Plasma-derived from human umbilical cord blood restores ovarian function and improves serum reproductive hormones levels in mice with premature ovarian insufficiency (POI) through cytokines and growth factors.

Premature ovarian insufficiency (POI) patients experience a decline in ovarian function and a reduction in serum reproductive hormones, leading to a significant impact on the outcomes of assisted reproductive technology. Despite the absence of an effective clinical treatment to restore fertility in POI patients, recent research has indicated that cord blood plasma (CBP) derived from human umbilical cord blood (hUCB) may offer therapeutic benefits for various degenerative diseases. The primary aim of this study is to explore approaches for enhancing ovarian function and serum reproductive hormones through the administration of CBP in a murine model. Initially, hUCB was utilized to obtain CBP (CBP), which was subsequently analyzed for cytokine and growth factor profiles in comparison to adult blood plasma (ABP) by use of flow cytometry. Subsequently, POI mouse models were established through the induction of 4-vinylcyclohexene diepoxide, followed by the injection of CBP into the tail. At 7, 14, and 21 days posttreatment, mouse ovaries and blood were collected, and their estrus cycle, body weight, and ovarian weights were evaluated using precise electronic balance. Finally, ovarian morphology and follicle number were assessed through HE staining, while serum levels of anti-Müllerian hormone (AMH), estradiol (E2) and follicle-stimulating hormone (FSH) were determined by ELISA. Our study revealed that individuals with CBP exhibited significantly lower concentrations of proinflammatory cytokines, including IL-ß (p < 0.01) and IL-2 (p < 0.05), while displaying elevated levels of anti-inflammatory cytokines and chemokines, such as IL-2, IL-4, IL-6, IL-8, IL-12P70, IL-17A, IP-10, interferon-γ, and tumor necrosis factor-α (p < 0.01). Furthermore, CBP demonstrated remarkably higher levels of growth factors, including transforming growth factor-ß1, vascular endothelial growth factor, and insulin-like growth factor-1 (p < 0.01) than ABP. Notably, our investigation also revealed that CBP restored the content of serum reproductive hormones, such as AMH, E2, and FSH (p < 0.05), and increased the number of primordial and primary follicles (p < 0.01) and decreased the number of luteal and atretic follicles (p < 0.01) in vivo. Our findings suggested that CBP-secreted cytokines and growth factors could be restored POI ovarian function, enhanced serum reproductive hormones and rescued follicular development in vivo. These findings further support the potential of CBP as a promising strategy in clinical applications for POI related infertility.

Effects of sericin on oxidative stress and PI3K/AKT/mTOR signal pathway in cryopreserved mice ovarian tissue.

Ovarian tissue cryopreservation is an effective fertility protective strategy for preadolescent female cancer patients, whose tumor treatment cannot be delayed. In the present study, the effects of sericin, as an antioxidant, on mice ovarian tissue freezing and thawing were investigated. Mice ovarian tissues were cryopreserved and thawed in medium containing 0.5% or 1%sericin (w/v), and 0.1 mM melatonin. Then, the follicular morphology was observed. The levels of antioxidant enzymes were determined, including glutathione (GSH), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) and catalase (CAT). Moreover, the levels of nitric oxide (NO), malondialdehyde (MDA) and lactate dehydrogenase (LDH) were also tested. Besides, apoptosis-related proteins Bcl-2 and Bax were determined. Our results showed that 1% sericin maintained follicular morphology, inhibited apoptosis, decreased MDA and NO levels, and boosted endogenous antioxidant enzyme levels, while had no significant effect on LDH levels. Furthermore, these effects may be related with the activation of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of Rapamycin (mTOR) signaling pathway, as demonstrated by increased PI3K, p-AKT and mTOR levels. These findings demonstrate that 1% sericin may reduce oxidative stress and protect ovarian tissues during freezing and thawing via PI3K/AKT/mTOR signaling pathway.

The status and comparison of ovarian reserve between fertile and infertile healthy Chinese women of reproductive age.

ABSTRACT: We aimed to investigate ovarian reserve status, and explore differences in ovarian reserve between fertile and infertile healthy Chinese women of reproductive age.We recruited 442 fertile women aged 23 to 49 years (mean: 35.22 ±â€Š4.91 years) as subjects, and 196 infertile women aged 23 to 46 years (mean: 32.34 ±â€Š4.34 years) as controls. For all participants, a number of parameters were tested on days 2 to 4 of a spontaneous cycle, including basal serum follicle-stimulating hormone (FSH), estradiol (E2), luteinizing hormone (LH), total testosterone, anti-Müllerian hormone (AMH), ovarian response prediction index (ORPI), and antral follicle count (AFC).There were significant differences in terms of AFC, serum AMH levels, and ORPI among subject subgroups (10.58 ±â€Š5.80; 2.533 ±â€Š2.146 ng/mL; 1.28 ±â€Š1.87; respectively), and among control subgroups (12.44 ±â€Š5.69; 3.189 ±â€Š2.551 ng/mL; 1.88 ±â€Š2.68; respectively) (P < .01 for all). For both subjects and controls, AFC, AMH levels, and ORPI decreased gradually with increasing age, and presented with similar age-related trends; there were positive correlations between AMH and AFC (P < .001), and negative correlations between age and AFC, AMH, ORPI (P < .05 for all). There was a significant difference in age (P < .001), serum E2 (P < .01), and AMH (P < .01) levels between subjects and controls; however, when controlling for confounding factors (age, body mass index, total testosterone, and LH), we found no differences between the 2 groups with regards to the serum levels of AMH, FSH, E2, and AFC (P > .05 for all). Moreover, receiver operating characteristic curve analysis indicated that the significant variables of subjects and controls for evaluating ovarian reserve included age, AMH and ORPI, and ORPI was more valuable than other variables.A diminished ovarian reserve was one of the manifestations caused by female aging. When confounding factors were controlled for, we found no differences in ovarian reserve when compared between fertile and infertile women, and no correlation with infertility.

Melatonin Inhibits Oxidative Stress and Apoptosis in Cryopreserved Ovarian Tissues via Nrf2/HO-1 Signaling Pathway.

In the field of assisted reproductive technology, female fertility preservation, particularly ovarian tissue cryopreservation in adolescent cancer patients, has attracted much attention. Melatonin (MLT) is well known for its antioxidative and anti-apoptotic properties; however, whether it can ameliorate the cryoinjury and inhibit the generation of reactive oxygen species (ROS) in cryopreserved ovarian tissues (OTs) has not yet been reported. Here, we demonstrated that MLT could protect follicular integrity; prevent cell apoptosis; decrease ROS, malondialdehyde (MDA), and nitric oxide (NO) levels; and increase activities of glutathione peroxidases (GSH-Px), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) in cryopreserved OTs. Furthermore, these effects may be related with the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, as evidenced by increased mRNA levels of Nrf2 downstream genes, including heme oxygenase-1 (HO-1), glutathione S-transferase M1 (GSTM1), SOD, and CAT. In summary, MLT can not only directly scavenge ROS but also significantly induce the activation of antioxidative enzymes via the Nrf2 signaling pathway, which is a new mechanism underlying the protection effects of MLT on cryopreserved OTs.

Antioxidative effect of melatonin on cryopreserved ovarian tissue in mice.

Cryopreservation of ovarian tissues (OTs) has become the most effective way to preserve the fertility of female cancer patients. However, cryopreservation of OTs is still relatively at an experimental stage. The aim of study is to examine the effect of melatonin (MTL) on cryopreserved-thawed OTs. Fragments of OTs were cryopreserved in medium containing different concentrations (0 mM, 0.001 mM, 0.01 mM, 0.1 mM and 1 mM) of MLT. The endogenous enzymes (GSH-PX, GSH, SOD, CAT and T-AOC), MDA and ROS levels were all evaluated after cryopreservation. Our results showed that the 0.1 mM of MLT significantly improved the survival and diameter of follicles (P < 0.001). Meanwhile, the antioxidant enzymes activities (including GSH-PX, GSH, SOD, CAT and T-AOC) were enhanced and MDA content were significantly decreased in 0.1 mM of MLT group compared to other groups (P < 0.001). Additionally, compared to the control group, MTL of 0.1 mM resulted in a significantly lower ROS level. In conclusion, MLT protects the quality of cryopreserved OTs by decreasing oxidative stress level and the optimal concentration is 0.1 mM.

Melatonin Reduces Androgen Production and Upregulates Heme Oxygenase-1 Expression in Granulosa Cells from PCOS Patients with Hypoestrogenia and Hyperandrogenia.

BACKGROUND/AIMS: Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by abnormal hormone levels in peripheral blood and poor-quality oocytes. PCOS is a pathophysiological syndrome caused by chronic inflammation and oxidative stress. The aim of this study was to investigate the mechanism of melatonin regulation on androgen production and antioxidative damage in granulosa cells from PCOS patients with hypoestrogenia and hyperandrogenia. METHODS: Cumulus-oocyte complexes were collected from PCOS patients who had low levels of estrogen in follicular fluids. RESULTS: Melatonin triggered upregulation of cytochrome P450 family 19 subfamily A member 1 (CYP19A1) expression via the extracellular signal-regulated kinase pathway in luteinized granulosa cells. As a result, conversion of androgen to 17ß-estradiol was accelerated. We also found that melatonin significantly reduced the levels of inducible nitric oxide (NO) synthetase and NO in luteinized granulosa cells. Levels of transcripts encoding NF-E2-related factor-2 and its downstream target heme oxygenase-1 were also increased, leading to anti-inflammatory and antioxidant effects. We also found that melatonin could improve oocyte development potential. CONCLUSION: Our preliminary results showed that melatonin had a positive impact on oocyte quality in PCOS patients with hypoestrogenia and hyperandrogenia.

Melatonin reduces oxidative damage and upregulates heat shock protein 90 expression in cryopreserved human semen.

Sperm cells can be damaged during the semen cryopreservation process, decreasing their fertilizing ability. Physical damage and oxidative stress may occur during the freeze-thawing process. Antioxidants such as the native antioxidant melatonin can potentially improve cryopreservation outcomes. In this study, we added melatonin to cryoprotectant to examine its effect on frozen-thawed human sperm. We found that adding 0.1mM melatonin to cryoprotectant significantly increased sperm viability (24.80 ± 0.46% vs. 20.97 ± 1.27%, P < 0.05) and membrane integrity (P < 0.05), and decreased intracellular reactive oxygen species and lipid peroxidation damage. Furthermore, mRNA levels of the transcription factor NF-E2-related factor-2 and its downstream genes were significantly increased. Resistance to oxidative stress was enhanced and expression of the antiapoptotic gene Bcl-2 was increased by inclusion of 0.1mM melatonin in the cryoprotectant. Moreover, 0.1mM melatonin upregulated the expression of heat shock protein 90 (HSP90), which confers resistance to stressors in frozen-thawed sperm. Results obtained upon addition of inhibitors of melatonin receptors (luzindole and 4-P-PDOT) and an HSP90 inhibitor (geldanamycin) in the cryoprotectant demonstrated that melatonin promoted HSP90 translation via the melatonin receptor MT1 and increased adenosine triphosphate levels, thus increasing the viability of thawed sperm.

Merotelic kinetochore attachment in oocyte meiosis II causes sister chromatids segregation errors in aged mice.

Mammalian oocyte chromosomes undergo 2 meiotic divisions to generate haploid gametes. The frequency of chromosome segregation errors during meiosis I increase with age. However, little attention has been paid to the question of how aging affects sister chromatid segregation during oocyte meiosis II. More importantly, how aneuploid metaphase II (MII) oocytes from aged mice evade the spindle assembly checkpoint (SAC) mechanism to complete later meiosis II to form aneuploid embryos remains unknown. Here, we report that MII oocytes from naturally aged mice exhibited substantial errors in chromosome arrangement and configuration compared with young MII oocytes. Interestingly, these errors in aged oocytes had no impact on anaphase II onset and completion as well as 2-cell formation after parthenogenetic activation. Further study found that merotelic kinetochore attachment occurred more frequently and could stabilize the kinetochore-microtubule interaction to ensure SAC inactivation and anaphase II onset in aged MII oocytes. This orientation could persist largely during anaphase II in aged oocytes, leading to severe chromosome lagging and trailing as well as delay of anaphase II completion. Therefore, merotelic kinetochore attachment in oocyte meiosis II exacerbates age-related genetic instability and is a key source of age-dependent embryo aneuploidy and dysplasia.

[Nuclear matrix proteins differentially expressed in human prostate cancer cell lines and benign prostatic hyperplasia epithelial cell line].

OBJECTIVE: To compare the expression of nuclear matrix proteins (NMPs) in benign prostatic hyperplasia (BPH) epithelial cell line BPH-1 versus those in androgen-dependent human prostate cancer cell line LNCap and androgen-independent prostate cancer cell line PC-3. METHODS: We isolated NMPs from the BPH-1, LNCap and PC-3 cell lines by 2-dimensional electrophoresis (2-DE), analyzed the differentially expressed proteins by matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS), and identified them by peptide mass fingerprint and database searching. RESULTS: We successfully obtained well-resolved reproducible 2-DE patterns of NMPs in human prostate cancer cell lines, identified 12 differentially expressed NMPs including enzymes, regulatory proteins, RNA-binding protein and various other factors, 3 up-regulated and 9 down-regulated in prostate cancer cell lines. CONCLUSION: There are obvious differences in the expressions of NMPs between human prostate cancer cell lines and benign prostatic hyperplasia epithelial cell line.