Judy F.C. Chow

Identification of novel genes expressed during spermatogenesis in stage-synchronized rat testes by differential display

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.

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct.

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.

Different Testicular Gene Expression Patterns in the First Spermatogenic Cycle of Postnatal and Vitamin A-Deficient Rat Testis

Abstract 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.

Preimplantation genetic diagnosis for monogenic diseases

Preimplantation genetic diagnosis (PGD) was first reported in 1990. Thereafter, more and more indications for PGD, including monogenic diseases (MGD) and translocations, are presently available, and the list of indications of PGD is expanding from early-onset and serious conditions to late-onset diseases. Polymerase chain reaction has been used for PGD of MGD, while newer techniques, including karyomapping and next-generation sequencing, emerge in recent decade. The limitations of various methods for PGD are discussed in this review.

Preimplantation genetic diagnosis and screening by array comparative genomic hybridisation: experience of more than 100 cases in a single centre

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.

Preimplantation genetic diagnosis for hereditary cancer syndrome: local experience.

A 33-year-old woman was referred for consideration of PGD because she was a BRCA2 gene mutation carrier. She had cancer of the right breast at the age of 24 years and underwent modified radical mastectomy with axillary dissection and immediate latissimus dorsi flap reconstruction. Adjuvant chemoradiotherapy was given and she was prescribed tamoxifen for 5 years after the operation. Her paternal grandmother had breast cancer diagnosed at the age of 60 years. Genetic screening was performed and confirmed the patient to be a BRCA2 mutation carrier. Her elder brother and her father underwent the spot test and were found to carry a BRCA2 mutation but her younger sister was not affected. Laparoscopic ovarian cystectomy was performed for a hyperechoic cyst noted over the right ovary, which was confirmed to be an endometriotic cyst. After a multidisciplinary meeting of clinical geneticists, breast surgeons, oncologists, gynaecologists, psychologists, nurses, and academics in the ethics department, followed by psychological assessment and also counselling, she was offered in-vitro fertilisation (IVF) and PGD. Her IVF and PGD cycle was performed in 2011, using an antagonist protocol with letrozole co-treatment. Fifteen oocytes were retrieved and 12 were fertilised following intracytoplasmic sperm injection (ICSI). Blastomere biopsy was performed on eight goodquality cleaving embryos and five were confirmed to be free of the BRCA2 mutation. Two unaffected blastocysts were transferred, resulting in a singleton pregnancy and one unaffected blastocyst was cryopreserved. She delivered a baby boy at term by caesarean section. Postnatal cord blood confirmed Hong Kong Med J 2016;22:289–91 DOI: 10.12809/hkmj144499

Experience of more than 100 preimplantation genetic diagnosis cycles for monogenetic diseases using whole genome amplification and linkage analysis in a single centre.

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.

Live birth following double-factor pre-implantation genetic diagnosis for both reciprocal translocation and alpha- thalassaemia

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.

Evaluation of preimplantation genetic testing for chromosomal structural rearrangement by a commonly used next generation sequencing workflow

OBJECTIVES To evaluate the applicability of a commonly used next generation sequencing workflow in detecting unbalanced meiotic segregation products for reciprocal translocation and inversion carriers. STUDY DESIGN All preimplantation genetic testing treatment cycles performed for reciprocal translocation or inversion carriers from 2012 to April 2017 were included. Three hundreds and forty-two archived whole genome amplified DNA, which had previously analyzed by array comparative genomic hybridization (aCGH), were retrospectively analyzed by next generation sequencing (NGS). Concordance on overall diagnosis and segmental aneuploidies related to the translocation/inversion breakpoints between aCGH and NGS were determined. RESULTS Retrospective analysis of 287 blastomere biopsies and 55 trophectoderm (TE) biopsies showed that the concordance rate on the overall diagnosis between aCGH and NGS on abnormal samples was 100% (266/266), irrespective to the type of biopsy. The concordance rates of normal biopsies were 98.4% (61/62) on blastomere and 78.6% (11/14) on TE biopsies. NGS detected a de novo segmental aneuploidy on one blastomere biopsy and three possible low level mosaic aneuploidies on 3 TE biopsies, which were previously concluded as euploid by aCGH. Using the karyotype of reciprocal translocation/inversion carriers, size of anticipated segmental aneuploidies could be calculated and be used to predict the applicability of NGS before proceeding to treatment. CONCLUSION This is the first report to evaluate the applicability of a commercial NGS-based workflow for preimplantation testing for reciprocal translocations/inversions. Our study demonstrated that NGS can diagnose unbalanced translocation/inversion products with the same efficiency as aCGH. The applicability of NGS, with respect to individual karyotype, can be predicted before proceeding to treatment.

Comparison between fluorescent in-situ hybridisation and array comparative genomic hybridisation in preimplantation genetic diagnosis in translocation carriers.

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 womens 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.