Optimizing non-invasive preimplantation genetic testing: investigating culture conditions, sample collection, and IVF treatment for improved non-invasive PGT-A results.

PURPOSE: This study aimed to optimize the non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) in the laboratory by comparing two collection timing of the spent culture medium (SCM), two embryo rinsing protocols, and the use of conventional insemination instead of intracytoplasmic sperm injection (ICSI). METHODS: Results of two embryo rinsing methods (one-step vs sequential) and SCM collected on day 5 vs day 6 after retrieval were compared against trophectoderm (TE) biopsies as reference. Results from day 6 SCM in cycles fertilized by conventional insemination were compared with PGT-A using ICSI. RESULTS: The rate of concordance was higher in day 6 samples than in day 5 samples when the sequential method was used, in terms of total concordance (TC; day 6 vs day 5: 85.0% vs 60.0%, p = 0.0228), total concordance with same sex (TCS, 82.5% vs 28,0%, p < 0.0001), and full concordance with same sex (FCS, 62.5% vs 24.0%, p = 0.0025). The sequential method significantly out-performed the one-step method when SCM were collected on day 6 (sequential vs one-step, TC: 85.0% vs 64.5%, p = 0.0449; TCS: 82.5% vs 54.8%, p = 0.0113; FCS: 62.5% vs 25.8%, p = 0.0021). There was no significant difference in niPGT-A results between cycles fertilized by the conventional insemination and ICSI. CONCLUSION: We have shown a higher concordance rate when SCM was collected on day 6 and the embryos were rinsed in a sequential manner. Comparable results of niPGT-A when oocytes were fertilized by conventional insemination or ICSI. These optimization steps are important prior to commencement of a randomized trial in niPGT-A.

Randomised double-blind controlled trial of non-invasive preimplantation genetic testing for aneuploidy in in vitro fertilisation: a protocol paper.

INTRODUCTION: The success rate of in vitro fertilisation (IVF) treatment for couples with infertility remains low due to lack of a reliable tool in selecting euploid embryos for transfer. This study aims to compare the efficacy in embryo selection based on morphology alone compared with non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) and morphology in infertile women undergoing IVF. METHODS AND ANALYSIS: This is a randomised double-blind controlled trial conducted in two tertiary assisted reproduction centres. A total of 500 infertile women will be recruited and undergo IVF as indicated. They will be randomly assigned on day 6 after oocyte retrieval into two groups: the intervention group using morphology and niPGT-A and the control group based on morphology alone. In the control group, blastocysts with the best quality morphology will be replaced first. In the intervention group, blastocysts with the best morphology and euploid result of spent culture medium will be replaced first. The primary outcome is a live birth per the first embryo transfer. The statistical analysis will be performed with the intention to treat and per protocol. ETHICS AND DISSEMINATION: Ethics approval was sought from the institutional review board of the two participating units. All participants will provide written informed consent before joining the study. The results of the study will be submitted to scientific conferences and peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04474522.

Wireless physical activity monitor use among adults living with HIV in a community-based exercise intervention study: a quantitative, longitudinal, observational study.

OBJECTIVES: Our aim was to examine wireless physical activity monitor (WPAM) use and its associations with contextual factors (age, highest education level, social support and mental health) among adults living with HIV engaged in a community-based exercise (CBE) intervention. DESIGN: Quantitative, longitudinal, observational study. SETTING: Toronto YMCA, Ontario, Canada. PARTICIPANTS: Eighty adults living with HIV who initiated the CBE intervention. INTERVENTION: Participants received a WPAM to track physical activity during a 25-week CBE intervention involving thrice-weekly exercise, supervised weekly (phase 1) and a 32-week follow-up involving thrice-weekly exercise with no supervision (phase 2), completed in December 2018. OUTCOME MEASURES: Uptake was measured as participants who consented to WPAM use at initation of the intervention. Usage was defined as the proportion of days each participant had greater than 0 steps out of the total number of days in the study. We measured contextual factors using a baseline demographic questionnaire (age, highest education level), and median scores from the bimonthly administered Medical Outcomes Study-Social Support Scale and Patient Health Questionnaire (mental health), where higher scores indicated greater social support and mental health concerns, respectively. We calculated Spearman correlations between WPAM usage and contextual factors. RESULTS: Seventy-six of 80 participants (95%) consented to WPAM use. In phase 1, 66% of participants (n=76) and in phase 2, 61% of participants (n=64) used the WPAM at least 1 day. In phase 1, median WPAM usage was 50% (25th, 75th percentile: 0%, 87%; n=76) of days enrolled and in phase 2, 23% (0%, 76%; n=64) of days. Correlation coefficients with WPAM usage ranged from weak for age (ρ=0.26) and mental health scores (ρ=-0.25) to no correlation (highest education level, social support). CONCLUSIONS: Most adults living with HIV consented to WPAM use, however, usage declined over time from phase 1 to phase 2. Future implementation of WPAMs should consider factors to promote sustained usage by adults living with HIV. TRIAL REGISTRATION NUMBER: NCT02794415.

Expanded Potential Stem Cells from Human Embryos Have an Open Chromatin Configuration with Enhanced Trophoblast Differentiation Ability.

Human expanded potential stem cells (hEPSC) have been derived from human embryonic stem cells and induced pluripotent stem cells. Here direct derivation of hEPSC from human pre-implantation embryos is reported. Like the reported hEPSC, the embryo-derived hEPSC (hEPSC-em) exhibit a transcriptome similar to morula, comparable differentiation potency, and high genome editing efficiency. Interestingly, the hEPSC-em show a unique H3 lysine-4 trimethylation (H3K4me3) open chromatin conformation; they possess a higher proportion of H3K4me3 bound broad domain (>5 kb) than the reported hEPSC, naive, and primed embryonic stem cells. The open conformation is associated with enhanced trophoblast differentiation potency with increased trophoblast gene expression upon induction of differentiation and success in derivation of trophoblast stem cells with bona fide characteristics. Hippo signaling is specifically enriched in the H3K4me3 broad domains of the hEPSC-. Knockout of the Hippo signaling gene, YAP1 abolishes the ability of the embryo-derived EPSC to form trophoblast stem cells.

Reference change value of global longitudinal strain in clinical practice: A test-rest quality implementation project.

BACKGROUND: Reference change value (RCV) is used to assess the significance of the difference between two measurements after accounting for pre-analytic, analytic, and within-subject variability. The objective of the current study was to define the RCV for global longitudinal strain (GLS) using different semi-automated software in standard clinical practice. METHODS: Using a test-retest study design, we quantified the median coefficient of variation (CV) for GLS using AutoStrain and Automated Cardiac Motion Quantification (aCMQ) by Philips. Triplane left-ventricular ejection fraction (LVEF) was measured for comparison. Multivariable regression analysis was performed to determine factors influencing test-retest CV including image quality and the presence of segmental wall motion abnormalities (WMA). RCV was reported using a standard formula assuming two standard deviations for repeated measurements; results were also translated into Bayesian probability. Total measurement variation was described in terms of its three different components: pre-analytic (acquisition), analytic (measuring variation), and within-subject (biological) variation. RESULT: Of the 44 individuals who were screened, 41 had adequate quality for strain quantification. The mean age of the cohort was 56.4 ± 16.8 years, 41% female, LVEF was 55.8 ± 9.8% and the median and interquartile range for LV GLS was -17.2 [-19.3 to -14.8]%. Autostrain was more time efficient (80% less analysis time) and had a lower total median CV than aCMQ (CV = 7.4% vs. 17.6%, p < .001). The total CV was higher in patients with WMA (6.4% vs. 13.2%, p = .035). In non-segmental disease, the CV translates to a RCV of 15% (corresponding to a probability of real change of 80%). Assuming a within-subject variability of 4.0%, the component analysis identified that inter-reader variability accounts for 3.7% of the CV, while acquisition variability accounts for 4.0%. CONCLUSION: Using test-retest analysis and CVs, we find that an RCV of 15% for GLS represents an optimistic estimate in routine clinical practice. Based on our results, a higher RCV of 17%-21% is needed in order to provide a high probability of clinically meaningful change in GLS in all comers. The methodology presented here for determining measurement reproducibility and RCVs is easily translatable into clinical practice for any imaging parameter.

Effect of surgical intervention for mild childhood obstructive sleep apnoea on attention and behavioural outcomes: A randomized controlled study.

BACKGROUND AND OBJECTIVE: We evaluated inattention and behavioural outcomes following surgery versus watchful waiting (WW) in school-aged children with mild obstructive sleep apnoea (OSA). METHODS: A prospective randomized controlled study was performed in pre-pubertal children aged 6-11 years with polysomnography (PSG)-confirmed mild OSA. They were assigned randomly to early surgical intervention (ES) or WW. The surgical intervention consisting of tonsillectomy with or without adenoidectomy and turbinate reduction was carried out within 4-6 weeks after randomization. Both groups underwent PSG, attention and behavioural assessment and review by an otorhinolaryngologist at baseline and 9-month follow-up. The primary outcome was omission T score from Conners' continuous performance test (CPT). Secondary outcomes were parent-reported behaviours, quality of life, symptoms and PSG parameters. RESULTS: A total of 114 participants were randomized. Data of 35 subjects from the ES and 36 from the WW group were available for final analysis. No significant treatment effect could be found in all CPT parameters and behavioural outcomes. Nevertheless, significantly greater reductions were seen in PSG parameters (obstructive apnoea-hypopnoea index [-1.4 ± 2.0 cf. +0.3 ± 4.1/h, p = 0.038] and arousal index [-1.3 ± 4.4 cf. +1.4 ± 4.5/h, p = 0.013]) and OSA-18 total symptom score (-17.3 ± 19.7 cf. -3.6 ± 14.1, p = 0.001) in the ES group. Subjects who underwent surgery also had significantly greater weight gain (+3.3 ± 2.1 cf. +2.2 ± 1.5 kg, p = 0.014) and increase in systolic blood pressure (+5.1 ± 12.4 cf. -1.2 ± 8.7 mm Hg, p = 0.016). CONCLUSION: Despite improvements in PSG parameters and parent-reported symptoms, surgical treatment did not lead to parallel improvements in objective attention measures in school-aged children with mild OSA.

Distinguishing between carrier and noncarrier embryos with the use of long-read sequencing in preimplantation genetic testing for reciprocal translocations.

Balanced reciprocal translocation carriers are usually phenotypically normal but are at an increased risk of infertility, recurrent miscarriage or having affected children. Preimplantation genetic testing on chromosomal structural rearrangement (PGT-SR) offers a way to screen against unbalanced embryos. Here, we demonstrated a new method to distinguish carrier from noncarrier embryos. Translocation breakpoints were first delineated by nanopore sequencing followed by polymerase chain reaction (PCR) across breakpoints. High-resolution breakpoint mapping was successful in all (9/9) balanced reciprocal translocation carriers. Retrospective analysis of their embryo biopsies with breakpoint PCR showed 100% concordant results with PGT-SR on trophectoderm biopsies (40/40) and 53% concordance on blastomere biopsies (8/15). The low concordant rate in blastomeres was due to failure in the amplification of derivative chromosomes involving large deletions. Breakpoint PCR also showed 100% concordant results with prenatal/postnatal outcomes on 5 pregnancies, indicating that our new method can accurately distinguish carrier from noncarrier embryos.

High-resolution mapping of reciprocal translocation breakpoints using long-read sequencing.

Long-read nanopore sequencing enables direct high-resolution breakpoint mapping on balanced carriers of reciprocal translocation. The mean sequencing depth on the translocated chromosomes to achieve accurate mapping of breakpoints ranged from 2.5-fold to 6.2-fold. To speed up determination of the breakpoints from long-read sequencing data, alignment reads on the translocated chromosomes were extracted before piped into NanoSV. Checking the position of breakpoints on Interactive Genomics Viewer (IGV) was crucial to successful design of breakpoint PCR primers, especially when large deletion was involved at the breakpoints. •Long-read sequencing enables accurate breakpoint mapping with base-pair resolution•Splitting bam files by translocated chromosomes drastically speeded up the breakpoint determination•IGV helps to identify the breakpoint positions and facilitate the design of breakpoint PCR primers.

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.

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.

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.

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 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.

Array comparative genomic hybridization analyses of all blastomeres of a cohort of embryos from young IVF patients revealed significant contribution of mitotic errors to embryo mosaicism at the cleavage stage.

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.

Preimplantation genetic diagnosis using combined strategies on a breast cancer patient with a novel genomic deletion in BRCA2.

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.

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.

Singleton birth after preimplantation genetic diagnosis for Huntington disease using whole genome amplification.

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.

Comparison of three-dimensional echocardiography to two-dimensional echocardiography and fluoroscopy for monitoring of endomyocardial biopsy.

Real-time 3-dimensional echocardiography (RT3DE) offers the rapid acquisition of quantitative and qualitative anatomic data without the use of geometric assumptions. This study was designed to test the feasibility and potential superiority of RT3DE versus 2-dimensional echocardiography (2DE) and standard fluoroscopy for monitoring endomyocardial biopsies (EMBs). Thirty-eight consecutive EMBs performed under fluoroscopic guidance in 26 patients were monitored using 2DE and RT3DE alternately. Two reviewers scored each biopsy pass for visualization of the tip of the bioptome and location of the actual biopsy. Overall image quality was noted as good or poor, and the effect of image quality on tip localization was analyzed. A total of 243 biopsy attempts were made during 38 EMBs. The location of the biopsy was determined in 74% of the biopsies monitored with RT3DE, whereas 2DE demonstrated the location with certainty in only 43% of the biopsies (p <0.0001). On a procedure-by-procedure comparison, RT3DE was found to show the bioptome tip better in 23 of 38 biopsies, compared with 1 of 38 for 2DE (p = 0.001). In 14 of 38 EMBs, neither method was clearly better. In conclusion, RT3DE improves the ability to see the location of the bioptome during EMB compared with 2DE and fluoroscopy.

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.