Diagnosis of aneuploidy in archival, paraffin-embedded pregnancy-loss tissues by comparative genomic hybridization.

OBJECTIVE: To evaluate the detection of aneuploidy in archival tissues from miscarriages by a method that uses microdissection and DNA extraction of villus cells from paraffin blocks, followed by universal DNA amplification and comparative genomic hybridization (CGH). DESIGN: Retrospective analysis. SETTING: Academic medical center. PATIENT(S): Nine archival tissues from cases of spontaneous abortion with trisomy 16 (two cases), trisomy 21 (three cases), trisomy 22 (two cases), triploidy (one case), and monosomy X (one case). INTERVENTION(S): Villus DNA was extracted from microdissected, formalin-fixed, paraffin-embedded tissues. Aneuploidy was detected by CGH after universal amplification of the DNA with the use of degenerate oligonucleotide-primed polymerase chain reaction. MAIN OUTCOME MEASURE(S): Detection of aneuploidy in archival pregnancy-loss tissues using CGH. RESULT(S): In all nine cases, DNA was successfully extracted from the microdissected tissues and was of sufficient quantity and quality for evaluation by CGH. In six of nine cases, the chromosomal abnormality detected by conventional cytogenetic analysis was identified by CGH: trisomy 16 (2/2), trisomy 21 (3/3), and trisomy 22 (1/2). One case of each of the following was not detectable: triploidy (1/1), monosomy X (1/1), and trisomy 22 (1/2). CONCLUSION(S): We propose CGH as a method for determination of aneuploidy in pregnancy-loss archival tissues when conventional cytogenetic analysis is unsuccessful or when it was not performed when fresh tissue was available.

Risk scoring, fetal fibronectin, and bacterial vaginosis to predict preterm delivery.

OBJECTIVE: To determine the value of markers for predicting spontaneous preterm birth. METHODS: One hundred forty asymptomatic gravidas were recruited from 20-24 weeks' gestation. Risk score was assessed, vaginal swabs were analyzed for bacterial vaginosis, and cervical and vaginal swab were tested for fetal fibronectin FDC-6, X18A4, and CAF. Univariate analysis was used to determine potential predictors (and combinations of predictors) of outcome. Multiple logistic regression was done to identify independent predictors of spontaneous preterm birth. Sensitivity, specificity, positive and negative predictive values; and odds and likelihood ratios were calculated for significant predictors. RESULTS: Predictors significantly associated with the primary outcome were preterm birth-risk score and vaginal fetal fibronection FDC-6 (logistic regression odds ratio [OR] 16.9 [95% confidence interval (CI) 3.1, 92.8]) and 8.0 ([95% CI 1.6, 38.2], respectively). Bacterial vaginosis, fetal fibronectin X18A4, fibronectin CAF, and cervical fetal fibronectin FDC-6 were not associated with spontaneous preterm birth; however, the statistical power to assess these variables was limited. The combination of positive preterm birth-risk score and vaginal fetal fibronectin FDC-6 had a sensitivity of 44.4%, specificity of 97.7%, positive predictive value of 57.1%, negative predictive value of 96.2%, and a significant likelihood ratio for a positive test of 19.4 (95% CI 5.1, 73.8). CONCLUSION: The combination of preterm birth-risk score and vaginal fetal fibronectin FDC-6 predicted spontaneous preterm birth. Intervention trials are required to determine whether a combination of screening tests will reduce rates of spontaneous preterm birth.

Cytogenetic diagnosis of "normal 46,XX" karyotypes in spontaneous abortions frequently may be misleading.

OBJECTIVE: To use the molecular identification of Y chromosome material in products of conception cytogenetically diagnosed as "46,XX" to confirm the occurrence of inaccurate cytogenetic test results most likely attributable to maternal cell contamination. DESIGN: Retrospective analysis. SETTING: Academic medical center. PATIENT(S): Thirty-four archival tissues from cases of spontaneous abortion with a "46,XX" karyotype based on cytogenetic analysis. INTERVENTION(S): Maternal and villus DNA were extracted from microdissected, formalin-fixed, paraffin-embedded archival tissues. The presence of the X and Y chromosomes was detected with the use of polymerase chain reaction assays and confirmed with fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): Accuracy of cytogenetic evaluation of products of conception. RESULT(S): Four (29%) of 14 first trimester and 1 (5%) of 20 second trimester "46,XX" pregnancy losses contained Y chromosome-specific DNA and demonstrated a single X chromosome-specific allele by polymerase chain reaction analysis consistent with an "XY" karyotype. Fluorescence in situ hybridization was confirmatory in 4 of 5 samples that demonstrated single X and Y signals in villus cells. CONCLUSION(S): Inaccuracy exists in the cytogenetic analysis of early products of conception that most likely is due to maternal cell contamination. In the absence of confirmatory testing, such as with a "DNA fingerprinting" assay, reports of a "46,XX" karyotype should be used cautiously in patient counseling and management.

Reliability of intraobserver and interobserver sonographic endometrial stripe thickness measurements.

OBJECTIVE: To determine the intraobserver and interobserver reliability of endometrial stripe thickness measurements in women undergoing controlled ovarian hyperstimulation. DESIGN: Prospective blinded study. SETTING: Tertiary care, university hospital. PATIENT(S): Sixty-three patients undergoing controlled ovarian hyperstimulation and being monitored with transvaginal ultrasound were studied. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Intraobserver and interobserver variability of endometrial stripe thickness measurements between 2 blinded observers, with 2 observations made by each observer. RESULT(S): A statistically significant correlation was detected between the 2 measurements of each observer. The mean (+/-SD) interobserver difference between the average of the 2 measurements performed by both observers was 1.02 +/- 0.82 mm. A statistically significant correlation between the measurements of the 2 observers was detected. For both observers A and B, who used < or =6 mm as an abnormal endometrial thickness, an excellent level of agreement was detected between the 2 measurements made on each patient. When comparing the average values obtained by the 2 observers for each of the patients, an excellent level of agreement was detected. CONCLUSION(S): There is an excellent correlation between intraobserver and interobserver measurements of endometrial stripe thickness.

Identification of oncofetal fibronectin in patients with advanced epithelial ovarian cancer: detection in ascitic fluid and localization to primary sites and metastatic implants.

BACKGROUND: The mechanisms by which metastatic ovarian cancer adheres to peritoneal surfaces are not well understood. A role for tumor-derived extracellular matrix adhesive molecules such as fibronectin (FN) has been proposed. Because oncofetal fibronectin (onfFN) isoforms function in the adhesion of trophoblasts and have been identified in association with several malignancies, we sought to study onfFN in patients with advanced epithelial ovarian cancer. METHODS: Total FN was identified with the nonspecific anti-FN monoclonal antibody CAF. OnfFN was identified using the specific monoclonal antibodies FDC-6 and X18A4. These antibodies were applied to: 1) ascitic fluid from advanced epithelial ovarian cancer patients and peritoneal fluid from patients without pathologic conditions and 2) tissue sections of primary lesions and metastatic ovarian cancer implants. Comparative histologic specimens included normal ovarian tissue and small bowel implants of endometriosis. RESULTS: When measured by sandwich enzyme-linked immunoadsorbent assay, all peritoneal fluids (32 malignant and 32 benign) contained marked quantities of total (CAF reactive) FN, although malignant ascites had higher concentrations than benign samples (173.2 +/- 36.8 microg/mL vs. 76.4 +/- 31.8 microg/mL; P = 0.001). Malignant ascites also had significantly higher levels of onfFN than benign peritoneal fluid (FDC-6: 3.4 +/- 0.6 vs. 0.9 +/- 0.2 microg/mL; and X18A4: 5.1 +/- 1.3 vs. 1.1 +/- 0.4 microg/mL; P = 0.0001). Immunohistochemical staining of malignant lesions revealed prominent localization of both CAF reactive FN and onfFN to the stroma surrounding epithelial tumor nests. More delicate fibrillar staining within tumor nests also was evident. In contrast, implants of endometriosis revealed strong stromal staining for CAF reactive FN but not for onfFN. CONCLUSIONS: These results demonstrate the presence of onfFN in advanced ovarian malignancies. We speculate that onfFN may participate in tumor-associated peritoneal adhesive interactions.

Hydrosalpinx fluid enhances human trophoblast viability and function in vitro: implications for embryonic implantation in assisted reproduction.

OBJECTIVE: To assess the effects of hydrosalpinx fluid on human cytotrophoblast viability and function in vitro. DESIGN: Human cytotrophoblasts obtained from third-trimester placentas were cultured in vitro with hydrosalpinx fluid, and cell viability and protein production were assayed. SETTING: A university hospital. PATIENT(S): Ten hydrosalpinx fluid samples obtained from seven women with clearly diagnosed hydrosalpinges. INTERVENTION(S): Recovery of hydrosalpinx fluid by transvaginal aspiration or at the time of surgery. MAIN OUTCOME MEASURE(S): Cell viability was assessed by the XTT assay. Secretion of trophoblast oncofetal fibronectin (tropho-uteronectin) and beta-hCG by cultured trophoblasts was determined by Western blot and ELISA of the culture media. RESULT(S): With increasing concentrations of hydrosalpinx fluid from 0% to 20%, there was a significant increase in trophoblast cell viability (1.63-fold increase in 20% hydrosalpinx fluid). Likewise, both Western blot and ELISA assays demonstrated a significant increase in tropho-uteronectin production by trophoblasts with increasing hydrosalpinx fluid concentrations (3.76-fold increase in 20% hydrosalpinx fluid). beta-Human chorionic gonadotropin production also increased significantly in the presence of hydrosalpinx fluid (3.31-fold increase in 20% hydrosalpinx fluid). CONCLUSION(S): These findings suggest that hydrosalpinx fluid improves human trophoblast viability in vitro and enhances the production of tropho-uteronectin and beta-hCG by these cells.

Recurrent pregnancy loss: a review.

Recurrent pregnancy loss (RPL) is a frustrating problem for both the patient and the clinician. The causes of RPL are diverse and may be associated with genetic, anatomic, microbiologic, endocrine, or immunologic factors. When a couple is ready for an evaluation, the nurse practitioner needs to be able to discuss possible causes, aspects of diagnostic testing, and available options. Reassurance and clear information about prognosis are important and emotional support must be offered throughout the investigation and subsequent pregnancy.

Fibronectinase activity in cultured human trophoblasts is mediated by urokinase-type plasminogen activator.

OBJECTIVE: Human trophoblast proteolytic activity is believed to have implications for early implantation events and maintenance of chorionic structural integrity later in gestation. Abnormal release of chorion-derived extracellular matrix proteins such as fibronectin may identify patients at risk for preterm labor and delivery. The aim of this study was to characterize the enzyme(s) potentially responsible for trophoblast-mediated proteolysis of fibronectin. STUDY DESIGN: Human term cytotrophoblasts were analyzed for their capacity to cleave fibronectin into discrete proteolytic fragments. Selective protease inhibitors were used to characterize trophoblast-derived enzymes with fibronectinase activity. Analysis and quantitation of fibronectin fragment release was determined by Western immunoblots and enzyme-linked immunosorbent assays. RESULTS: Fibronectinase activity in trophoblast cultures was found to be both cell mediated and secreted, with the release of discrete fibronectin fragments into the media. Cell-mediated proteolytic activity could be partially inhibited by serum, whereas conditioned media containing fibronectinase activity was completely inhibited by serum, a serine protease inhibitor, and a selective inhibitor of urokinase-type plasminogen activator. Digestion of fibronectin with pure urokinase produced a similar pattern of fibronectin fragments compared with fibronectinase-generated fragments. Immunodepletion of urokinase from trophoblast media abolished fibronectinase activity. CONCLUSIONS: Trophoblast-derived urokinase-type plasminogen activator has significant proteolytic activity in vitro with the capability of cleaving fibronectin into discrete fragments. In early pregnancy this activity could be part of the enzymatic cascade leading to uterine extracellular matrix remodeling and implantation. Later in pregnancy trophoblast derived urokinase could promote normal or inflammation-induced changes in the chorionic extracellular matrix.

Oncofetal fibronectin: new insight into the physiology of implantation and labor.

Oncofetal fibronectin is a newly studied protein produced by the trophoblast and is present in plasma and cervicovaginal secretions of pregnant women as labor approaches or when they have certain complications of pregnancy. Alterations in levels of oncofetal fibronectin occur in preterm labor, postterm pregnancy, and pregnancy-induced hypertension. Determining the presence or absence of full-term and preterm labor often is difficult in the clinical setting, and decision-making sometimes is hindered by the lack of a specific biochemical marker for labor. Oncofetal fibronectin may become a clinical indicator or predictor of true labor, preterm labor, or some complications of pregnancy. Assay techniques that identify clinically meaningful levels of oncofetal fibronectin are being developed and investigated and soon may be available to screen and identify pregnant women at risk. Research findings suggest new paths for investigation that may lead to important interventions in health care directed at identifying and decreasing maternal and neonatal morbidity and mortality. This article reviews pertinent aspects of placentation and the rationale for using oncofetal fibronectin detection as a clinical marker for abnormal pregnancy states.

Recurrent pregnancy loss: a review.

Recurrent pregnancy loss (RPL) is a frustrating problem for both the patient and the clinician. The causes of RPL are diverse and may be associated with genetic, anatomic, microbiologic, endocrine, or immunologic factors. When a couple is ready for an evaluation, the nurse practitioner needs to be able to discuss possible causes, aspects of diagnostic testing, and available options. Reassurance and clear information about prognosis are important and emotional support must be offered throughout the investigation and subsequent pregnancy.

The effect of leukemia inhibitory factor (LIF) on trophoblast differentiation: a potential role in human implantation.

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein strongly associated with normal implantation in the mouse. We have recently determined that LIF is expressed in the human endometrium in a menstrual cycle dependent manner. Maximal expression is observed between days 19 and 25 of the menstrual cycle, coinciding with the time of human implantation. In this study we have utilized purified cultures of human cytotrophoblasts to examine the effects of LIF on several morphologic and biochemical markers of the trophoblastic differentiation. We purified human cytotrophoblasts from term placentae and cultured them with and without LIF (10 ng/mL). The secretion of human CG, oncofetal fibronectin, and progesterone were measured at 24, 48, 72, and 96 h. Northern blot analysis was used to assess messenger RNA (mRNA) expression of beta hCG and oncofetal fibronectin. We found that LIF markedly decreased trophoblast production of hCG protein at 72 and 96 h, as well as expression of beta hCG mRNA. LIF also significantly increased the expression of oncofetal fibronectin mRNA and secretion of the protein. LIF did not affect steroidogenic activity of cultured trophoblasts, as determined by progesterone production. These biochemical changes are characteristic of cytotrophoblast differentiation toward an anchoring extravillous phenotype. Thus, LIF appears to be an important regulator of human embryonic implantation by directly modulating trophoblast differentiation.

Extracellular matrix interactions in early human embryos: implications for normal implantation events.

OBJECTIVE: To study the role of the extracellular matrix (ECM) proteins in supporting the development and implantation competence of human embryos. DESIGN: Expression of an implantation site adhesive glycoprotein, oncofetal fibronectin, and basement membrane collagen-degrading matrix metalloproteinase-2 and -9 were studied in cultured human embryos. The ability of exogenously added laminin and fibronectin to enhance hatching and matrix metalloproteinase-2 expression was quantitated also. PATIENTS: Fifty-four women with tubal factor infertility enrolled in the IVF program at the University Hospital of Oulu agreed to participate by providing 20 residual oocytes and 227 residual early embryos for this study. MAIN OUTCOME MEASURES: The presence of oncofetal fibronectin immunoreactive protein was assayed by immunocytochemical staining with two specific monoclonal antibodies, FDC-6 and X18A4. These antibodies bind to specific and distinct epitopes within tumor and trophoblast-derived oncofetal fibronectin. Changes in embryo matrix metalloproteinase-2 production were measured by zymography and confirmed by immunocytochemical staining. RESULTS: Intracellular oncofetal fibronectin was identified within blastomeres of early stage embryos. The immunoreactivity of oncofetal fibronectin in the zona pellucida was associated with fragmentation and dissolution of the zona. Exogenously added laminin or adult-type fibronectin significantly increased the hatching rate of the cultured embryos. Embryos cultured with added adult-type fibronectin or trophoblast-derived oncofetal fibronectin stimulated the matrix metalloproteinase-2 production (72-kd type IV collagenase) by 2.25 +/- 0.16-fold when compared with control embryos (mean +/- SD). CONCLUSIONS: Embryonic production of specific ECM proteins, such as oncofetal fibronectin, appears to be important for the morphological and biochemical development of human preimplantation embryos. Moreover, ECM proteins promote acquisition of the adhesive and degradative properties required by human embryos for successful implantation.

Monoclonal antibody X18A4 identifies an oncofetal fibronectin epitope distinct from the FDC-6 binding site.

OBJECTIVE: Oncofetal fibronectin reactive with antibody FDC-6 has been associated with trophoblastic implantation and chorion structural stability. Abnormal release of this fibronectin into cervical and vaginal secretions has identified patients at risk for preterm labor and delivery. The aim of this study was to determine whether trophoblast-derived oncofetal fibronectin contains other novel epitopes distinct from the FDC-6 binding site. STUDY DESIGN: Antitrophoblast fibronectin hybridomas were generated and screened by comparative immunoassays. One specific monoclonal antibody, X18A4, was identified and compared with antibody FDC-6 by immunocytochemical and immunoblot analyses. Both antibodies were also evaluated in "sandwich"-type double monoclonal immunosorbent assays. RESULTS: X18A4 and FDC-6 bind avidly and noncompetitively to distinct epitopes within oncofetal fibronectin. They exhibit similar immunohistochemical staining of the extracellular matrix within placental tissue, ovarian epithelial tumors, and cultured trophoblasts. However, in contrast to FDC-6, X18A4 has no detectable binding activity to human plasma fibronectin, and its binding to oncofetal fibronectin was unaffected by enzymatic deglycosylation. Immunoblot analyses of oncofetal fibronectin proteolytic digests suggest that X18A4 binds near or within the alternatively spliced type III connecting segment domain. CONCLUSIONS: X18A4 identifies and binds with high affinity to a new epitope within oncofetal fibronectin, distinct from the FDC-6 binding site. Because X18A4 displays no detectable binding to plasma fibronectin, it could be used as an important adjunctive antibody for enhancing the specificity of clinically based oncofetal fibronectin diagnostic assays.

A mucin-like glycoprotein identified by MAG (mouse ascites Golgi) antibodies. Menstrual cycle-dependent localization in human endometrium.

Human endometrial glands synthesize and secrete a high molecular weight mucin-like glycoprotein in a menstrual cycle-dependent fashion. A novel moiety within this Golgi-associated glycoprotein is strongly reactive with IgG antibodies in numerous murine ascites, and has been termed MAG (mouse ascites Golgi). Immunohistochemical staining of 201 endometrial biopsies revealed the following patterns: MAG first appeared in the Golgi on cycle day 5, peaked on day 15, was present on the surface of the luminal epithelium between days 17 and 19, and was no longer detectable after day 19. MAG was also present in cervical, prostate, seminal vesicle, and lacrimal glands, pancreatic acinar cells, gall bladder and bile duct epithelium, and certain cells of the salivary and sweat glands. Interestingly, only tissues from blood group A individuals exhibited this staining. As a common link among all these cell types is the expression of mucins, we speculated that the MAG epitope could be a mucin-associated blood group A-related epitope. This hypothesis was tested by absorption experiments with a variety of glycoconjugates and erythrocytes and by immunoblots of MAG-rich material. The absorption studies demonstrated that only type III porcine mucin (< 1% sialic acid) and blood type A or AB erythrocytes were able to absorb the anti-MAG antibody. Inasmuch as N-acetyl-galactosamine alone, the terminal blood group A carbohydrate, did not block MAG antibody binding, the MAG epitope appears to involve N-acetylgalactosamine plus other determinants. Immunoblots of endometrial extracts and saliva from blood type A individuals revealed MAG-reactive material with a molecular weight > 200 kd under reducing conditions. Because the MAG epitope appears on the endometrial surface during the purported implantation window, we speculate that mucin-like epitopes could play a role in the earliest apposition phases of conceptus-endometrial interaction.

Monoclonal antibody FDC-6 exhibits binding to human plasma fibronectin: a caveat for cervicovaginal oncofetal fibronectin testing?

OBJECTIVE: Monoclonal antibody FDC-6 has been used clinically to measure cervicovaginal fibronectin and to predict patients at risk for preterm labor. Because bleeding is often a complicating factor during pregnancy, the aim of this study was to determine whether FDC-6 reactive fibronectin is present in normal human plasma. STUDY DESIGN: Quantitative Western immunoblots and a commercially available enzyme-linked immunosorbent assay were used to measure FDC-6 binding to nonpregnant human and bovine plasma fibronectin. FDC-6 binding to whole sera was also assessed, as was the effect of enzymatic deglycosylation of human plasma fibronectin. RESULTS: Both assays demonstrated measurable FDC-6-reactive fibronectin in nonpregnant human blood, with plasma concentrations ranging from 3 to 12 micrograms/ml (1% to 4% of total plasma fibronectin). FDC-6 had no detectable binding activity to bovine plasma fibronectin. De-O-glycosylated human plasma fibronectin had markedly reduced binding activity for FDC-6. CONCLUSIONS: FDC-6 binds specifically to a clinically significant percentage of circulating O-glycosylated human plasma fibronectin isoforms that are not associated with pregnancy. This caveat should be considered when pregnant patients are evaluated for the presence of FDC-6 reactive oncofetal fibronectin in the cervix or vagina.

Transforming growth factor-beta stimulates trophoblast oncofetal fibronectin synthesis in vitro: implications for trophoblast implantation in vivo.

In pregnancy tissues, oncofetal fibronectin (onfFN) has been localized specifically to the extracellular matrix (ECM) surrounding extravillous anchoring trophoblasts of the placental-uterine junction and chorion. When isolated from first or third trimester placentas, human cytotrophoblasts in culture secrete and deposit onfFN in the ECM. In addition, onfFN synthesis is significantly up-regulated in response to serum stimulatory factor(s). The goal of this study was to examine the role of transforming growth factor-beta (TGF beta), a cytokine present in uterine decidua, as a stimulator of trophoblast onfFN production. Our initial insight into the significance of TGF beta resulted from the serendipitous use of cord serum from a neonate with severe alloimmune thrombocytopenia. Trophoblasts cultured in medium containing this serum underwent normal morphological differentiation, but produced markedly less onfFN. In an analogous fashion, trophoblasts cultured in normal serum preincubated with anti-TGF beta neutralizing antibodies also produced significantly less onfFN. Exogenously added TGF beta 1 restored the ability of trophoblasts to produce onfFN by a factor of 4- to 5-fold in medium containing thrombocytopenic serum. In platelet-poor serum derived from human or bovine plasma, TGF beta 1 also induced onfFN synthesis, as assayed both in the conditioned medium and by immunocytochemical localization of onfFN in cell-associated ECM fibrils. Dose-response analysis demonstrated that the onfFN stimulatory response is sensitive to TGF beta, with an ED50 of 0.1-0.2 ng/ml. In a reciprocal fashion, TGF beta inhibited beta hCG secretion 3- to 4-fold. Our results demonstrate that TGF beta is a significant stimulator of trophoblast onfFN production. Furthermore, TGF beta appears to modulate trophoblast differentiation by up-regulating the expression of an anchoring trophoblast marker (onfFN) and down-regulating a phenotypic marker of villous syncytiotrophoblast (hCG beta). We speculate that trophoblast responsiveness to TGF beta in the implantation milieu contributes to trophoblast adhesion by stimulating the production of a trophoblast-derived implantation site fibronectin.

Preeclampsia, trisomy 13, and the placental bed.

Genetic predisposition and abnormal trophoblastic function are thought to contribute to the development of preeclampsia. A multipara developed severe preeclampsia and subsequently delivered a live growth-retarded infant with trisomy 13. Biopsy of the placental bed taken immediately after delivery demonstrated inadequate trophoblastic remodeling of the maternal uterine vasculature, with an absence of normal physiologic changes in the spiral arteries. This case suggests that fetal trisomy 13 can be associated with preeclampsia in multiparous women and that abnormal trophoblastic invasion may contribute to the pathophysiology.