Shu Hashimoto

In brief: We describe how NAD+ precursors in embryo culture media promote embryonic development into blastocysts by enhancing NAD+ level in embryos. Nicotinamide adenine dinucleotide (NAD+) is an important regulator of cellular physiological processes. However, in the culture environment, NAD+ levels are predicted to decrease below physiological requirements. Therefore, we examined the effects of the NAD+ precursor nicotinamide mononucleotide (NMN) on NAD+ levels and viability of mammalian fertilized ova. NAD+ levels decreased during in-vitro culture, but this decrease was alleviated by NMN. Nicotinamide mononucleotide also increased the blastocyst formation rate and accelerated the developmental rate. Transcriptome analysis showed that NMN induced significant changes in the expression of genes involved in mitochondrial function in morula, and that NMN increased mitochondrial membrane potential, oxygen consumption rate and mitochondrial DNA copy number of morula while reducing reactive oxygen species levels. The addition of NMN to the embryo culture medium increased NAD+ levels in embryos, boosted mitochondrial function at the morula stage, and enhanced their ability to develop into blastocysts.

We examined the effect of niacin, a water-soluble vitamin essential for the metabolism of carbohydrates, lipids, and other substrates, on the developmental potential of oocytes grown in vitro, as well as the number and activity of mitochondria and reduced glutathione (GSH) content in oocytes. Porcine oocytes were cultured in medium containing 0–10 mM niacin for 12 days. Subsequently, in vitro maturation was performed, followed by in vitro fertilization using intracytoplasmic sperm injection, and the ability to develop into blastocysts was examined. Niacin treatment (10 mM) increased the mitochondrial, GSH, and glutamate–cysteine ligase catalytic subunit contents in porcine oocytes grown in vitro. Furthermore, niacin treatment enhanced male pronuclear formation and blastocyst development rates in porcine oocytes grown in vitro. Niacin increased the mitochondrial content and GSH levels in porcine oocytes and enhanced the ability of oocytes to form male pronuclei, which in turn promoted embryonic development.

In brief: Mammalian zygotes often fail to unify their genomes. This study showed that the position of the centrosome is crucial for regulation of pronuclear apposition and genome unification. After the sperm enters the ovum, male and female pronuclei carrying the genetic information of both parents are formed. These pronuclei migrate to the center of the ovum, followed by genome unification upon mitosis onset. However, the genomes of some gametes fail to unify. The mechanism by which parental genetic information fails to unify is not yet fully understood. Here, we used immunofluorescence staining and live imaging techniques to study pronuclear migration in fertilized bovine ova. Microtubules play a more important role in pronuclear migration than actin. Centrosomal microtubules regulate the localization of motor dynein bound to the pronuclear membrane. The presence of at least one centrosome between the male and female pronuclei is necessary for apposition of the two pronuclei and genome unification. Our data highlight the importance of centrosome positioning in the apposition of pronuclei and parental genome unification.

Nicotinamide adenine dinucleotide (NAD(H)) and its metabolites function as crucial regulators of physiological processes, allowing cells to adapt to environmental changes such as nutritional deficiencies, genotoxic factors, disruptions in circadian rhythms, infections, inflammation, and exogenous substances. Here, we investigated whether elevated NAD(H) levels in oocytes enhance their quality and improve developmental competence following in vitro fertilization (IVF). Bovine cumulus-oocyte complexes (COCs) were matured in a culture medium supplemented with 0–100 μM nicotinamide mononucleotide (NMN), a precursor of NAD(H). The addition of NMN caused an increase in intracellular NAD(H) and nicotinamide adenine dinucleotide phosphate levels, leading to enhanced competence for development to the blastocyst stage after IVF. The increase in intracellular NAD(H) levels led to changes in the expression of mitochondria function-related genes. As a result, NMN supplementation increased the ratio of MitoTracker Orange fluorescence to nonyl acridine orange fluorescence, as well as adenosine triphosphate levels, while decreasing reactive oxygen species levels in the oocytes. NMN also lowered chromosome lagging during anaphase. These results suggest that increased NAD(H) levels in oocytes following NMN treatment enhances post-fertilization developmental competence through improved mitochondrial function.

Purpose In our previous study, we confirmed that the supplementation of vitrified-warmed murine oocytes with autologous adipose stem cell (ASC)-derived mitochondria during intracytoplasmic sperm injection enhances post-fertilization developmental competence in mice. To ensure the safety of this technology, we conducted a thorough study in mice to investigate the potential presence of specific malformations in offspring developed from this approach. Methods A transgenerational comparative analysis was conducted on founder mice from embryos that developed after mitochondrial supplementation, and two subsequent generations. Reproductive performance, body growth rate, histopathological parameters, hematological parameters, daily activity patterns, and daily body temperature changes in male and female mice across these three generations were assessed in comparison to wild-type mice of the same age. Results Both male and female animals in all three generations showed comparable reproductive performance to the control group. Additionally, body growth rate by the age of 8 weeks were found to be comparable to controls across all three generations. Notably, no significant histopathological abnormalities were detected in vital organs, including the brain, heart, liver, kidneys, lungs, ovaries, and testes, in any individuals from the studied cohorts. The blood parameters were consistent with the control data. The continuous monitoring of activity and body temperature changes (both day and night) over a 1-week period revealed a pattern closely resembling that observed in the control animals. Conclusion Injection of ASC-mitochondria into oocytes may be a promising technique to support developmental potential without causing adverse epigenetic events in the offspring in mice. However, before considering clinical application, additional safety screening using larger animals or non-human primates is essential.

Endometriomas (chocolate cysts) are cystic lesions that can develop on ovaries, and are characterized by the presence of ectopic endometrial tissue or similar tissue. Such lesions can cause a decline in the number and quality of oocytes, and lead to implantation failure. In this study, we retrospectively assessed the efficacy of repeated endometrioma aspiration and dienogest combination therapy in patients suffering endometriosis-associated infertility with endometriomas. A comparison was made between a treated group that underwent combination therapy followed by controlled ovarian hyperstimulation (COH) (n = 30) and a control group that did not undergo treatment (n = 40), at the IVF Osaka Clinic from September 2019 to September 2021. There were no differences in patient background between the two groups. A reduction in endometrioma size continued for 12 months after treatment. The numbers of follicles that developed to 15 mm or greater in size following COH and mature oocytes were significantly lower in the treated group compared to those in the control group. The levels of inflammatory cytokines in the follicular fluid significantly decreased in the treated group (p < 0.05). In patients in the treated group who underwent a second ova retrieval, the results were compared between those in the first ova retrieval (immediately after the end of treatment) and those in the second ova retrieval (four months after the first retrieval). The numbers of follicles following COH, retrieved, mature and fertilized ova were significantly increased in the second ova retrieval.

One of the most critical issues to be solved in reproductive medicine is the treatment of patients with multiple failures of assisted reproductive treatment caused by low-quality embryos. This study investigated whether mitochondrial transfer to human oocytes improves embryo quality and provides subsequent acceptable clinical results and normality to children born due to the use of this technology. We transferred autologous mitochondria extracted from oogonia stem cells to mature oocytes with sperm at the time of intracytoplasmic sperm injection in 52 patients with recurrent failures (average 5.3 times). We assessed embryo quality using the following three methods: good-quality embryo rates, transferable embryo rates, and a novel embryo-scoring system (embryo quality score; EQS) in 33 patients who meet the preset inclusion criteria for analysis. We also evaluated the clinical outcomes of the in vitro fertilization and development of children born using this technology and compared the mtDNA sequences of the children and their mothers. The good-quality embryo rates, transferable embryo rates, and EQS significantly increased after mitochondrial transfer and resulted in 13 babies born in normal conditions. The mtDNA sequences were almost identical to the respective maternal sequences at the 83 major sites examined. Mitochondrial transfer into human oocytes is an effective clinical option to enhance embryo quality in recurrent in vitro fertilization-failure cases.

Purpose: The MiOXSYS system is a new technique to analyze the semen oxidative reduction potential (ORP) that may use to classify the level of sperm DNA integrity. It does not clearly explain how the semen ORP values could help to change the IVF outcomes. We have analyzed correlations between semen ORP value and the IVF results.Methods: Four hundred and thirty couples were enrolled. The male counterparts were divided into two groups according to their semen ORP values and compared the fertilization rate, cell cleavage rate, and embryo quality, following the IVF procedures. The relations between ORP values and the clinical pregnancy, live birth, and abortion rates were analyzed.Results: The ORP values show negative and positive correlations with some conventional semen parameters. The fertilization and the cleavage rate did not show any differences in those two groups, but the transferable embryo rate was significantly high in patients with high semen ORP. However, the patients with high ORP show a tendency to lower clinical pregnancy with a low abortion rate compared to the low ORP group.Conclusion: The main purpose of measuring the ORP value in semen is still questionable and shows controversial results.

Background: Female fertility declines with age, due to increased chromosomal aneuploidy and possible reduced mitochondrial function in the embryo.Methods: This review outlines how mitochondrial function in human embryos, as predicted from oxygen consumption rate (OCR) measurements, changes in preimplantation stage, and what factors, particularly maternal age, affect mitochondrial function in embryos.Main findings: The structure of the mitochondrial inner membrane and its respiratory function developed with embryo development, while the copy number of mitochondrial DNA per specimen was transiently reduced compared with that of the oocyte. The undifferentiated state of the inner cell mass cells appears to be associated with a low OCR. In contrast, the copy number of mitochondrial DNA increased in trophoblast cells and mitochondrial aerobic metabolism increased.The OCRs at morulae stage decreased with maternal age, but there was no relationship between maternal age and the copy number of mitochondrial DNA at any stages. The higher oxygen spent at the morula stage; the shorter time was needed for development to the mid‐stage blastocyst.Conclusions: The mitochondrial respiratory function of human embryos developed along with embryonic growth. Mitochondrial function at morula stage declined with their maternal age and reduced mitochondrial function decreased the rate of development from morula to blastocyst.

Purpose The latest treatments do not sufficiently prevent miscarriage and fetal growth restriction (FGR) in pregnant women. Here, we assessed the effects of a human protein, CTRP6, that specifically inhibits the activation of the alternative complement pathway on miscarriage, fetal and placental development. Methods Pregnant CBA/J mice mated with DBA/2 male mice as a model of spontaneous abortion and FGR were randomly divided into the control and CTRP6 groups. In the CTRP6 group, the mice were intravenously administered CTRP6 on days 4.5 and 6.5 post-conception (dpc). The abortion rate and fetal and placental weights on 14.5 dpc were examined. Remodeling of the spiral artery was also assessed. Results The abortion rate in the CTRP6 group (13%) was reduced compared to the control group (21%), but there was no statistical difference. The placental and fetal weights in the CTRP6 group were also heavier than those in the control (P < 0.05). Moreover, the thickness of the blood vessel wall in the CTRP6 group was significantly thinner than that in the control (P < 0.05) and comparable to that in the non-abortion model (CBA/J x BALB). The ratio of the inner-per-the-outer diameter of the spiral artery increased more in the CTRP6 group than that in the control (P < 0.05). As well, the Th1/Th2 cytokine ratio was significantly reduced by CTRP6 treatment. Conclusions Taken together, the supplementation with a protein that regulates the alternative complement pathway in vivo improves FGR and promotes spiral artery remodeling in a mouse model of miscarriage and FGR.