New phenotype with generalized platyspondyly, large mandible, hypoplastic teeth, strabismus, hyperopia and low cholesterol levels.

A sporadic, adult male patient with generalized platyspondyly, large mandible, hypoplastic teeth, strabismus, and low serum cholesterol levels is presented. Some of the patient's features resemble brachyolmia, Spondylo-epiphyseal dysplasia tarda, Kenny-Caffey and Stickler syndromes. Based on literature review, possible diagnoses are discussed. In conclusion, this patient can have a variant of brachyolmia or Spondylo-epiphyseal dysplasia tarda. However, we cannot exclude that this constellation of clinical features may represent a new syndrome.

Pregnancy following intracytoplasmic sperm injection and preimplantation genetic diagnosis after the conservative management of endometrial cancer.

A rare case of a patient with conservatively treated endometrial carcinoma who conceived and delivered a healthy baby after the transfer of embryos with intracytoplasmic sperm injection (ICSI) and preimplantation genetic diagnosis (PGD) is presented. A 41-year-old woman had an office hysteroscopy in the infertility work-up and stage I endometrial adenocarcinoma was diagnosed. After conservative treatment, the patient underwent ICSI and PGD. She achieved pregnancy with two normal embryos. Two gestational sacs were observed but one of them was blighted. The patient subsequently delivered a healthy female infant. Repeated office hysteroscopy and endometrial sampling was performed after delivery. The appearance of the endometrium was normal on hysteroscopy, and the histology report was normal. The principal concern with medical therapy is that the lesion cannot be fully evaluated until the hysterectomy is performed, the nodes palpated, and the uterus is sectioned. The patient was referred to a gynaecological oncologist for definitive surgery.

Relationship between granular cytoplasm of oocytes and pregnancy outcome following intracytoplasmic sperm injection.

Couples undergoing intracytoplasmic sperm injection (ICSI) for male infertility using oocytes with centrally located granular cytoplasm (CLCG) were evaluated for fertilization, embryo development, implantation and pregnancy rate. CLCG is a rare morphological feature of the oocyte, that is diagnosed as a larger, dark, spongy granular area in the cytoplasm. Severity is based on both the diameter of granular area and the depth of the lesion. Twenty-seven couples with 39 cycles presenting CLCG in >50% of retrieved oocytes were evaluated. A total of 489 oocytes was retrieved, out of which 392 were at MII. CLCG was observed in 258 of the MII oocytes (65. 8%); 66.7% of these oocytes had slight and 33.3% had severe CLCG. The overall fertilization rate was 72.2% and no statistical significant difference was found between normal and CLCG oocytes and between the oocytes representing slight and severe CLCG. The development and quality of embryos was the same in normal and CLCG oocytes. In nine cycles, preimplantation genetic diagnosis was executed to evaluate a possible accompanying chromosomal abnormality. Out of 44 blastomeres biopsied, 23 had chromosomal abnormality (52. 3%). Eleven pregnancies were achieved in 39 cycles (28.2%), six pregnancies resulted in abortion (54.5%). The implantation rate was found to be 4.2%. Only five ongoing pregnancies were achieved in 39 cycles (12.8%). Couples with CLCG oocytes should be informed about poor on-going pregnancy rates even if fertilization, embryo quality and total pregnancy rates are normal. Furthermore, a high aneuploidy rate may be linked to a high abortion rate.

Healthy births and ongoing pregnancies obtained by preimplantation genetic diagnosis in patients with advanced maternal age and recurrent implantation failure.

Preimplantation genetic diagnosis (PGD) and subsequent embryo development was evaluated in 72 couples presenting at our centre for intracytoplasmic sperm injection (ICSI) due to severe male factor. The embryo biopsies were performed in Ca(2+)/Mg(2+)-free medium. These patients were further divided into those with advanced maternal age (AMA, n = 49) and those with recurrent implantation failure (RIF, n = 23). Fluorescence in-situ hybridization (FISH) was carried out on 329 blastomeres (91.3%) with probes for the X, Y, 13, 18 and 21 chromosomes. The chromosomal abnormality rate was 41.3% with no significant difference between the AMA and RIF groups. Aneuploidy accounted for the majority (72.8%) of chromosomal abnormalities. Out of 329 embryos, 84.2% had cleaved after 24 h and 15.1% had arrested. Embryos were transferred in 70 patients and 22 pregnancies were achieved (31.4% with an ongoing pregnancy rate of 28.5%). There were no significant differences between the pregnancy rates of the AMA and RIF groups (32.5 and 30% respectively). Therefore PGD should be offered to patients with AMA and RIF. Furthermore, the use of Ca(2+)/Mg(2+)-free medium during the blastomere biopsy facilitates the procedure, while further embryo cleavage, ongoing pregnancies and healthy births are possible.

Improvements of preimplantation diagnosis of aneuploidy by using microwave hybridization, cell recycling and monocolour labelling of probes.

Recent studies indicate that preimplantation genetic diagnosis of aneuploidy significantly reduces spontaneous abortions and increases pregnancy rates in women of advanced maternal age undergoing in-vitro fertilization. A new protocol was developed involving cell recycling (sequential hybridizations) and microwave hybridization of repetitive probes in order to obtain in a few hours enumeration results for chromosomes X, Y, 1, 13, 15, 16, 18, 21 and 22 in single blastomeres. The error rate using this protocol was significantly lower than in previous protocols and the analysis of nine chromosomes can be achieved in a shorter time than before.

Chromosome abnormalities in 1255 cleavage-stage human embryos.

The relationship was examined between chromosome abnormalities in cleavage stage human embryos and maternal age, embryo morphology and development rate. Embryos that were classified as suboptimal for transfer from patients undergoing IVF treatment were disaggregated, and all or most of their cells were fixed for analysis by fluorescence in-situ hybridization. Chromosomes X, Y, 13, 18 and 21, and in some instances 16 were examined. A total of 731 non-viable embryos was analysed. An increase in chromosome abnormalities with decreasing embryo competence and increasing maternal age was shown. Compared with an earlier study, the major difference was that polyploidy (P<00.01) and aneuploidy were previously more common. After pooling results, it was found that aneuploidy increased with maternal age, from 3.1% in embryos from 20-34 years old patients to 17% in patients 40 years or older. Also, aneuploidy occurred more frequently in embryos with good morphology and development rate than in embryos developing poorly. In contrast, dysmorphic and slowly developing or arrested embryos had significantly more polyploidy and mosaicism than normally developing embryos. Clear associations between maternal age and aneuploidy, and between cleavage anomalies and mosaicism have been established in non-viable embryos. Arrested embryos were mostly polyploid. Moreover, polyploidy was found more frequently in embryos analysed on day 4, suggesting that developmentally compromised embryos became arrested in extended culture. A slightly higher aneuploidy rate in the earlier study may be attributed to differences in hormonal stimulation, which also resulted in different numbers of oocytes recruited and matured.

Preimplantation genetic diagnosis of aneuploidy: were we looking at the wrong chromosomes?

PURPOSE: Our purpose was to study aneuploidy frequencies of chromosomes 1, 4, 6, 7, 14, 15, 17, 18, and 22 in cleavage-stage embryos. These frequencies were compared to spontaneous abortion data to determine differences in survival rate of their aneuploidies. METHODS: One hundred ninety-four embryos were analyzed with multicolor fluorescence in situ hybridization. Embryos were divided into three maternal age groups: 20 to 34.9 years, (2) 35 to 39.9 years, and (3) 40 years and older. Embryos were also divided into two developmental and morphological groups; arrested and nonarrested embryos. RESULTS: The rate of aneuploidy was 14.51%, 14.10%, and 31.48% for age groups 1, 2, and 3, respectively (P < 0.005). The chromosomes most frequently involved in aneuploidy events were 22, 15, 1, and 17. CONCLUSIONS: The chromosomes most involved in spontaneous abortions are not necessarily the ones causing a decrease in implantation rates with maternal age. Other aneuploidies, such as for chromosomes 1 and 17, may seldom implant or die shortly after implantation.

Spontaneous abortions are reduced after preconception diagnosis of translocations.

PURPOSE: Preimplantation genetic diagnosis of translocations has seldom been attempted. Recently, a genetic test based on analyzing polar bodies at the methaphase stage, following fluorescent in situ hybridization with commercially available whole-chromosome painting DNA probes has been presented. Here we report the use of this method in seven couples in whom the female was a carrier of one of these balanced translocations: 45,XX,der (13q;14q)(q10;q10) (two cases), 46,XX,t(4;14)(p15.3;q24), 45,XX,der(14q;21q) (q10;q10), 46,XX,t(7;20)(q22;q11.2), 46,XX,t(9,11)(p24;q12), 46,XX,t(14;18)(q22;q11), and 46,XX,t(3;8)(q11;q11). METHODS: The original method was improved in two ways. First, centromeric probes for one or both chromosomes involved in the translocation were added to avoid misdiagnosis caused by possible confusion of first polar body monovalent chromosomes (with two chromatids each) with single chromatids. Second, for cases with terminal translocations where commercially available probes do not cover telomere sequences, a telomere probe labeling the translocated fragment was added. RESULTS: A total of 26 abnormal, 18 balanced, and 22 normal eggs was detected. Nine normal and seven balanced embryos were transferred, resulting in eight (50%) implanting, of which one spontaneously aborted. To date, the remainder have produced karyotypically normal or balanced babies and ongoing pregnancies. The rate of spontaneous abortions after preimplantation genetic diagnosis (12.5%) was significantly reduced (P < 0.001) compared to natural cycles in the same patients (95%). CONCLUSIONS: With the above improvements, the test can characterize any translocation of maternal origin and produce a high pregnancy rate and an apparently low frequency of spontaneous abortion.

Case report: chromatid exchange and predivision of chromatids as other sources of abnormal oocytes detected by preimplantation genetic diagnosis of translocations.

Preimplantation genetic diagnosis of translocations can be performed on first polar bodies (PB) at metaphase stage using FISH with whole-chromosome painting DNA probes. Here we report the use of this method in a couple in which the female was a carrier of a balanced translocation 46,XX,t(11;16)(q21;q22). This case unusual in that two polar bodies showed recombination events between the homologue chromosomes of 11 and 16 pairs, resulting in M-II oocytes with monovalent chromosomes having a normal and a derivative chromatid. For this type of case, PGD analysis on polar bodies cannot give a useful result, because, at the second meiotic division, either of these chromatids could remain in the oocyte, resulting in a normal, balanced or unbalanced embryo. PGD analysis on blastomeres can provide a solution. 11 previous cases of PGD of translocations performed by metaphase PB analysis are reviewed.

Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22.

The present preimplantation diagnosis test is able to screen for the most common aneuploidies from single blastomeres in about five hours with a 15 per cent misdiagnosis. This means that the risk of spontaneous abortion and trisomic offspring for women of advanced maternal age could be reduced to the same level as younger women for whom prenatal diagnosis is usually not necessary. Better probes and more fluorochromes could improve the success rate of the test.

P53, p15INK4B, p16INK4A and p57KIP2 mutations during the progression of chronic myeloid leukemia.

The occurrence of acute transformation during the treatment of chronic myeloid leukemia (CML) is still a poorly understood mechanism. In this disease p53, p16INK4A, p15INK4B, p57KIP2 mutations and p15INK4B/p16INK4A homo/hemizygous deletions were analyzed in the initial diagnosis phase and during the treatment phase of twelve CML cases, in order to establish whether there was a consistent molecular genetic alteration in its progression. During the treatment period, four of twelve cases had blastic crisis. All the mutations observed in p53, p16INK4A and p15INK4B cumulated in three out of four CML cases who had blastic crises. In one case, p53 codon 282 mutation (CGG-->TGG; arg-->trp) were observed in initial diagnosis. Seven months later, G-->C transition in the 3' side of p15 cDNA (778. nucleotide) was observed in the accelerated phase with the same p53 codon 282 mutation. Thirteen months later, this patient died as a result of blastic crisis. The patient in blastic crises in the initial diagnosis phase had a mis-sense point mutation in p16 codon 69 (ACT-->AGT; thr-->ser) and a polymorphism in codon 68 (GCC-->GCG). Six months later, this patient also died. In one case, p53 codon 237 mutation (ATG-->ATA; met-->ile) were observed in the initial diagnosis phase. Then months later, the patient died as a result of blastic crises. No p15INK4B/p16INK4A homo/hemizygous deletion and p57KIP2 gene mutation which was described in the same pathway were observed in CML progression. These results indicate that p15INK4B and p16INK4A gene alterations may have an affect on the progression of CML-like p53 mutation. A correlation was found with the progression of CML and p53, p15INK4B and p16INK4A somatic mutations. Finding p15INK4B and p16INK4A gene alteration as well as p53 mutations may be a prognostic marker in patients with CML.

Application of the 'Apt test' in prenatal diagnosis to evaluate the fetal origin of blood obtained by cordocentesis: results of 30 pregnancies.

This study aimed to set up a practical lab-side approach to discriminate fetal from maternal blood in samples obtained by cordocentesis. To determine the fetal origin of the blood, a modified Apt test was applied to 30 cases of prenatal diagnosis. A change of colour of the fetal and adult blood during the procedure was the hallmark to assess fetal origin. At the end of 60 s of the test, fetal blood yielded a pink colour whereas adult blood was dark green-brown. The test was repeated in mixtures of fetal and adult blood. The results suggest that the modified Apt test is a practical, quick, inexpensive, and efficient test to determine the origin of blood samples obtained by cordocentesis. However, it should be kept in mind that samples containing a mixture of both fetal and adult blood could also yield a fetal blood reaction. When maternal contamination is suspected, we propose that at least 30 metaphases from different slides should be counted. This could yield fetal as well as maternal chromosomes.