULT(S) The remainder of the pregnancy was uncomplicated and the patient had a full-term cesarean delivery for footling breech of a healthy male infant. CONCLUSION(S) Cervical heterotopic pregnancy is a very rare event that almost universally results from infertility treatment. We present a case where we were able to remove the cervical ectopic and tamponade the bleeding, thus preserving the intrauterine pregnancy for this subfertile couple, and we review the existing literature.

Trends in fetal echocardiography and implications for clinical practice: 1985 to 2003.

Objective. The purpose of this study was to determine whether patterns of referral for fetal echocardiography (FE) and the subsequent yield for structural congenital heart disease (CHD) have changed between 1985 and 2003. Methods. All FE performed between 1985 and 2003 at Yale–New Haven Hospital was reviewed. The primary indication for study and the presence of structural CHD were recorded, and data were analyzed for trends. Linear regression with Pearson coefficient calculation and Mantel‐Haenszel χ2 analysis were performed (P < .05 significant). Results. Between 1985 and 2003, 10,806 patients had FE at Yale–New Haven Hospital, and 774 cases of structural CHD were detected. The annual number of studies and rate of detected structural CHD remained constant through the study period. There was a significant increase in the proportion of studies for diabetes, maternal structural CHD, suspicious 4‐chamber heart, and family history of cardiac disease. There was a significant decrease in the proportion of studies for a previous child with structural CHD, cardiac teratogen exposure, other fetal anomalies, aneuploidy, fetal arrhythmia, and nonimmune hydrops. The percentage of structural CHD detected by indication remained constant through the study period. Subgroup analysis of diabetes revealed an increase in class B diabetes, while classes C and D remained stable. Conclusions. This is one of the largest series of FE and suggests that the pattern of indications has changed since 1985. Specifically, referral for diabetes (mostly class B) has increased without a change in yield of structural CHD by indication for sonography. The changing referral patterns reflect a change in obstetric demographics and has implications for obstetric care.

Nuchal translucency measurement plus non-invasive prenatal testing to screen for aneuploidy in a community-based average-risk population

Beginning in June 2012, the division of maternal-fetal medicine at South Shore Hospital in Massachusetts, a community hospital south of Boston with 3359 deliveries in 2012, began offering first-trimester nuchal translucency (NT) ultrasound measurement followed by non-invasive prenatal testing (NIPT) to all women who presented for evaluation in the first trimester. During 2011, standard analyte screens indicated increased risk in 96 cases (4.6%), resulting in 41 invasive procedures that allowed identification of three cases of aneuploidy (positive predictive value, 3%). We hypothesized that our new protocol would allow us to offer immediate invasive testing to patients with thickened NT, cystic hygroma or other structural abnormalities, while reducing the high false-positive rate of conventional screening. Women received genetic counseling and were offered NT measurement and NIPT (Harmony, Ariosa Diagnostics, San Jose, CA, USA) or invasive diagnostic testing. All patients’ insurance policies reimbursed NIPT, irrespective of risk status (Medicaid did not reimburse, but patients were not charged for the test). Invasive diagnostic testing was again offered to women with a fetal NT ≥ 3 mm, cystic hygroma or other ultrasound findings associated with chromosomal abnormalities, and to women with an NIPT or quad screen result indicative of high risk. Outcome data were collected at birth. We obtained institutional review board approval for the study. Between 4 June 2012 and 31 January 2013, 1228 women were seen for routine first-trimester screening. Their median age was 31.5 years and 71% were under 35 years of age. All women underwent NT measurement, which was normal in 96.4% of cases. Thickened NT or cystic hygroma was diagnosed in 37 (3.0%) cases (Figure S1), and other fetal structural abnormalities were diagnosed in seven (0.6%) women. Six of these seven cases had a normal karyotype and, in one, NIPT indicated a high risk for trisomy 18 that was confirmed by cytogenetic analysis. Of the 1184 women with a normal ultrasound result, 1153 underwent NIPT. Thirty-one patients declined all further screening (Figure S2). NIPT failed to yield a result after two attempts in 14 patients (1.2%). Approximately half of the patients in whom two tests failed had pregnancy complications or comorbidities (Table S1) and in one case triploidy was diagnosed later in pregnancy. Overall, 16 major abnormalities were detected, including six cases of anatomic malformation, 10 cases of chromosomal abnormalities and one case of both chromosomal and structural abnormalities. Our clinical protocol detected 15 of the 16 major abnormalities. Ultrasound results were abnormal in nine of the 11 cases of aneuploidy. Aneuploidy was observed in seven average-risk women. Eight of the 11 cases were diagnosed prior to 14 weeks of gestation. Our paper is, to our knowledge, the first to report on the use of first-trimester NT screening followed by NIPT in a general screening population. NT measurement and NIPT, used concurrently, optimized detection of more abnormal fetuses earlier in pregnancy than either modality alone. During the 8-month period described in this paper, we performed four invasive diagnostic tests to confirm false-positive NIPT results, compared to 30 procedures during the same 8-month period in the prior year. There were 62 false-positive analyte screens during that prior period but only 30 patients opted for invasive testing.