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Introduction: Preimplantation genetic screening has been proposed to improve the in-vitro fertilisation outcome by screening for aneuploid embryos or blastocysts. This study aimed to report the outcome of 133 cycles of preimplantation genetic diagnosis and screening by array comparative genomic hybridisation., Methods: This study of case series was conducted in a tertiary assisted reproductive centre in Hong Kong. Patients who underwent preimplantation genetic diagnosis for chromosomal abnormalities or preimplantation genetic screening between 1 April 2012 and 30 June 2015 were included. They underwent in-vitro fertilisation and intracytoplasmic sperm injection. An embryo biopsy was performed on day-3 embryos and the blastomere was subject to array comparative genomic hybridisation. Embryos with normal copy numbers were replaced. The ongoing pregnancy rate, implantation rate, and miscarriage rate were studied., Results: During the study period, 133 cycles of preimplantation genetic diagnosis for chromosomal abnormalities or preimplantation genetic screening were initiated in 94 patients. Overall, 112 cycles proceeded to embryo biopsy and 65 cycles had embryo transfer. The ongoing pregnancy rate per transfer cycle after preimplantation genetic screening was 50.0% and that after preimplantation genetic diagnosis was 34.9%. The implantation rates after preimplantation genetic screening and diagnosis were 45.7% and 41.1%, respectively and the miscarriage rates were 8.3% and 28.6%, respectively. There were 26 frozen-thawed embryo transfer cycles, in which vitrified and biopsied genetically transferrable embryos were replaced, resulting in an ongoing pregnancy rate of 36.4% in the screening group and 60.0% in the diagnosis group., Conclusions: The clinical outcomes of preimplantation genetic diagnosis and screening using comparative genomic hybridisation in our unit were comparable to those reported internationally. Genetically transferrable embryos replaced in a natural cycle may improve the ongoing pregnancy rate and implantation rate when compared with transfer in a stimulated cycle.

Objective: To report the outcomes of more than 100 cycles of preimplantation genetic diagnosis for monogenetic diseases., Design: Case series., Setting: Tertiary assisted reproductive centre in Hong Kong, where patients needed to pay for the cost of preimplantation genetic diagnosis on top of standard in-vitro fertilisation charges., Patients: Patients undergoing preimplantation genetic diagnosis for monogenetic diseases at the Centre of Assisted Reproduction and Embryology, Queen Mary Hospital-The University of Hong Kong between 1 August 2007 and 30 April 2014 were included., Interventions: In-vitro fertilisation, intracytoplasmic sperm injection, embryo biopsy, and preimplantation genetic diagnosis., Main Outcome Measures: Ongoing pregnancy rate and implantation rate., Results: Overall, 124 cycles of preimplantation genetic diagnosis were initiated in 76 patients, 101 cycles proceeded to preimplantation genetic diagnosis, and 92 cycles had embryo transfer. The ongoing pregnancy rate was 28.2% per initiated cycle and 38.0% per embryo transfer, giving an implantation rate of 35.2%. There were 16 frozen-thawed embryo transfer cycles in which, following preimplantation genetic diagnosis, cryopreserved embryos were replaced resulting in an ongoing pregnancy rate of 37.5% and implantation rate of 30.0%. The cumulative ongoing pregnancy rate was 33.1%. The most frequent indication for preimplantation genetic diagnosis was thalassaemia, followed by neurodegenerative disorder and cancer predisposition. There was no misdiagnosis., Conclusions: Preimplantation genetic diagnosis is a reliable method to prevent couples conceiving fetuses severely affected by known genetic disorders, with ongoing pregnancy and implantation rates similar to those for in-vitro fertilisation for routine infertility treatment.

Objectives: To compare the pregnancy outcome of the fluorescent in-situ hybridisation and array comparative genomic hybridisation in preimplantation genetic diagnosis of translocation carriers., Design: Historical cohort., Setting: A teaching hospital in Hong Kong., Patients: All preimplantation genetic diagnosis treatment cycles performed for translocation carriers from 2001 to 2013., Results: Overall, 101 treatment cycles for preimplantation genetic diagnosis in translocation were included: 77 cycles for reciprocal translocation and 24 cycles for Robertsonian translocation. Fluorescent in-situ hybridisation and array comparative genomic hybridisation were used in 78 and 11 cycles, respectively. The ongoing pregnancy rate per initiated cycle after array comparative genomic hybridisation was significantly higher than that after fluorescent in-situ hybridisation in all translocation carriers (36.4% vs 9.0%; P=0.010). The miscarriage rate was comparable with both techniques. The testing method (array comparative genomic hybridisation or fluorescent in-situ hybridisation) was the only significant factor affecting the ongoing pregnancy rate after controlling for the women's age, type of translocation, and clinical information of the preimplantation genetic diagnosis cycles by logistic regression (odds ratio=1.875; P=0.023; 95% confidence interval, 1.090-3.226)., Conclusion: This local retrospective study confirmed that comparative genomic hybridisation is associated with significantly higher pregnancy rates versus fluorescent in-situ hybridisation in translocation carriers. Array comparative genomic hybridisation should be the technique of choice in preimplantation genetic diagnosis cycles in translocation carriers.

Purpose: To perform Preimplantation Genetic Diagnosis (PGD) on a paternal Brca2 unknown mutation carrier with early-onset breast cancer, whose paternal grandmother and mother had breast cancer at 60s., Method: Elucidating the linkage via single sperm haplotyping on patient's carrier brother, and identifying the genomic deletion via BLAST followed by PCR screening. PGD was subsequently conducted., Result: The mutant allele was found by using 4 microsatellite and 2 intragenic SNP markers. Recombination was detected in 8% of sperms. BLAST was utilized to locate putative hairpin structure(s), followed by PCR screening with seven sets of primers. A novel 2,596 bp deletion containing exon 15 ~ 16 was identified. Due to the severity of phenotype and the integrity of exon 11 encoding RAD51 binding domain, and the fact that the patient's mother also had breast cancer at her 60s, we speculate a possible coexistence of maternal breast cancer risk allele(s). Embryo biopsy was performed on day 3. Unaffected morula and blastocyst were replaced on day 5, resulting in a singleton livebirth. A breast lump appeared in the patient after delivery without the presence of malignant cells., Conclusion: Concerning the assisted reproductive option for breast cancer patients, the possibility of coexistence of multiple familial risk alleles and the significance of each mutation to the phenotype should be evaluated. To eliminate misdiagnosis resulting from recombination and/or allelic drop-out, both direct mutation detection and linkage analysis approaches may be necessary. BLAST is a very useful and cost-effective tool for identifying large genomic deletion.

Background: Embryos produced by in vitro fertilization (IVF) have a high level of aneuploidy, which is believed to be a major factor affecting the success of human assisted reproduction treatment. The aneuploidy rate of cleavage stage embryos based on 1-2 biopsied blastomeres has been well-reported, however, the true aneuploidy rate of whole embryos remain unclear because of embryo mosaicism. To study the prevalence of mosaicism in top quality IVF embryos, surplus embryos donated from young patients (aged 28-32) in the assisted reproduction program at Queen Mary Hospital, Hong Kong were used., Methods: Thirty-six good quality day 2 embryos were thawed. Out of the 135 blastomeres in these embryos, 121 (89.6%) survived thawing. Twelve of these embryos without lysed blastomeres and which cleaved to at least seven cells after a 24-h culture were dissembled into individual blastomeres, which were analysed by array comparative genomic hybridization and microsatellite marker analysis by fluorescent PCR., Results: Out of 12 day-3 embryos, 2 (16.7%) were normal, 3 (25%) were diploid/aneuploidy with <38% abnormality, 4 (33.3%) were diploid/aneuploidy mosaic with > =38% abnormality, and three (25%) were mosaic aneuploids. Conclusive chromosomal data were obtained from a high percentage of blastomeres (92.8%, 90/97). Microsatellite marker analysis performed on blastomeres in aneuploid embryos enabled us to reconstruct the chromosomal status of the blastomeres in each cleavage division. The results showed the occurrence of meiotic errors in 3 (25%) of the studied embryos. There were 16 mitotic errors (18.8%, 16/85) in the 85 mitotic divisions undertaken by the studied embryos. The observed mitotic errors were mainly contributed by endoreduplication (31.3%, 5/16), non-disjunction (25%, 4/16) and anaphase lagging (25%, 4/16). Chromosome breakages occurred in 6 divisions (7.1%, 6/85)., Conclusions: Mosaicism occurs in a high percentage of good-quality cleavage stage embryos and mitotic errors contribute significantly to the abnormality.

We report a live birth from a couple with two genetic diseases, namely: reciprocal translocation carrier and alpha-thalassaemia trait, following pre-implantation genetic diagnostic tests. This is the first case in Hong Kong in which the technique of using one blastomere biopsy for two diseases was established, using array comparative genomic hybridisation and polymerase chain reaction.

Objective: To report a successful case of preimplantation genetic diagnosis (PGD) for Huntington disease using whole genome amplification., Design: Case report., Setting: University assisted reproduction unit., Patient(s): A couple with family history of Huntington disease: The husband was carrying the expanded allele of the IT15 gene, and the wife had the normal allele., Intervention(s): Preimplantation genetic diagnosis with whole genome amplification for identification of genetically normal embryos., Main Outcome Measure(s): Live birth., Result(s): In an IVF cycle, 15 oocytes were retrieved, of which 13 were mature and 11 were fertilized. On day 3, embryo biopsy and PGD were performed on ten good-quality embryos. Multiple displacement amplification was conducted, followed by polymerase chain reaction with fluorescence primers. Three pairs of primers were used for the amplification of the IT15 gene at the: 1) trinucleotide expansion site; 2) trinucleotide expansion site plus the polymorphic site situated on its 3'-end; and 3) polymorphic marker located downstream of the trinucleotide repeats. Two normal blastocysts were replaced on day 5 and another two good-quality blastocysts were cryopreserved. The woman gave birth to a normal baby girl whose normal genetic status was confirmed by prenatal diagnosis., Conclusion(s): Whole genome amplification by multiple displacement amplification can be used for PGD of Huntington disease.

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene-expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo-containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo-pregnant mice when assayed by real-time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.

Spermatogenesis is a complicated process of germ cell differentiation, involving programmatic expression of diverse cell type- and developmental stage-specific genes. To date, the vitamin-A-deficiency (VAD) rats and postnatal rats are two models commonly used to study spermatogenesis. In the present study, we studied the expression of 1185 known genes in the vitamin-A-deficient and retinol-reinitiated spermatogenesis of rat testis using Clontech Atlas rat cDNA expression arrays. The mRNA expression patterns of post-vitamin-A (PVA) testis on Days 15 and 35 were compared with those of the spermatogenic arrested rat testis on Day 0. About 9% (110/1185) of the genes studied were highly expressed. When compared with VAD rat testis on Day 0, 20 and 31 genes were differentially expressed by a factor of twofold or greater on Days 15 and 35, respectively. Four genes (cytochrome P450 17, sulfated glycoprotein 2, protein kinase inhibitor, and cathepsin L) that play important roles in spermatogenesis were selected and their gene expression patterns were confirmed by semiquantitative reverse transcription-polymerase chain reaction. Comparison of the expression patterns of these genes between PVA-VAD and postnatal rat testis in developmentally matched stages revealed substantial differences during the early stages of spermatogenesis. This discrepancy could be caused by either the presence of arrested but mature somatic cells in the PVA-VAD testis that may contribute to a unique gene expression pattern in this model or the direct effect of retinol on spermatogenesis. Therefore, caution is needed in interpreting the gene expression data using the PVA-VAD and postnatal rat models in studying spermatogenesis in rat testes.

The molecular mechanism regulating spermatogenesis at different developmental stages remains largely unknown. In a vitamin A-deficiency (VAD) rat model, five distinct histologically defined, stage-synchronized testes: (i) resting spermatogonia and preleptotene spermatocytes at Day 0 of post-vitamin A treatment (PVA); (ii) early pachytene spermatocytes at Day 7 PVA; (iii) late pachytene at Day 15 PVA; (iv) round spermatids at Day 25 PVA; and (v) elongated spermatids at Day 35 PVA were used to study gene expression profiles by mRNA differential display. Twenty-four differentially expressed cDNA fragments were identified and cloned; oligonucleotide sequence analyses indicated that there are 12 novel gene sequences, half of which share no apparent match in current GenBank/EMBL databases. Other 12 VAD clones share sequence homology to membrane channel and transport, transcription and translation, cell cycle and morphogenesis, inducer and transducer, surface or secreted glycoproteins or enzymes, and other miscellaneous molecules. Semi-quantitative RT-PCR analyses against different stages of VAD testes demonstrated: (i) restricted expression of VAD1.2 and 1.3 (novel) on Day 25 PVA when round spermatids form; (ii) escalating pattern of VAD12 (Cx43) in Sertoli cells; and (iii) relative constant levels of VAD4 (A5D3), VAD26.1 (ribonuclease), and VAD27 (GRP8) in spermatogenesis.

We hypothesized that transforming growth factor beta1 (TGFbeta(1)) and its receptors play a role in the interaction between the preimplantation embryo and the reproductive tract. To investigate this hypothesis, TGFbeta 1 mRNA in mouse embryos was quantified by competitive reverse transcription-polymerase chain reaction using an RNA mimic. TGFbeta 1 was first detected in the unfertilized oocyte, disappeared after fertilization and was expressed again at the 2-cell stage (4410 +/- 1330 transcripts/embryo). Its expression increased gradually, peaked at the 8-cell stage (58 600 +/- 17 300 transcripts/embryo) and declined rapidly after the morula stage reaching a concentration of 1520 +/- 546 transcripts/embryo at the blastocyst stage. The mRNA levels of TGFbeta 1 at the 8-cell and morula stages were significantly higher than that at other cell stages (P < 0.05). The expression of TGF receptors in embryos and in the reproductive tract was also investigated. Both TGFbeta(1) type I (ALK-5) and type II TGFbeta receptors were detected in embryos from 1-cell to blastocyst stage by immunohistochemistry. Northern hybridization and immunohistochemistry showed a constant expression of both TGFbeta receptors in the oviduct from day 1 to day 4 of pregnancy, whilst in the uterus there was a marked increase in the expression of TGFbeta type I receptor on day 3. Expression of TGFbeta type II receptor in the uterus remained unaltered throughout the study period. This study has shown that preimplantation mouse embryos produce TGFbeta(1) and that both the embryos and the reproductive tract are responsive to TGFbeta(1) in the preimplantation period.

The objectives of this study were to compare the mRNA expression patterns in early mouse embryos in different culture conditions by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Embryos developed in vivo, cultured in vitro, and cocultured with human oviductal epithelial cells were studied at the 2-cell, 4-cell, 8-cell/morula, and blastocyst stages. Messenger RNA profiles were displayed by DDRT-PCR using downstream T11VV (V = A, C, or G) and upstream decamer primers. Total cDNA banding patterns were highly conserved in the three groups studied. Some fragments are unique in different culture conditions. Thirteen out of the 40 selected differentially expressed clones were characterized. The DNA sequence analyses of these clones displayed high sequence homology with cDNA sequences in the mouse expressed sequence tag database. Using semiquantitative RT-PCR, we confirmed differential expression of these DD amplicons in the three groups of embryos. The temporal expression of some of the selected DD amplicons during preimplantation development were studied in the three groups of embryos. In conclusion, DDRT-PCR is an effective tool for contrasting gene expression patterns and characterizing mRNA transcripts in mouse embryo.

A young woman is described in whom symptomatic porphyria cutanea tarda (PCT) developed during copper chelation therapy for Wilson's disease. Termination of ethanol ingestion and oral contraceptive use resulted in cessation of blistering skin lesions and reduction in urinary porphyrin excretion. This is the first recorded coincidence of these two rare hepatic diseases. Therapeutic implications are discussed.