Preimplantation genetic diagnosis of chromosome translocations by analysis of polymorphic short tandem repeats.

INTRODUCTION: We aimed to develop and implement a short tandem repeat (STR) polymerase chain reaction alternative to fluorescence in situ hybridisation (FISH) for the preimplantation genetic diagnosis (PGD) of chromosomal translocations. METHODS: Selected informative STRs located on translocated arms of relevant chromosomes were used to discriminate between normal and unbalanced chromosome states in each embryo. RESULTS: PGD cycles were performed on five couples where one spouse carried a balanced translocation. 27 embryos were analysed, of which 12 were normal/balanced, 12 were abnormal/unbalanced and three were indeterminate. Four PGD cycles proceeded to embryo transfer, of which two led to pregnancy. The first pregnancy showed a normal male karyotype, and a healthy baby was delivered at term. A second pregnancy unexpectedly miscarried in the second trimester from unknown causes. CONCLUSION: STR analysis is a simple and suitable alternative to FISH for detecting unbalanced chromosomal states in preimplantation embryos.

Ex vivo expansion of conjunctival and limbal epithelial cells using cord blood serum-supplemented culture medium.

PURPOSE: Conventional cell culture methods use fetal bovine serum (FBS) as a growth supplement. The purpose of this study was to develop a xenobiotic-free culture system using umbilical cord blood serum (CBS) as an alternative growth supplement for the cultivation of human conjunctival and limbal epithelial cells. METHODS: Human conjunctival and limbal epithelial cells were cultivated in varying concentrations of CBS-supplemented medium and compared with FBS-supplemented medium. Bromodeoxyuridine (BrdU) ELISA proliferation assay, colony-forming efficiency (CFE), and a number of cell generations were analyzed. Cytokeratin expression of cultured cells was evaluated (K3, K4, K12, K13, K14, K15, K19, and PanCK). The authors compared the cytokine and growth factor levels in CBS, FBS, and adult serum using antibody array assays. RESULTS: Conjunctival and limbal cells cultivated in 0.25% CBS- and 0.5% CBS-supplemented culture media demonstrated the highest proliferative capacity in terms of BrdU proliferation assay, CFE, and number of cell generations. These results were comparable to FBS-supplemented medium. Cultured epithelial cells retained their normal cytokeratin expression. Cytokines brain-derived neurotrophic factor, growth-related oncogene, and leptin and growth factors EGF, HGF, FGF-6, IGF-1, PDGF, and IGFBP were present in higher concentrations in CBS than in FBS and adult serum. CONCLUSIONS: CBS-supplemented culture medium supported the proliferation and differentiation of conjunctival and limbal epithelial cells. CBS contained a higher concentration of growth factors and cytokines than FBS and adult serum. CBS may be a viable and safer alternative to FBS as a growth supplement in the culture medium for culturing epithelial cells, which may have important clinical implications when bioengineering tissues for clinical use.

Simplified PGD of common determinants of haemoglobin Bart's hydrops fetalis syndrome using multiplex-microsatellite PCR.

The high incidence of double-gene deletions in α-thalassaemia increases the risk of having pregnancies with homozygous α(0)-thalassaemia, the cause of the lethal haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. However, the current gap-PCR based PGD protocol for deletional α-thalassaemia requires specific primer design for each specific deletion. A universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome has been developed. Microsatellite markers 16PTEL05 and 16PTEL06 within the α-globin gene cluster were co-amplified with a third microsatellite marker outside the affected region in a multiplex-PCR reaction and analysed by capillary electrophoresis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. A total of 47 embryos were analysed. Three pregnancies were achieved from three couples, with the births of two healthy babies and one ongoing pregnancy. This work has successfully adapted an earlier protocol and developed a simple and reliable single-cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of type of deletion. Alpha-thalassaemia is one of the most common inheritable disorders worldwide. It is a blood disorder that, in its lethal form caused by deletion of all four copies of the α-globin gene, results in the demise of the affected fetus, a condition referred to as haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. Current PGD protocols for deletional α-thalassaemia utilize a strategy called gap-PCR, which requires the different assays for different deletion types. We have developed a universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome based on microsatellite marker analysis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. Forty-five embryos were analysed in total. Three pregnancies were achieved from three couples, with the births of two healthy babies and one pregnancy still ongoing. We have successfully adapted our earlier protocol and developed a simple and reliable single cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of the type of deletion.

Successful preimplantation genetic diagnosis of Hb Bart's hydrops fetalis in Singapore after fresh and frozen embryo replacement cycles.

INTRODUCTION: We report the fi rst successful preimplantation genetic diagnosis (PGD) for Hb Bart's hydrops fetalis in Singapore, involving both fresh and frozen embryo replacement cycles. CLINICAL PICTURE: Two couples who were carriers of the Southeast Asian type double gene deletion (--(SEA) deletion carriers) requested for PGD. Couple A had 2 previous affected pregnancies, while couple B have a child of unknown genotypic status. TREATMENT: One PGD cycle was performed for each couple. The --(SEA) deletion was detected using a gap-PCR strategy. Couple A had 1 fresh-embryo replacement cycle while couple B underwent 2 frozen-embryo replacement cycles. OUTCOME: Couple A achieved a twin pregnancy. Second trimester complications resulted in premature delivery, where 1 baby girl survived. Couple B achieved a singleton pregnancy resulting in delivery of a healthy baby boy. Genotype analysis of all babies confirmed the PGD results consistent with clinically unaffected status. CONCLUSIONS: We have successfully performed PGD to avoid Hb Bart's hydrops fetalis syndrome.

First successful preimplantation genetic diagnosis in Singapore--avoidance of beta-thalassaemia major.

INTRODUCTION: We report on the first successful preimplantation genetic diagnosis (PGD) in Singapore. CLINICAL PICTURE: A couple who are beta-thalassaemia carriers and have an affected daughter requested for PGD. TREATMENT: Two cycles of PGD were performed on the couple. Beta-thalassaemia mutations were detected using a nested PCR and minisequencing strategy, and unaffected embryos were selected for transfer. OUTCOME: A singleton pregnancy was achieved in the second PGD cycle, resulting in the birth of a healthy baby boy with carrier genotype. CONCLUSIONS: This case report documents the first successful PGD in Singapore, involving a couple at-risk of transmitting beta-thalassaemia major.

Embryo donation: a review.

BACKGROUND: Embryo donation is currently an uncommon procedure associated with many ethical, legal and psychosocial implications. It is, however, the only answer to infertility in some patients. This article summarizes the indications for embryo donation as well as the procedure and considerations in the preparation of both the donors and the recipients. METHODS: A MEDLINE search was conducted for all English articles using the keywords embryo donation, in vitro fertilization and gamete donation. CONCLUSION: Embryo donation might indeed be the answer to many infertile couples who would otherwise have to resort to childlessness or adoption. The advent of in vitro fertilization and cryopreservation of the spare embryos make these embryos a ready source for potential donors. The success rates of both fresh and freeze-thawed embryo transfers have been encouraging and there do not seem to be any long-term clinical implications for the offspring. However, there remain many legal and psychosocial issues associated with embryo donation.