Project description:Background: In vitro culture of preimplantation mouse embryos is associated with changes in gene expression. It is however not known if the method of fertilization affects the global pattern of gene expression. Method: We compared gene expression and development of mouse blastocysts produced by intra-cytoplasmic sperm injection and cultured in Whitten’s medium (ICSIWM) or KSOM medium with amino acids (ICSIKSOMaa) with control blastocysts flushed out of the uterus on post coital day 3.5 (In vivo). Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. In addition we compared gene expression in embryos generated in IVF or ICSI using WM. Results: Blastocysts resulting from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared to in vivo generated embryos. Approximately 1000 genes are different between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after in vitro culture. Unexpectedly, only 41 genes were statistically different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOMaa). Conclusion: The method of fertilization plays a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.
Project description:Background: In vitro culture of preimplantation mouse embryos is associated with changes in gene expression. It is however not known if the method of fertilization affects the global pattern of gene expression. Method: We compared gene expression and development of mouse blastocysts produced by intra-cytoplasmic sperm injection and cultured in Whitten’s medium (ICSIWM) or KSOM medium with amino acids (ICSIKSOMaa) with control blastocysts flushed out of the uterus on post coital day 3.5 (In vivo). Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. In addition we compared gene expression in embryos generated in IVF or ICSI using WM. Results: Blastocysts resulting from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared to in vivo generated embryos. Approximately 1000 genes are different between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after in vitro culture. Unexpectedly, only 41 genes were statistically different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOMaa). Conclusion: The method of fertilization plays a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used. 16 samples were used in 4 treatment groups (4 replicate samples per treatment)
Project description:BackgroundUterine adenomyosis is a benign disease, common among women in their 40 and 50 s, characterised by ectopic endometrial tissue in the uterine myometrial layer. Adenomyosis causes infertility and has a negative effect on the outcomes of in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) embryo transfer (ET) cycles. It has also been reported to have different characteristics depending on the adenomyotic lesion localisation. The effect of its localisation on IVF/ICSI-ET outcomes is unclear. This study aimed to investigate whether adenomyotic lesion localisation, assessed using magnetic resonance imaging (MRI), was associated with outcomes of IVF/ICSI-ET cycles.MethodsThis multicentre, joint, retrospective cohort study analysed the medical records of 67 infertile patients with adenomyosis who underwent IVF/ICSI with fresh and frozen-thawed ET at five participating facilities from January 2012 to December 2016 and for whom MRI data were available. Fifteen patients were excluded; therefore, the MRI data of 52 patients were evaluated by two radiologists. We assessed the localisation of and classified adenomyotic lesions into advanced (invades the full thickness of the uterine myometrium), extrinsic (localised on the serosal side), and intrinsic (localised on the endometrial side) subtypes.ResultsThere were 40 advanced, nine extrinsic, and three intrinsic cases, and the outcomes of 100, 27, and nine ET cycles, respectively, were analysed. Pregnancy loss/clinical pregnancy and live birth rates of the advanced, extrinsic, and intrinsic groups were 64 % (16/25) and 9 % (9/100), 33.3 % (3/9) and 22.2 % (6/27), and 50 % (1/2) and 11.1 % (1/9), respectively. A logistic regression analysis adjusted for age, prior miscarriage, and body mass index showed that the extrinsic group had fewer pregnancy losses (odds ratio 0.06; 95 % confidence interval [CI]: 0.00-0.54, p = 0.026) and more live births (odds ratio 6.05; 95 % CI: 1.41-29.65, p = 0.018) than the advanced group.ConclusionsAdenomyotic lesions exert different effects on IVF/ICSI-ET outcomes. Thus, MRI assessments of adenomyosis in infertile patients are beneficial. Establishment of treatment plans based on adenomyotic lesion localisation should be considered.
Project description:Recently, genome-editing tools have come into common use in the field of rat research, and consequently, many genetically modified rat strains have been preserved and archived as frozen embryos. Although there have been many reports published on the topic of rat sperm cryopreservation, no report has yet provided satisfactory and acceptable protocols for the cryopreservation of rat sperm. In this study, we developed methods for both the cryopreservation of transgenic rat sperm and in vitro fertilisation using frozen sperm, which yielded high fertilisation rates.
Project description:Whole-genome sequencing of preimplantation human embryos to detect and screen for genetic diseases is a technically challenging extension to preconception screening. Combining preconception genetic screening with preimplantation testing of human embryos facilitates the detection of de novo mutations and self-validates transmitted variant detection in both the reproductive couple and the embryo's samples. Here we describe a trio testing workflow that involves whole-genome sequencing of amplified DNA from biopsied embryo trophectoderm cells and genomic DNA from both parents. Variant prediction software and annotation databases were used to assess variants of unknown significance and previously not described de novo variants in five single-gene preimplantation genetic testing couples and eleven of their embryos. Pathogenic variation, tandem repeat, copy number and structural variations were examined against variant calls for compound heterozygosity and predicted disease status was ascertained. Multiple trio testing showed complete concordance with known variants ascertained by single-nucleotide polymorphism array and uncovered de novo and transmitted pathogenic variants. This pilot study describes a method of whole-genome sequencing and analysis for embryo selection in high-risk couples to prevent early life fatal genetic conditions that adversely affect the quality of life of the individual and families.
Project description:PurposeTo determine if the use of ICSI in women of advanced maternal age with non-male factor infertility increases chances of live birth.MethodsRetrospective data analysis of 10 years of cycle data from a single Australian IVF clinic (Repromed). First cycle patients only of an advanced maternal age (≥ 35 years) with non-male factor infertility utilising standard IVF or ICSI insemination and having at least three oocytes collected at egg pick up were assessed for live birth following transfer of single genetically unscreened blastocyst (N = 577). Subanalysis of clinical pregnancy, miscarriage, fertilisation, embryo utilisation rate and having a blastocyst for transfer were considered. Unadjusted, covariate adjusted and propensity score weighted analysis were performed.ResultsThe use of standard IVF insemination in women ≥ 35 years with non-male factor infertility increased the chance of a live birth compared with ICSI insemination (unadjusted OR = 2.72, 95% CI [1.78, 4.17]; adjusted OR = 2.64, 95% CI [1.64, 4.27] and weighted OR = 2.26, 95% CI [1.72, 2.98] 31% vs 14%). All other outcomes (fertilisation rate, embryo utilisation, blastocyst for embryo transfer and miscarriage rate) were unaffected.ConclusionIn couples with advanced maternal age and non-male factor infertility, standard IVF insemination appears to increase the chance of a live birth compared with ICSI. As such, the results of this study support the use of routine IVF as the preferred insemination technique for older women in non-male factor infertility. However, future randomised controlled trials are still required to assess this policy.