Efficacy of dehydroepiandrosterone to overcome the effect of ovarian ageing (DITTO): a proof of principle randomised controlled trial protocol

Introduction: Dehydroepiandrosterone (DHEA) has been proposed to improve pregnancy rates in women with diminished ovarian reserve undergoing in vitro fertilisation (IVF) treatment. However, evidence regarding its efficacy is supported by a limited number of randomised controlled trials (RCTs). This doubleblinded RCT aims to measure the effect of DHEA supplementation prior to and during controlled ovarian hyperstimulation on ovarian response prior to IVF treatment in women predicted to have poor ovarian reserve. Methods and analysis: Sixty women with ovarian antral follicle count ≤10 and serum anti-Mullerian hormone ≤5 pmol/L undergoing IVF/intracytoplasmic sperm injection (ICSI) treatment at the Nurture fertility clinic, Nottingham will be recruited. They will be randomised to either receive DHEA capsule 75 mg/day or placebo for at least 12 weeks before egg collection. All participants will undergo standard long down regulation protocol using human menopausal gonadotropin 300 IU/day. Serum samples and follicular fluids at the time of egg collection will be collected for hormonal immunoassays. For ICSI participants, cumulus cells stripped from oocyte will be collected for cumulus gene expression analyses regarding oocyte competence. Microdrops of oocyte culture media before the time of ICSI will be assessed for glucose, pyruvate and lactate utilisation. Embryo transfer will be performed on day 2, 3 or 5 based on the number and quality of the embryos available. Pregnancy will be defined as urine pregnancy test positive (biochemical pregnancy) and 6–8 weeks ultrasound scan with fetal heart beat (clinical pregnancy) and live birth. It is planned to perform the molecular and nutritional fingerprint analyses in batches after finishing the clinical phase of the study. Ethics and dissemination: The approval of the study was granted by the NHS Research Ethics Committee (Ref number NRES 12/EM/0002), the Medicines and Healthcare products Regulatory Agency (MHRA), and the Nottingham University Hospitals Trust Research and Development department. All participants shall provide written informed consent before being randomised into allocated treatment groups

Nuclear transfer in ruminants

Ruminants were the first mammalian species to be cloned successfully by nuclear transplantation. Those experiments were designed to multiply high merit animals (Willadsen, Nature 320(6057):63–65, 1986; Prather et al., Biol Reprod 37(4):859–866, 1987; Wilmut et al., Nature 385(6619):810–813, 1997). Since then, cloning has provided us with a vast amount of knowledge and information on the reprogramming ability of somatic cells to different cell types which became an important basis for stem cell research and human medicine. Nowadays, the goals of most nuclear transfer work vary widely but in most cases the micromanipulation procedures remain the same. However, differences between species require different technical considerations. In this chapter, we describe in detail somatic cell nuclear transfer which is the foremost method for cloning ruminants with specific reference to sheep and cattle

Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term

<p>Abstract</p> <p>Background</p> <p>Genome reprogramming in early mouse embryos is associated with nuclear reorganization and particular features such as the peculiar distribution of centromeric and pericentric heterochromatin during the first developmental stage. This zygote-specific heterochromatin organization could be observed both in maternal and paternal pronuclei after natural fertilization as well as in embryonic stem (ES) cell nuclei after nuclear transfer suggesting that this particular type of nuclear organization was essential for embryonic reprogramming and subsequent development.</p> <p>Results</p> <p>Here, we show that remodeling into a zygotic-like organization also occurs after somatic cell nuclear transfer (SCNT), supporting the hypothesis that reorganization of constitutive heterochromatin occurs regardless of the source and differentiation state of the starting material. However, abnormal nuclear remodeling was frequently observed after SCNT, in association with low developmental efficiency. When transient treatment with the histone deacetylase inhibitor trichostatin A (TSA) was tested, we observed improved nuclear remodeling in 1-cell SCNT embryos that correlated with improved rates of embryonic development at subsequent stages.</p> <p>Conclusion</p> <p>Together, the results suggest that proper organization of constitutive heterochromatin in early embryos is involved in the initial developmental steps and might have long term consequences, especially in cloning procedures.</p

Early epigenetic reprogramming in fertilized, cloned, and parthenogenetic embryos

Despite ongoing research in a number of species, the efficiency of embryo production by nuclear transfer remains low. Incomplete epigenetic reprogramming of the nucleus introduced in the recipient oocyte is one factor proposed to limit the success of this technique. Nonetheless, knowledge of reprogramming factors has increased—thanks to comparative studies on reprogramming of the paternal genome brought by sperm on fertilization—and will be reviewed here. Another valuable model of reprogramming is the one obtained in the absence of sperm fertilization through artificial activation—the parthenote—and will also be introduced. Altogether the objective of this review is to have a better understanding on the mechanisms responsible for the resistance to reprogramming, not only because it could improve embryonic development but also as it could benefit therapeutic reprogramming research

Steroids and miRNAs in assessment of ovarian tissue damage following cryopreservation

Ovarian cortical tissue cryopreservation is a relatively novel approach to preserving fertility in women diagnosed with cancer. However, the effects of freezing-thawing are not fully understood, mainly due to the lack of suitable methods to assess tissue’s survival after thawing. Disparities in steroid production have been associated with ovarian failure by disrupting folliculogenesis, ovulation and oocyte apoptosis. Moreover, specific miRNAs, identified in human ovarian follicles, are thought to play a fundamental role in folliculogenesis. In this study, we investigated the possible interplay between the ovariansteroidal production and miRNA expression patterns in spent culture media, as potential non-invasive markers for ovarian tissue damage after cryopreservation. Cryopreservation of ovarian cortical tissue decreased (P < 0.05) both steroid production (oestradiol and progesterone) and expression of miRNA-193b and 320A in spent culture media over 5 days; however, expression of miRNA-24 increased (P < 0.05). The number of primordial follicles was also reduced (P < 0.05) in fresh-cultured and cryopreserved-cultured cortical tissues when compared with fresh tissues. Downregulation of miRNA-193b and miRNA-320A together with upregulation of miRNA-24 could have a synergistic role in cell apoptosis, and consequently leading to reduced oestradiol and progesterone production.Thus, there appears to be an interplay between these miRNAs, ov arian steroid production and cell damage, which can be further explored as novel non-invasive markers of cell damage following cryopreservation

Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage

Study questionDoes cryoprotection of spermatozoa using a vitrification protocol with improved cryoprotective agents and a novel device for large storage lead to better outcomes than conventional slow freezing?Summary answerVitrification of human sperm using sucrose and dextran-based cryoprotectant (CPA4) with a new vitrification device resulted in significantly better sperm motility and progressive motility and improved DNA integrity with lower DNA fragmentation compared with conventional slow freezing.What is known alreadyA major limitation to clinical implementation of vitrification is the right balance between the volume of spermatozoa suspension cryopreserved and a standardised use of CPAs for survival of spermatozoa.Study design, size, durationThis was a control versus current clinical practice study using 30 fresh human semen samples to carry out the different cryoprotectant analyses followed by a further 23 semen samples to test the novel vitrification protocol.Participants/materials, setting, methodsAll human specimens fulfilled the following criteria: > 5 million spermatozoa/mL, > 20% total motility, ≥ 1.8 mL in volume, with all participants falling within the age range of 25–45 inclusively. The concentration, progressive motility, non-progressive motility, immotility, and various morphokinetic variables including DAP, DCL, DSL, LIN, and STR were then determined using the IVOS II™ Clinical CASA system (Hamilton Thorne, Beverly, MA, USA) on the basis of the 5th Edition of WHO Laboratory Manual for the Examination and Processing of Human Semen.Main results and the role of chanceAmong the 6 cryopreservation methods in this study, vitrification with the funnel-shaped device using CPA4 best preserves the 13 sperm parameters evaluated by CASA system. Conventional slow freezing and vitrification with the device using seminal plasma also protects sperm quality, but the overall motilities are statistically lower in comparison with the novel vitrification approach with cryoprotective media using the device. DNA fragmentation significantly increased after cryopreservation through the method of conventional slow freezing (p = 0.07). There was no significant difference in DNA fragmentation between fresh control and vitrification (p = 1.000).Limitations, reasons for cautionExtensive training is required to minimise the human error in using the vitrification device to perform cryopreservation. Each operator can only handle one sample at a time with device vitrification, whereas several samples can be processed without the need for special training with conventional slow freezing.Wider implications of the findingsThe presented study shows that a new vitrification method could improve survival sperm rate. Human sperm vitrification using our novel protocol gives higher motility and progression and lower percentage of DNA fragmentation than conventional slow freezing. Our findings indicate that this method could supersede the current clinical practice in particular for patients with oligospermia as it reduces osmotic damage, time, and cost

Partially-constrained sex allocation and the indirect effects of assisted reproductive technologies on the human sex ratio

Infertility affects around 15% of human couples and in many countries approximately 1–4% of babies are born following Assisted Reproductive Technologies (ART). Several ART techniques are used and these differentially affect the sex ratio of offspring successfully produced. These direct effects on sex ratio also have the potential to influence, indirectly, the sex ratios of offspring born to untreated couples. This is of concern because human sex ratio bias may adversely affect public health. Here the extent of indirect effects of ART that could operate, via Fisherian frequency-dependent natural selection, on the progeny sex ratio of unassisted members of a population is heuristically modelled. Given the degrees to which ART techniques bias sex ratios directly, it is predicted that well over 20% of couples would have to reproduce via ART for there to be any discernible effect on the sex ratios produced, in response, by the remainder of the population. This value is greater than the estimated prevalence of infertility problems among human couples. It is concluded that providing ART to couples with fertility problems does not currently generate significant ethical issues or public health concern in terms of indirect effects on the offspring sex ratios of untreated couples

Bio-electrosprayed human sperm remain viable

© 2019 Elsevier Ltd In our previous investigations, we demonstrated that certain living cells exposed to bio-electrosprays remained viable, and behaved as expected in comparison to control cells. These studies also extended to post-bio-electrosprayed cells being transplanted into mice, which demonstrated no rejection, and in fact they were seen to integrate with the surrounding host tissues. Therefore, highlighting bio-electrosprays as a front running bioplatform for engineering functional tissues for repair, replacement and rejuvenation of damaged and/ageing tissues. In the present studies, we take bio-electrosprays further into human health, investigating the possibility of this platform biotechnology to directly handle the smallest and most highly specialized cell in the human body, the spermatozoon. These studies demonstrated the ability for bio-electrosprays to directly handle human sperm without compromising their viability, while also demonstrating the technology's capacity to encapsulate human sperm. These investigations reported herein present interesting implications to human reproductive science and medicine, while also having promising applicability to areas such as the agriculture and aquaculture industries

Effects of assisted reproductive technologies on human sex ratio at birth

ObjectiveTo investigate the effect of assisted reproductive technology (ART) treatments on the sex ratio of babies born.DesignAssessment of direct effects of assisted conception through retrospective data analysis on the progeny sex ratio of treated women in the United Kingdom.SettingThe study uses the anonymized register of the Human Fertilisation and Embryology Authority.Patient(s)A total of 106,066 babies of known gender born to 76,994 treated mothers and 85,511 treatment cycles between 2000 and 2010 in the United Kingdom.Intervention(s)Intrauterine insemination, IVF, or intracytoplasmic sperm injection (ICSI).Main Outcome Measure(s)Sex ratio of babies born.Result(s)Intrauterine insemination, IVF, and ICSI lead to different sex ratios, highest after IVF (proportion male = mean 0.521 ± confidence interval 0.0056) and lowest under ICSI embryo transfer (0.493 ± 0.0031). In addition, for both ICSI and IVF, transferring embryos at a later stage (blastocyst) results in approximately 6% more males than after early cleavage-stage ET.Conclusion(s)Because the cumulative number of IVF babies born is increasing significantly in Britain and elsewhere, more research is needed into the causes of gender bias after ART and into the public health impact of such gender bias of offspring born observed on the rest of the population

Regulatory mechanisms for natriuretic peptide signalling in sheep granulosa cells

Natriuretic peptides (NPs) have been reported to have critical roles in follicular development and oocyte maturation in rodents. This study aimed to extend our current understanding of NP-mediated signalling pathways and mechanisms of action in the follicles of a monovulatory species. Ovine granulosa cells (GCs) and theca cells (TCs) were cultured under conditions designed to allow gonadotrophin-stimulated cell differentiation. Gene expression analysis was performed by qualitative (q)PCR for NPs and NPRs (between 16 and 96 h of culture) and VEGF120 and VEGF164 (between 16 and 144 h of culture). A qualitative analysis of the production of NP/NPR family members and NP ligand/receptor associations was carried out utilising a highly sensitive immunological approach known as ‘proximity ligation assay’ (PLA). All NPRs were observed in GCs, while NPRA was absent in TCs. In GCs, gene expression of NPRA, NPRB and NPRC was apparent but only active BNP and CNP and not ANP, were detected. Also in GCs, ANP but not CNP was able to significantly (P [less than] 0.05) reduce oestradiol and increase (P [less than] 0.05) progesterone. Inhibition of VEGF164 by ANP and CNP (P [less than] 0.01) after 48 h of culture preceded up-regulation of VEGF120 by ANP (P [less than] 0.01) after 144 h, but not CNP. Taken together, these findings appear to demonstrate that NP responsiveness in the GC compartment of sheep follicles is multi-facilitated, utilising both autocrine and paracrine stimulation pathways