Preimplantation diagnosis and HLA typing for haemoglobin disorders.

Haemoglobin disorders are among the most frequent indications for preimplantation genetic diagnosis (PGD), introduced as an important option to couples at risk for producing offspring with thalassaemia and sickle cell disease. Previous experience mainly included PGD for beta-thalassaemia, while PGD for alpha-thalassaemia resulting in an unaffected pregnancy has not been reported. This study presents the results of the world's largest experience of 197 PGD cycles for haemoglobin disorders, which includes PGD for alpha-thalassaemia, resulting in 53 clinical pregnancies and birth of 45 healthy children, with five still ongoing. Fifty-four of these cycles were performed in combination with HLA typing, allowing the birth of thalassaemia-free children who were also HLA identical to the affected sibling, with successful stem cell transplantation in one case. As an increasing proportion of patients requesting PGD with HLA typing are of advanced reproductive age, aneuploidy testing was performed simultaneously with PGD. The results show that PGD has now become a practical approach for prevention of haemoglobin disorders, and is gradually being used also for improving access to HLA compatible stem cell transplantation for this group of diseases.

Sandhoff disease in Cyprus: population screening by biochemical and DNA analysis indicates a high frequency of carriers in the Maronite community.

In the last 15 years, four patients with the infantile form of Sandhoff disease were diagnosed in four different families in Cyprus (population 703,000, birth rate 1.7%). Three of these cases came from the Christian Maronite community (less than 1% of the population) and one from the Greek community (84% of the population). This relatively large number of patients prompted us to initiate an epidemiological study in order to establish the frequency of the mutant allele in Cyprus. Carrier detection was initially based on the measurement of beta-hexosaminidase A and B in both leucocytes and serum. Using the enzyme test, 35 carriers were identified among 244 random Maronite samples and 15 among 28 Maronites with a family history of Sandhoff disease, but only one carrier was found out of 115 random samples from the Greek community. In parallel to the biochemical screening, DNA studies were undertaken in one of the three Maronite patients and in a Greek carrier related to the Greek patient. These studies resulted in the identification of two novel mutations, a deletion of A at nt76 and a G to C transversion at position 5 of the 5'-splice site of intron 8, which have been published. We subsequently screened the carriers detected in the biochemical study for these two mutations using PCR-based tests. Of 50 Maronite carriers examined, 42 were found to have the nt76 deletion. Eight Maronite samples, designated carriers from the biochemical results, were negative for both mutations. It is possible that these individuals were incorrectly classified as carriers since their enzyme values are equivocal, although the presence of another mutation has not been excluded. Two Greek Cypriot carriers and two obligate Lebanese carriers were negative for both mutations. We conclude that there is a high frequency of Sandhoff disease carriers in the Maronite community of Cyprus, approximately 1 in 7, and that a single mutation predominates in this population.

Birth of healthy children after preimplantation diagnosis of thalassemias.

BACKGROUND: Preimplantation genetic diagnosis (PGD) allows couples at risk of having children with thalassemia to ensure the healthy outcome of their pregnancy. METHODS: Seventeen PGD clinical cycles were initiated for Cypriot couples at risk of having children with different thalassemia mutations, including IVSI-110, IVSI-6, and IVS II-745. Unaffected embryos for transfer were selected by testing oocytes, using first and second polar body (PB) removal and nested polymerase chain reaction analysis followed by restriction digestion. RESULTS: Unaffected embryos were selected in 16 of 17 PGD cycles. Of 166 oocytes studied from these cycles, 110 were analyzed by sequential analysis of both the first and the second PB, resulting in preselection and transfer of 45 unaffected embryos. This resulted in seven pregnancies and in the birth of five healthy thalassemia-free children. The embryos predicted to have inherited the affected allele were not transferred. Analysis of these embryos confirmed the PB diagnosis. CONCLUSIONS: Sequential first and second PB testing of oocytes is reliable for PGD of thalassemia and is a feasible alternative to prenatal diagnosis in high-risk populations.

Transfusional transmitted viruses in pregnancy.

Blood has long been recognized as a vehicle for transmission of infectious organisms and as molecular laboratory technology has advanced, a seemingly endless array of infectious agents has occasionally been documented to be blood transmitted. Transfusion associated hepatitis (TAH) has been the most common serious consequence of blood transfusion although in recent years this has been significantly reduced (blood donor screening, blood processing, etc.). Thalassaemia major is classically associated with increased susceptibility to infections caused by those agents that are blood transmitted such as HBV, HCV, HIV, CMV, HPV B-19 (frequency rates vary from country to country). Monitoring the prevalence of transfusion transmitted infections in thalassaemics has been in recent years an indispensable part of their clinical management protocol. As a number of these viruses have been documented to be efficiently transmitted through the vertical route, the issue of blood transmitted viral infection monitoring becomes particularly important in order to provide protection or treatment both to the pregnant thalassaemic patient herself and to her foetus/newborn. Hepatitis (mainly B and C) and HIV in the obstetric thalassaemic is what the clinician is faced with most frequently. Although preventative measures have been very successful in the case of HBV infection and recently to an encouraging extent in the case of HIV (recommendations have been constructed), the mechanisms and frequency of HCV vertical transmission as well as the clinical outcome of children born to HCV carriers are not yet completely clarified. No vaccines are available and HIGB or antivirals do not appear to offer protection to the foetus against infection with HCV. Thalassaemics are frequently seropositive to markers of other transfusion transmitted viruses, such as CMV and HPV B-19, particularly by the age of pregnancy. Infection with a second or multiple strains as well as reactivation of existing CMV strain(s) are possible events in thalassaemics. However, the frequency of "recurrency" episodes, their implication in vertical transmission and clinical outcome for the foetus/newborn are issues requiring further investigation.

Cervical pregnancy ending in a live vaginal birth.

A case of cervical pregnancy followed by cataclysmic haemorrhage estimated at 8500 ml and necessitating total abdominal hysterectomy and replacement of 18 units of blood is reported. This pregnancy remained undiagnosed until 28 wk and is to our knowledge the first case described in the English literature in which the pregnancy ended in a live vaginal birth. Both mother and baby are now well.