Differential expression of parental alleles of BRCA1 in human preimplantation embryos

Gene expression from both parental genomes is required for completion of embryogenesis. Differential methylation of each parental genome has been observed in mouse and human preimplantation embryos. It is possible that these differences in methylation affect the level of gene transcripts from each parental genome in early developing embryos. The aim of this study was to investigate if there is a parent-specific pattern of BRCA1 expression in human embryos and to examine if this affects embryo development when the embryo carries a BRCA1 or BRCA2 pathogenic mutation. Differential parental expression of ACTB, SNRPN, H19 and BRCA1 was semi-quantitatively analysed by minisequencing in 95 human preimplantation embryos obtained from 15 couples undergoing preimplantation genetic diagnosis. BRCA1 was shown to be differentially expressed favouring the paternal transcript in early developing embryos. Methylation-specific PCR showed a variable methylation profile of BRCA1 promoter region at different stages of embryonic development. Embryos carrying paternally inherited BRCA1 or 2 pathogenic variants were shown to develop more slowly compared with the embryos with maternally inherited BRCA1 or 2 pathogenic mutations. This study suggests that differential demethylation of the parental genomes can influence the early development of preimplantation embryos. Expression of maternal and paternal genes is required for the completion of embryogenesis

Investigation of microRNA expression and DNA repair gene transcripts in human oocytes and blastocysts

PURPOSE: The aim of the study is to investigate the regulation of DNA repair genes by microRNAs (miRNAs). miRNAs are short non-coding RNAs that regulate transcriptional and post-transcriptional gene silencing. Several miRNAs that are expressed during preimplantation embryo development have been shown or are predicted to target genes that regulate cell cycle checkpoints and DNA repair in response to DNA damage. METHODS: This study compares the expression level of 20 miRNAs and 9 target transcripts involved in DNA repair. The statistical significance of differential miRNA expression between oocytes and blastocysts was determined by t test analysis using the GraphPad Prism v6 software. The possible regulatory roles of miRNAs on their target messenger RNAs (mRNAs) were analysed using a Pearson correlation test. RESULTS: This study shows for the first time that several miRNAs are expressed in human oocytes and blastocysts that target key genes involved in DNA repair and cell cycle checkpoints. Blastocysts exhibited statistically significant lower expression levels for the majority of miRNAs compared to oocytes (p < 0.05). Correlation analyses showed that there was both inverse and direct association between miRNAs and their target mRNAs. CONCLUSIONS: miRNAs target many mRNAs including ones involved in DNA repair mechanisms. This study suggests that miRNAs and their target mRNAs involved in DNA repair are expressed in preimplantation embryos. Similar to the miRNAs expressed in adult tissues, these miRNAs seem to have regulatory roles on their target DNA repair mRNAs during preimplantation embryo development

The impact of mosaicism in preimplantation genetic diagnosis (PGD): approaches to PGD for dominant disorders in couples without family history

OBJECTIVES: Mosaicism in certain dominant disorders may result in a 'non-Mendelian' transmission for the causative mutation. Preimplantation genetic diagnosis (PGD) is available for patients with inherited disorders to achieve an unaffected pregnancy. We present our experience for two female patients with different dominantly inherited autosomal disorders; neurofibromatosis type 1 (NF1) and tuberous sclerosis complex type 2 (TSC2). METHODS: PGD protocol development was carried out using single cells from the patients. PGD was carried out on polar bodies and different embryonic cells. RESULTS: Protocol development for NF1 using lymphocytes from the patient suggested mosaicism for the mutation. This was supported further by quantitative fluorescent-PCR performed on genomic DNA. During PGD, polar bodies and blastomeres lacked the mutation that probably was absent or present at very low levels in the patient's germline. Single lymphocyte analysis during protocol development for TSC2 did not indicate mosaicism; however, analysis of single buccal cells and multiple embryo biopsies across two consecutive IVF/PGD cycles confirmed gonosomal mosaicism. CONCLUSIONS: The trend in PGD is for blastocyst biopsy followed by whole genome amplification, eliminating single cell analysis. In the case of certain dominantly inherited disorders, pre-PGD single cell analysis is beneficial to identify potential mosaicism that ensures robust protocols. © 2016 John Wiley & Sons, Ltd

Preconceptual care for couples seeking fertility treatment, an evidence-based approach

There is accumulating evidence demonstrating that positive lifestyle modification and the optimization of the preconceptual period can influence the reproductive potential for both men and women. However, a large percentage of couples attending fertility clinics with potential to improve preconception habits may not always receive appropriate preconceptual advice. In addition, supplements and adjuncts that promise to increase fertility treatment success rates are marketed to infertile patients despite lack of convincing evidence supporting their benefit. This review aims to identify possible associations between lifestyle factors for couples seeking fertility treatment and fertility treatment outcomes and to offer possible explanations of the biological basis of these associations. An electronic search was conducted from 1978 through July 2019 linking preconceptual behaviors for women and men with the outcome of fertility treatment. The literature search explored the importance of numerous factors, including smoking, caffeine, alcohol, obesity, physical exercise, recreational drugs, stress, diet, supplements, alternative medicine, environmental factors, and pollutants. Some associations were found to be more significant than others. The preconceptual period is undeniably a delicate and important window which should not be overlooked during fertility counseling. Simple lifestyle modifications could positively influence fertility treatment outcomes. Fertility teams, consisting of clinicians, fertility nurses, dieticians, psychologists, exercise advisors and others, should dedicate time to offer evidence-based preconceptual advice and targeted interventions to couples seeking fertility treatment

Next Generation Sequencing Detects Premeiotic Errors in Human Oocytes

Autosomal aneuploidy is the leading cause of embryonic and foetal death in humans. This arises mainly from errors in meiosis I or II of oogenesis. A largely ignored source of error stems from germinal mosaicism, which leads to premeiotic aneuploidy. Molecular cytogenetic studies employing metaphase fluorescence in situ hybridization and comparative genomic hybridisation suggest that premeiotic aneuploidy may affect 10–20% of oocytes overall. Such studies have been criticised on technical grounds. We report here an independent study carried out on unmanipulated oocytes that have been analysed using next generation sequencing (NGS). This study confirms that the incidence of premeiotic aneuploidy in an unselected series of oocytes exceeds 10%. A total of 140 oocytes donated by 42 women gave conclusive results; of these, 124 (88.5%) were euploid. Sixteen out of 140 (11.4%) provided evidence of premeiotic aneuploidy. Of the 140, 112 oocytes were immature (germinal vesicle or metaphase I), of which 10 were aneuploid (8.93%); the remaining 28 were intact metaphase II-first polar body complexes, and six of these were aneuploid (21.4%). Of the 16 aneuploid cells, half contained simple errors (one or two abnormal chromosomes) and half contained complex errors. We conclude that germinal mosaicism leading to premeiotic aneuploidy is a consistent finding affecting at least 10% of unselected oocytes from women undergoing egg collection for a variety of reasons. The importance of premeiotic aneuploidy lies in the fact that, for individual oocytes, it greatly increases the risk of an aneuploid mature oocyte irrespective of maternal age. As such, this may account for some cases of aneuploid conceptions in very young women

The origin and significance of additional aneuploidy events in couples undergoing preimplantation genetic diagnosis for translocations by array comparative genomic hybridization

Diagnostic application of array comparative genomic hybridization (aCGH) in preimplantation genetic diagnosis for reciprocal and Robertsonian translocations has revealed 55–65% embryos with additional aneuploidies with or without translocation-related imbalances. The occurrence of these extra abnormalities with the balanced form of the translocation reduces the number of embryos suitable for transfer. Eighty-three embryos were followed up on days 5–7 of development from 23 infertile or sub-fertile carriers for whole chromosome and segmental aneuploidies present in addition to the balanced or unbalanced translocations detected on aCGH diagnosis. Embryos were analysed by fluorescence in-situ hybridization (n = 63) and aCGH (n = 20). Meiotic aneuploidy affected 35% of embryos and 47% had mitotic events; 15% had both types. Meiotic and mitotic events were almost equal (60 versus 64), 97 affected whole chromosomes (58 meiotic, 39 mitotic) and 27 were segmental (two meiotic, 25 mitotic). In 85.5% of embryos with whole chromosome additional aneuploidies, the aneuploidy was present throughout or in more than 50% of cells. All embryos diagnosed as abnormal (translocation balanced or unbalanced) after aCGH diagnosis at cleavage stage would have remained unsuitable for transfer if tested at later stages of development. Additional aneuploidies merit full consideration when considering the choice of embryos to transfer

Reproductive outcomes from ten years of elective oocyte cryopreservation

Research question: To assess the relationship between the number of oocytes retrieved during elective oocyte cryopreservation (EOC) cycles with various clinical, biochemical, and radiological markers, including age, body mass index (BMI), baseline anti-Müllerian hormone (AMH), antral follicle count (AFC), Oestradiol level (E2) and total number of follicles ≥ 12 mm on the day of trigger. To also report the reproductive outcomes from women who underwent EOC. Methods: A retrospective cohort of 373 women embarking on EOC and autologous oocyte thaw cycles between 2008 and 2018 from a single London clinic in the United Kingdom. Results: 483 stimulation cycles were undertaken amongst 373 women. The median (range) age at cryopreservation was 38 (26–47) years old. The median numbers of oocytes retrieved per cycle was 8 (0–37) and the median total oocytes cryopreserved per woman was 8 (0–45). BMI, E2 level and number of follicles ≥ 12 mm at trigger were all significant predictors of oocyte yield. Multivariate analysis confirmed there was no significant relationship between AFC or AMH, whilst on univariate analysis statistical significance was proven. Thirty six women returned to use their cryopreserved oocytes, of which there were 41 autologous oocyte thaw cycles undertaken. There were 12 successful livebirths achieved by 11 women. The overall livebirth rate was 26.8% per cycle. No livebirths were achieved in women who underwent EOC ≥ 40 years old, and 82% of all livebirths were achieved in women who had done so between 36 and 39 years old. Conclusion: Clinical, biochemical and radiological markers can predict oocyte yield in EOC cycles. Reproductive outcomes are more favourable when cryopreservation is performed before the age of 36, with lower success rates of livebirth observed in women aged 40 years and above

Factors influencing the outcome of intrauterine insemination (IUI): age, clinical variables and significant thresholds

The aim was to investigate the influence of various biological factors upon the outcome of intrauterine insemination (IUI). The total IUI history (856 cycles) of 352 couples was studied. Live-birth showed a strong negative correlation with female age but no correlation with male age. Antimullerian hormone (AMH) and antral follicle count (AFC) correlated negatively with female age, and follicle stimulating hormone (FSH) correlated positively. Significant thresholds were found for all three variables, and also for total motile count (TMC) in the prepared sperm. Calculating pregnancy losses per positive pregnancy showed a strong correlation with increasing female age. This was highly significant for biochemical losses but not for fetal heart miscarriages. Male age had no effect on rate of pregnancy loss. In conclusion, female age, FSH, AMH and TMC are good predictive factors for live-birth and therefore relate to essential in vivo steps in the reproductive process

Live birth rate is associated with oocyte yield and number of biopsied and suitable blastocysts to transfer in preimplantation genetic testing (PGT) cycles for monogenic disorders and chromosomal structural rearrangements

OBJECTIVES: To investigate whether live birth (LB) is associated with oocyte yield and number of biopsied and suitable blastocyst to transfer following preimplantation genetic testing (PGT) for monogenic disorders (PGT-M) and chromosomal structural rearrangements (PGT-SR). STUDY DESIGN: All couples underwent controlled ovarian stimulation, blastocyst biopsy, vitrification and transfer of suitable embryo(s) in a frozen embryo transfer (FET) cycle. RESULTS: Of 175 couples who underwent PGT treatment, 249 oocytes retrievals were carried out and 230 FET were subsequently undertaken. 122/230 (53%, 95% CI 47–59) FET resulted in a LB and 16/230 (7%, 95% CI 4–11) have resulted in ongoing pregnancies. 21/230 (9%, 95% CI 6–14) FET resulted in miscarriage and 69/230 (30%, 95% CI 24–36) concluded with failed implantation. Two (1%, 95% CI 0–3) transfers underwent termination for congenital malformation, with no evidence of misdiagnosis by prenatal testing. The relationship between number of oocytes retrieved and number of blastocysts biopsied and suitable embryos to transfer were significant (p = 0.00; Incidence rate ratio (IRR) 1.05; 95% 1.04–1.06; p = 0.00; IRR 1.04; 95%, 1.03–1.06), respectively. The number of oocytes collected (p = 0.007; OR 1.06; 95% CI 1.01–1.10), the number of blastocysts biopsied (p = 0.001; OR 1.14; 95% 95% CI 1.06–1.23) and the number of suitable embryos to transfer (p = 0.00; OR 1.38; 95% CI 1.17–1.64) were all significantly associated with the odds of achieving a LB. There is a 14% and 38% increased chance of a LB per additional blastocyst biopsied and suitable embryo to transfer, respectively. CONCLUSIONS: PGT-M and PGT-SR outcomes are significantly associated with egg yield, number of blastocysts to biopsy and suitable embryos to transfer

In assisted reproduction by IVF or ICSI, the rate at which embryos develop to the blastocyst stage is influenced by the fertilization method used: a split IVF/ICSI study

PURPOSE: To compare in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) in regard to post-fertilization development and outcome with the purpose of ascertaining the most effective fertilization method for assisted reproduction. METHODS: A retrospective cohort study of 136 split IVF/ICSI cycles (where sibling oocytes are fertilized by two different methods using the same sperm sample). RESULTS: IVF-derived embryos developed to the blastocyst stage at a significantly faster rate than ICSI-derived embryos. There was no significant difference in fertilization or livebirth rates between the two fertilization methods. CONCLUSIONS: For patients with sperm progressive motility ≥ 1.0 × 106/ml (who usually constitute the majority of patients), no significant difference between the two fertilization methods was found in regard to fertilization rate or livebirth rate. Remaining factors influencing choice between the two methods appear to be restricted to convenience, financial considerations and concern with regard to possible perpetuation of genetically linked infertility to future generations