Alida C Knegt

Embryonic stem cell-like cells derived from adult human testis

BACKGROUND Given the significant drawbacks of using human embryonic stem (hES) cells for regenerative medicine, the search for alternative sources of multipotent cells is ongoing. Studies in mice have shown that multipotent ES-like cells can be derived from neonatal and adult testis. Here we report the derivation of ES-like cells from adult human testis. METHODS Testis material was donated for research by four men undergoing bilateral castration as part of prostate cancer treatment. Testicular cells were cultured using StemPro medium. Colonies that appeared sharp edged and compact were collected and subcultured under hES-specific conditions. Molecular characterization of these colonies was performed using RT-PCR and immunohistochemistry. (Epi)genetic stability was tested using bisulphite sequencing and karyotype analysis. Directed differentiation protocols in vitro were performed to investigate the potency of these cells and the cells were injected into immunocompromised mice to investigate their tumorigenicity. RESULTS In testicular cell cultures from all four men, sharp-edged and compact colonies appeared between 3 and 8 weeks. Subcultured cells from these colonies showed alkaline phosphatase activity and expressed hES cell-specific genes (Pou5f1, Sox2, Cripto1, Dnmt3b), proteins and carbohydrate antigens (POU5F1, NANOG, SOX2 and TRA-1-60, TRA-1-81, SSEA4). These ES-like cells were able to differentiate in vitro into derivatives of all three germ layers including neural, epithelial, osteogenic, myogenic, adipocyte and pancreatic lineages. The pancreatic beta cells were able to produce insulin in response to glucose and osteogenic-differentiated cells showed deposition of phosphate and calcium, demonstrating their functional capacity. Although we observed small areas with differentiated cell types of human origin, we never observed extensive teratomas upon injection of testis-derived ES-like cells into immunocompromised mice. CONCLUSIONS Multipotent cells can be established from adult human testis. Their easy accessibility and ethical acceptability as well as their non-tumorigenic and autogenic nature make these cells an attractive alternative to human ES cells for future stem cell therapies.

Positive predictive values for detection of trisomies 21, 18 and 13 and termination of pregnancy rates after referral for advanced maternal age, first trimester combined test or ultrasound abnormalities in a national screening programme (2007–2009)

The objective of this article is to analyse the positive predictive value (PPV) of trisomies 21, 18 and 13 after referral for advanced maternal age (AMA), first trimester combined test or ultrasound findings to suggest improvements for clinical practice.

Inherited unbalanced structural chromosome abnormalities at prenatal chromosome analysis are rarely ascertained through recurrent miscarriage

To determine the mode of ascertainment of inherited unbalanced structural chromosome abnormalities detected at prenatal chromosome analysis.

Selective karyotyping in recurrent miscarriage: are recommended guidelines adopted in daily clinical practice?

BACKGROUND Couples with recurrent miscarriage (RM) have an increased risk of one of the partners carrying a structural chromosome abnormality. On the basis of four independent risk factors, an evidence-based model was developed, which allows limiting karyotyping to high-risk couples. The aim of this study was to assess the level of adoption of selective karyotyping, its clinical consequences and the factors at the patient and hospital level that determine adoption. METHODS A retrospective cohort study was performed in nine Departments of Obstetrics and Gynaecology, the Netherlands, in 2006. Selective karyotyping was defined as offering karyotyping to high-risk couples and refraining from karyotyping in low-risk couples. Data were collected for risk factors as described in the model for selective karyotyping, cytogenetic results as a measure for clinical consequences, and information about determinants and costs. RESULTS A total of 530 couples were included; 252 (48%) high-risk couples and 278 (52%) low-risk couples. Among the high-risk couples, 186 (74%) were offered karyotyping. Although not advised, karyotyping was still performed in 198 (71%) low-risk couples. Overall, selective karyotyping was offered to 50% of the couples. The main determinants for adoption of the model were maternal age, obstetric history, treatment by specialists in RM and the number of patients per centre. If selective karyotyping was adopted adequately, a potential reduction of 34% of all karyotyping tests performed is possible. CONCLUSION Selective karyotyping is applied in only half of the couples with RM in daily practice. Implementation of selective karyotyping should be a topic of future research.

Aiming at multidisciplinary consensus: what should be detected in prenatal diagnosis?

To determine expert consensus on which chromosomal abnormalities should and should not be detected in prenatal diagnosis, and for which abnormalities disagreement remains after structured discussion.

Genetic analysis of fetal nucleated red blood cells from CVS washings

Chorionic villus sampling (CVS) is an established invasive prenatal diagnostic method for the detection of fetal chromosome aberrations. In 1–2% the karyotype result of CVS is inconclusive and follow‐up confirmation will be required. To avoid another invasive procedure we examined fetal nucleated red blood cells (NRBCs) from CVS washings for genetic analysis. We analysed the washings of 20 chorionic villi samples of male fetuses. Fetal NRBCs were immunostained by an antibody against embryonic haemoglobin (HbE). FISH was performed with probes specific for the X and Y chromosome and the nucleus was counterstained with DAPI. Cells positive for the antibody, as well as for DAPI, were collected and stored by a semi‐automated microscope. An operator reviewed those cells for their FISH signals. In 19 out of 20 CVS washings we found nucleated cells positive for HbE together with XY FISH signals. In none of the washings HbE positive cells with two X signals were found. Our results indicate that anti‐HbE is a very specific antibody for identifying fetal NRBCs. NRBCs from CVS washings can be used as an additional fetal tissue for first trimester prenatal diagnosis. Copyright

Trisomy 13 or 18 (mosaicism) in first trimester cytotrophoblast cells: false-positive results in 11 out of 51 cases

OBJECTIVE The finding of full or mosaic trisomy 13 or 18 in first trimester chorionic villus sampling (CVS) may be a false-positive result. This report provides incidence and outcome information that may be helpful in counselling individual patients and in choosing adequate follow-up. STUDY DESIGN From a series of 6820 CVS cases, we retrospectively collected data on all patients (n=51) with full (n=30) or mosaic (n=5) trisomy 18, and full (n=13) or mosaic (n=3) trisomy 13 in cytotrophoblast cells. RESULTS Five false-positives were seen in patients with full trisomy 18 and three in the mosaic cases. One false-positive result was observed in full trisomy 13 and two false-positives in cases of mosaicism. No false-negative results were reported. CONCLUSION The diagnosis of trisomy 13 or 18 in cytotrophoblasts should be confirmed in other tissues, unless fetal abnormalities are seen at ultrasound. In case of mosaicism, follow-up amniocentesis is advised.