Faculty Bibliography

Pregnancy loss is probably the most common problem faced by women worldwide. There are differences in the rates of early and late pregnancy loss based on geography among the developing compared with the developed nations of the world. Most physicians worldwide have different criteria for treating pregnancy loss. Although pregnancy loss is not a disease, it might be best approached with a medical evaluation in order to define the cause and offer specific treatment. This report describes the results obtained by a multi-disciplinary pregnancy loss prevention center in the initial 104 patients. The most common diagnoses were Asherman syndrome (intrauterine adhesions), cervical insufficiency and uterine fibroids, accounting for 47% of the patients. When the diagnosis was not obtained, which occurred in 19% of the patients, in vitro fertilization (IVF) was the treatment provided. Specifically diagnosed and treated patients achieved a 91% success rate. The 19 patients without a specific diagnosis who were treated with IVF had a 60% success rate. Thus patients for whom it was possible to specifically diagnose and treat had better results (P<0.01 t-test). There was an overall success rate of 87% including patients lost to follow-up with this multidisciplinary medical approach. A pregnancy loss prevention center using the described multidisciplinary model can accomplish success rates of 85-90%. Preventing recurrent pregnancy loss we suggest can best be achieved by a dedicated center with a multidisciplinary medical approach.

Amniotic fluid cells (AFC) from 2nd trimester amniocentesis have been found to be a source of multipotent stem cells which might overcome the limitations of expansion, histocompatibility, tumorigenesis, and ethical issues associated with using human embryonic cells, umbilical cord, cord blood, bone marrow, and induced pluripotent cells. Previous work by our group and others demonstrated multipotency and the ability to grow well in culture. However, all these studies were done in media containing fetal calf serum. We sought to observe the properties of AFC grown in serum-free media as that would be required for clinical transplantation in humans. Fresh samples were obtained from three patients, and each sample divided into a culture whose cells were not exposed to fetal calf serum, and the other half into a standard culture medium containing fetal calf serum. Doubling time and stem cell marker expression by flow cytometry were assessed. Differentiation to neural, osteoid, and chondrogenic lineages was induced using appropriate media and confirmed by fluorescent microscopy, histology, and immunohistochemistry. There were no statistically significant differences between cells grown serum-free and in standard media in any of these parameters. The data supports the possibility of clinical use of AFC in stem cell transplantation.

INTRODUCTION: Trisomy is a common chromosomal abnormality associated with fetal loss and multi-systemic complications of survival in humans. Animal models cannot inform developmental pathophysiology in trisomies. Previous studies have shown slow cell kinetics and reliable differentiation into hematopoietic and neural lineages of trisomic human amniotic fluid cells (AFC). We investigated the behavior of trisomic AFCs compared with euploid AFCs in culture to guide better understanding of abnormal development. METHODS: Amniotic fluid was collected from women identified at risk for fetal abnormality at second trimester amniocentesis. Three samples were identified as trisomic (2x Trisomy21, 1x Trisomy18). AFCs were isolated and cultured to observe morphology and multiplication potential. Fluorescence activated cell sorting (FACS) was performed to evaluate cell surface markers of pluripotency, as euploid AFCs are known to be pluripotent. Cells were grown as 3D spheroids for differentiation into osteogenic, chondrogenic and neural lineages. Immunohistochemistry staining and fluorescent imaging was performed for analysis of lineage markers. Trisomic cultures were compared to matched euploid controls. RESULTS: Fetal Trisomy 18 and Trisomy 21 AFCs were morphologically indistinguishable from euploid samples. Trisomic AFCs exhibited rapid division for a 96 hr period, exceeding euploid pace. FACS analysis using antibodies for stem cell pluripotency markers SSEA4, TRA-1-60 and CD90 revealed expression patterns similar to euploid samples. Trisomy18 spheroids differentiated similarly to euploid samples into osteogenic, chondrogenic and neural lineages. Trisomy21 samples uniformly induced either more weakly or did not induce at all into all three lineages. CONCLUSIONS: We have shown rapid growth kinetics in Trisomy 21 and Trisomy 18 samples. Presence of stem-like pluripotency markers and inducibility characteristics are novel findings. Of interest, Trisomy21 showed reduced differentiation to neural lineage, which is tempting to relate to the clinical condition. AFC cultures may be useful for the study of developmental differences in aneuploid fetuses. (Figure Presented)