Tannin Supplementation Improves Oocyte Cytoplasmic Maturation and Subsequent Embryo Development in Pigs

To investigate the effects of tannins (TA) on porcine oocyte in vitro maturation (IVM), different concentrations of TA (0, 1, 10 and 100 μg/mL) were supplemented with a maturation medium and the COCs and subsequent embryonic development were examined. The results showed that 10 µg/mL TA significantly improved the cumulus expansion index (CEI), cumulus-expansion-related genes (PTGS1, PTGS2, PTX-3, TNFAIP6 and HAS2) expression and blastocyst formation rates after parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) compared to the control groups, but not oocyte nuclear maturation. Nevertheless, 10 µg/mL TA dramatically enhanced the mRNA expression of oocyte-development-related genes (BMP15, GDF9, CDC2 and CYCLIN B1), GSH, ATP, SOD1, PGC1α, BMP15, GDF9 and CDC2 levels and reduced intracellular ROS level in porcine oocytes. These results indicated that porcine oocyte cytoplasmic maturation was improved by 10 µg/mL TA treatment during IVM. In contrast, a high concentration of TA (100 μg/mL) significantly decreased the CEI and PTGS1, PTGS2, PTX-3 and HAS2 mRNA expressions in cumulus cells, and reduced oocyte nuclear maturation and the total cell numbers/blastocyst. In general, these data showed that 10 μg/mL TA supplementation has beneficial effects on oocyte cytoplasmic maturation and subsequent embryonic development in pigs.

P–192 Efficacy of postponement of intracytoplasmic sperm injection timing after spindle visualization for Metaphase I oocytes

Study question Does postponement of intracytoplasmic sperm injection (ICSI) timing after spindle visualization for Metaphase I (MI) oocytes improve developmental outcomes of embryos? Summary answer Postponement of ICSI timing after spindle visualization for MI oocytes improves blastocyst utility rates. What is known already Immature oocytes are generally considered poor developmental outcomes. Meanwhile, the timing of ICSI adjusted by using spindle visualization can improve clinically utilized embryos and live birth rates, but these outcomes remain inferior to those of mature oocytes. In in vitro maturation culture, nuclear maturation is thought to occur before the completion of cytoplasmic maturation, and in immature oocytes, synchronization of nuclear and cytoplasmic maturation may be insufficient for ICSI immediately after spindle visualization. Study design, size, duration Data for this retrospective cohort study were obtained 672 oocytes retrieved under mild stimulation cycles using letrozole, in patients aged younger than 39 years between April 2017 and October 2020.Written informed consent was obtained from all patients. This study was approved by the institutional review board. Participants/materials, setting, methods As a control group, 464 MetaphaseIIoocytes that underwent ICSI immediately after visualization of the spindle were used. In group A, 103 MI oocytes underwent ICSI immediately after the first polar body release and spindle visualization, and in group B, 105 oocytes underwent ICSI 2–3 hours after spindle visualization. The primary outcomes were fertilization rates, degeneration, cleavage, embryo blastocyst formation, and utility rates. Outcomes were compared among the three groups. Main results and the role of chance The baseline fertilization rates of each group (control, A, B) were 82.3% (382/464), 73.8% (76/103), and 83.8% (88/105), respectively. The rate was significantly lower in group A than in the control group (P < 0.05), and also tended to be lower in group A than in group B, although the difference was not significant. There was no significant difference in abnormal fertilization rates, oocyte degeneration rates, cleavage rates, and blastocyst formation rates among the three groups. [control, A, B: abnormal fertilization rate: 4.3% (20/464), 8.7% (9/103), 4.8% (5/105); oocyte degeneration rates: 3.0% (14/464), 1.9% (2/103), 3.8% (4/105); cleavage rates: 95.6% (307/321), 93.8% (61/65), 98.7% (74/75); blastocyst formation rates: 58.6% (177/302), 51.7% (31/60), 55.4% (41/74), respectively]. The blastocyst utility rates of control group and group B were significantly higher than in group A [41.7% (126/302), 45.9% (34/74), 26.7% (16/60), respectively] (P < 0.05). There were no significantly different outcomes between the control group and group B. Limitations, reasons for caution The optimal timing of ICSI for MI oocyte cannot be determined by the presence or absence of spindles. In addition, the postponement duration we set was based on reports which reported on final oocyte maturation, and further investigation is needed to establish the optimal ICSI timing for MI oocytes. Wider implications of the findings: In MI oocytes, postponement of ICSI timing after spindle visualization is essential for synchronization of the nucleus and cytoplasmic maturation. Trial registration number none

L-carnitine Supplementation Enhances Nuclear and Cytoplasmic Maturation Rates of Sheep Oocytes In Vitro

The aim of the present study was to determine the effectiveness of l-carnitine (LC) supplementation on nuclear and cytoplasmic maturation rates of sheep oocytes. In experiment 1, oocytes were maturated for 24 hours in tissue culture medium 199 supplemented with LC at doses of 0.3 mg/mL, 0.6 mg/mL, and 0.9 mg/mL. In experiment 2, oocytes were maturated and fertilized in a media supplemented with LC at a dose of 0.3 mg/mL and incubated with 5x106 sperm/mL for 12 hours. The treatment group consisted of LC supplementation only in maturation medium (P1), only in fertilization medium (P2), and in both maturation and fertilization media (P3). In experiment 3, sperm motility patterns were assessed using CASA after being exposed to fertilization medium supplemented with LC at a dose of 0.3 mg/mL for 0 and 3 hours. Our results showed that supplementation of LC at a dose of 0.3 mg/mL significantly (p<0.05) increased the percentage of oocytes reaching metaphase II (86.7±4.1%) compared to those supplemented with LA at doses of 0, 0.6, and 0.9 mg/mL (73.6±1.2%, 81.4±1.3%, and 70.5±1.6%, respectively). The LC treatment in the fertilization medium only did not influence the number of two pronuclear formations (62.1±2.5%) compared to supplementation either in the maturation medium only (72.0±4.7%) or a combination of both in maturation and fertilization media (68.2±2.7%) (p<0.05). Further results after 3 hours of incubation compared to the control group showed the total motility (24.8±2.04% vs. 17.49±2.37%), progressive motility (14.17±2.03% vs. 6.49±1.64%), and curvilinear velocity (VCL) (119.70±3.73% vs. 71.15±10.59%) (p<0.05) were increased in the fertilization medium containing LC but it did not improve the fertilization rate. It is concluded that supplementation of LC at a dose of 0.3 mg/mL in the maturation medium only could better improve the nuclear and cytoplasmic maturation rates of sheep oocytes.

Lysophosphatidic acid improves oocyte quality during IVM by activating the ERK1/2 pathway in cumulus cells and oocytes

Oocyte IVM technology is an option for fertility preservation in some groups of patients, such as those with polycystic ovary syndrome, patients with ovarian hyperstimulation syndrome, and for patients with cancer. However, the developmental potential of oocytes from IVM still needs to improve. Several previous studies have reported that lysophosphatidic acid (LPA) promotes glucose metabolism, cumulus cell (CC) expansion, and oocyte nuclear maturation. However, the effect of LPA on oocyte cytoplasmic maturation, particularly mitochondrial function, has rarely been studied and the underlying mechanism is largely unknown, which impedes (pre)clinical applications of LPA. In this study, cumulus-oocyte complexes (COCs) and cumulus-denuded germinal vesicle oocytes (DOs) were treated with various concentrations of LPA during IVM, in the presence or absence of the oxidative stressor cyclophosphamide (CTX). In both normal and CTX-damaged COCs, the 25 μM LPA group exhibited improved CC expansion capacity, a higher nuclear maturation rate, and superior mitochondrial function, compared to no LPA treatment. When the concentration of LPA was over 40 μM, detrimental effects of LPA on oocyte maturation occurred. Compared with COCs, the addition of LPA slightly improved oocyte nuclear and cytoplasmic maturation of DOs, but this was not statistically significant. We observed that LPA promotes the activation of ERK1/2, although this was not statistically significant in DOs. Furthermore, LPA could not reverse the negative effect of CC expansion and mitochondrial function after inactivation of ERK1/2 by U0126. RNA-Sequencing and RT-PCR results showed that LPA upregulated several ERK1/2 downstream genes related to CC expansion, such as Areg, Cited4, and Ptgs2. This study demonstrates that LPA improves oocyte quality during IVM through the activation of ERK1/2 pathway CCs and oocytes, which provides evidence for the potential addition of LPA to IVM medium.

Dissecting pathways to thrombocytopenia in a mouse model of visceral leishmaniasis

Visceral leishmaniasis is an important yet neglected parasitic disease caused by infection with Leishmania donovani or L infantum. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulation, and extensive hematological complications. Thrombocytopenia is a dominant hematological feature seen in both humans and experimental models, but the mechanisms behind this infection-driven thrombocytopenia remain poorly understood. Using a murine model of experimental visceral leishmaniasis (EVL), we demonstrated a progressive decrease in platelets from day 14 after infection, culminating in severe thrombocytopenia by day 28. Plasma thrombopoietin (TPO) levels were reduced in infected mice, at least in part because of the alterations in the liver microenvironment associated with granulomatous inflammation. Bone marrow (BM) megakaryocyte cytoplasmic maturation was significantly reduced. In addition to a production deficit, we identified significant increases in platelet clearance. L donovani–infected splenectomized mice were protected from thrombocytopenia compared with sham operated infected mice and had a greater response to exogenous TPO. Furthermore, infection led to higher levels of platelet opsonization and desialylation, both associated with platelet clearance in spleen and liver, respectively. Critically, these changes could be reversed rapidly by drug treatment to reduce parasite load or by administration of TPO agonists. In summary, our findings demonstrate that the mechanisms underpinning thrombocytopenia in EVL are multifactorial and reversible, with no obvious residual damage to the BM microenvironment.

Paraquat Reduces the Female Fertility by Impairing the Oocyte Maturation in Mice

Paraquat (PQ) is a widely used non-selective and oxidizing herbicide in farmland, orchards, flower nursery, and grassland. Overuse of PQ will accumulate in the body and affect the reproduction in mammals. In this study, we found that PQ could reduce the female fertility by oral administration for 21 days in mice. PQ exposure could impair the nuclear maturation by perturbing the spindle assembly and kinetochore–microtubule attachment to cause the misaligned chromosomes during meiosis. In the meantime, PQ exposure disturbed the mitochondrial distribution and enhanced the level of reactive oxygen species and early apoptosis, which thereby deteriorated the early embryo development. Also, PQ administration could cause some changes in epigenetic modifications such as the level of H3K9me2 and H3K27me3. Therefore, PQ administration reduces the female fertility by impairing the nuclear and cytoplasmic maturation of oocytes in mice.

Cyclic nucleotide in oocyte In vitro maturation in Assisted Reproductive Technology

In vitro maturation (IVM) is a promising assisted reproductive technology (ART) for human infertility treatment. However, when cumulus oocyte complexes (COCs) are removed from their follicular environment when manipulated in vitro, it can lead to a decrease of intra-oocyte cyclic adenosine 3’, 5’-monophosphare (cAMP) causing spontaneous nuclear maturation and an asynchrony with the oocytes’ cytoplasmic maturation, resulting in poor embryo developmental outcomes. Nuclear and cytoplasmic synchrony is important during oocyte maturation within antral follicles.It is maintained partially by the actions of c-type natriuretic peptide (CNP) binding with natriuretic peptide receptor 2 (NPR2), supporting high cAMP levels thus holding the oocyte in meiotic arrest. Addition of CNP to pre-IVM media has the capacity of maintaining cAMP levels and thus improve synchrony. Moreover, in women with advanced maternal age, successful IVM of aging oocytes faces significant challenges due to the morphological and cellular changes. Inhibiting initiation of nuclear maturation by cAMP modulator, CNP during pre-IVM period and thus improve oocyte developmental competence regardless of oocyte age.