Ronald F. Feinberg

The prevalence and biologic significance of lupus anticoagulant and antic ardiolipin antibodies in a general obstetric population

Circulating antibodies to negatively-charged phospholipids have been implicated in the genesis of adverse pregnancy outcomes. However, it has yet to be established that these antibodies are causative or that they are invariably associated with untoward perinatal outcomes. To address this issue, the prevalence of lupus anticoagulant and anticardiolipin antibodies was recorded in a low-risk obstetric population, and the outcome of untreated pregnancies were evaluated. Two of 737 patients (0.27%) had lupus anticoagulant documented by a prolonged activated partial thromboplastin time that did not correct this mixing studies. In comparison, greatly elevated concentrations of immunoglobulin M-anticardiolipin antibodies or immunoglobulin G-anticardiolipin antibodies were identified in 16/737 (2.2%) patients by means of an enzyme-linked immunosorbent assay. Spontaneous abortions occurred in both lupus anticoagulant-positive patients, suggesting that the activated partial thromboplastin time used was a relatively insensitive but specific marker for antiphospholipid antibody-associated adverse pregnancy outcomes. In contrast, although 12 of 16 anticardiolipin antibodies-positive pregnancies were complicated by perinatal loss, preterm delivery, or fetal growth retardation, four patients had uncomplicated pregnancies. Moreover, the distribution of anticardiolipin antibodies concentrations in these four patients was not clustered among the lowest anticardiolipin antibodies values, and anticardiolipin antibodies concentrations correlated weakly with adverse outcomes. These findings suggest that antiphospholipid antibodies are related to adverse pregnancy outcomes in a complex fashion and that therapy is not always required for acceptable outcomes in patients without other risk factors.

Human trophoblast-endometrial interactions in an in vitro suspension culture system

We developed an in vitro suspension co-culture system to examine the interaction of 1st, 2nd and 3rd trimester purified cytotrophoblasts with human endometrium. Endometrium explants were added to cytotrophoblast cell suspensions and placed on an angled gyrating platform in a 37 degrees C incubator. When endometrium was cultured alone it was able to remain viable for up to 3 days. When trophoblasts were cultured alone, they formed small and large aggregates, and occasionally spherical shells with hollow centers. When trophoblasts and endometrium were cultured together, the trophoblasts adhered to the exposed stromal surfaces of the tissue fragments. The surface epithelium was not receptive to trophoblast attachment except in one experiment when day 19 endometrium was used for the co-incubation, suggesting that surface attachment is usually restricted. A common finding was the presence of an acellular zone in the endometrium only adjacent to the attached trophoblasts. We speculate that this zone may be caused by proteolysis and resynthesis of ECM proteins by the trophoblasts. Based on our results, this in vitro suspension should prove useful for examining those factors which: (1) induce endometrial permissiveness, (2) promote paracrine effects on the endometrium, and (3) facilitate human trophoblast invasion.

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.

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.

Improved endometrial assessment using cyclin E and p27

OBJECTIVE To evaluate endometrial expression of cyclin E and p27 in fertile and infertile women. DESIGN Retrospective clinical study. SETTING University medical center and private practice. PATIENT(S) Thirty-three fertile volunteers, 83 women seeking infertility treatment, and 23 women undergoing mock cycles. INTERVENTION(S) Endometrial biopsy. MAIN OUTCOME MEASURE(S) Cyclin E and p27 immunohistochemistry. RESULT(S) Glandular cyclin E and p27 expression dramatically changed in intensity and subcellular localization throughout the menstrual cycle. In normal control biopsies, glandular cyclin E progressed from the basal to the lateral cytoplasm (midproliferative phase) to the nucleus (days 18 to 19) and was absent in biopsies after day 20. First appearing on days 17 to 19, p27 was found only in the nuclei. Cyclin E was more frequently seen after day 20 in infertility patients. In the hyperstimulated cycles, staining for cycle E in proliferative samples was more intense than in the natural cycles, but p27 staining was unchanged. CONCLUSION(S) Cyclin E and p27 may be clinically useful markers of development in the endometrium. As cell cycle regulators, cyclins reveal underlying biochemical processes driving endometrial progression and may partly represent the means by which estrogen and progesterone regulate this dynamic tissue.

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.

Should embryos developing to blastocysts on day 7 be cryopreserved and transferred: an analysis of pregnancy and implantation rates

OBJECTIVE To compare pregnancy rates (PRs) using blastocysts cryopreserved on day 7 with those cryopreserved on days 5 and 6. DESIGN Retrospective observational cohort study. SETTING Infertility center performing IVF. PATIENT(S) Eight hundred women with infertility undergoing frozen ET. INTERVENTION(S) Blastocysts cryopreserved on days 5, 6, and 7 after retrieval were thawed and transferred. MAIN OUTCOME MEASURE(S) Ongoing PRs (pregnancy developing appropriately into the second trimester). Thaw survival, implantation rates, and clinical PRs were also calculated. RESULT(S) A total of 1,406 embryos were thawed with a survival of 90.7% for day 5, 83.7% for day 6, and 78.7% for day 7. Implantation rates were 43.3%, 28.9%, and 28.9%, respectively. Ongoing PRs were 43.9%, 32.9%, and 26.7%, respectively. CONCLUSION(S) Blastocysts cryopreserved on day 7 have a lower, but clinically important potential. Embryos that do not achieve blastocyst stage on day 6 should not be universally discarded, but should be observed in culture 1 more day as 27% may result in an ongoing pregnancy.

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.

Identification of oncofetal fibronectin in patients with advanced epithelial ovarian cancer

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.

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.