Melatonin prevents cyclophosphamide-induced primordial follicle loss by inhibiting ovarian granulosa cell apoptosis and maintaining AMH expression.

Cyclophosphaty -45mide (Cyc) chemotherapy in young female cancer patients is associated with an increased risk of premature ovarian insufficiency (POI). This study was designed to investigate the protective role of melatonin (Mel) as an adjuvant against Cyc-induced POI. Female mice received a single intraperitoneal (i.p.) dose of Cyc (75 mg/kg). Mel protection was achieved in mice after i.p. injection of melatonin (50 mg/kg) every 24 h for four consecutive days prior to chemotherapy initiation and for 14 additional days. Ovarian reserve testing, hormonal assays for follicle-stimulating hormone, luteinizing hormone, and anti-Müllerian hormone (AMH), assessment of the oxidative stress status, and measurement of the relative expression of genes in PTEN/AKT/FOXO3a and mitochondrial apoptosis pathways were performed. The results showed that treatment with 50 mg/kg Mel significantly prevented Cyc-induced over-activation of primordial follicles by maintaining the plasma level of AMH and subsequently preventing litter size reduction in mice treated with Cyc chemotherapy. Importantly, Mel treatment significantly prevented ovarian granulosa cell loss by inhibiting the mitochondrial apoptotic pathway. Identifying the protective actions of Mel against Cyc-induced primordial follicle loss has important implications for fertility maintenance in young cancer patients undergoing chemotherapy.

Melatonin Improves Quality of Repeated-Poor and Frozen-Thawed Embryos in Human, a Prospective Clinical Trial.

Objective: In this study, two experiments were performed to assess the effect and the role of melatonin on human in vitro embryo quality. Methods: Experiment I: A total of 42 repeated-poor-quality-embryo patients were enrolled, with a total of 181 oocytes retrieval cycles. After IVF, for the same patient, the MT cycles group (10-7 M melatonin added to the culture medium; n=48) were compared with the previous non-MT cycles group (n=133), following by in vitro culture to blastocyst stage and embryo transfer. 31 patients were transplanted with 65 embryo transfer, including 24 MT embryo transfer, 41 non-MT embryo transfer. Cycle outcomes were compared between the two groups. Experiment II:A total of 143 supernumerary human cleavage-stage embryos (from non-repeated-poor-quality-embryo patients) vitrified on Day 3 after IVF were warmed and randomized into two groups: melatonin group (10-7 M melatonin added to the culture medium; n=71) and control group (n=72), and then cultured for 72 h. Rate of blastocyst and high-quality blastocyst, reactive oxygen species (ROS) levels of culture media as well as embryonic GPX1, CAT, Mn-SOD, Cu/Zn-SOD, BCL-2, BAX gene expression levels were analyzed. Results: Experiment I: Results showed that the rate of Day 3 high-quality embryos (29.6% vs.19.5%) in the MT cycles group was significantly higher than that in the non-MT cycles group (P<0.05). The rate of available blastocysts (17.1% vs.12.7%) and clinical pregnancy rate (25.0% vs.17.1%) were in tendency higher in the group treated with melatonin (P>0.05). Experiment II:Results showed that the blastocyst rates in the melatonin administered group were significantly higher than in control group (42.25% vs.26.38%, P<0.05). There were no significant differences in high-quality blastocyst rates. In addition, quantitative PCR showed that the expression of CAT was significantly upregulated by melatonin treatment (P<0.05), while there were no significant differences in the expression of GPX1, Mn-SOD, Cu/Zn-SOD, BAX and BCL-2 gene as well as the levels of ROS. Conclusion: These data showed that melatonin supplement in the culture medium will improve Day 3 high-quality embryos rate of repeated-poor-quality-embryo patients and improve blastocyst rate of vitrified-warmed cleavage-stage embryos, suggesting that melatonin intervention may provide a potential rescue strategy for IVF failures. Clinical Trial Registration: identifier [ChiCTR2200059773].

Aanat Knockdown and Melatonin Supplementation in Embryo Development: Involvement of Mitochondrial Function and DNA Methylation.

Aims: In addition to pineal gland, many cells, tissues, and organs also synthesize melatonin (N-acetyl-5-methoxytryptamine). Embryos are a group of special cells and whether they can synthesize melatonin is still an open question. However, melatonin application promoted embryo development in many species in in vitro condition. The purpose of this study was to investigate whether embryos can synthesize melatonin; if it is so, what are the impacts of the endogenously produced melatonin on embryo development and the associated molecular mechanisms. These have never been reported previously. Results: Melatonin synthesis was observed at different stages of embryonic development. Aanat (aralkylamine N-acetyltransferase), a rate-limiting enzyme for melatonin production, was found to mostly localize in the mitochondria. Aanat knockdown significantly impeded embryonic development, and melatonin supplementation rescued it. The potential mechanisms might be that melatonin preserved mitochondrial intact and its function, thus providing sufficient adenosine 5'-triphosphate for the embryo development. In addition, melatonin scavenged intracellular reactive oxygen species (ROS) and reduced the DNA mutation induced by oxidative stress. In the molecular level, Aanat knockdown reduced tet methylcytosine dioxygenase 2 (Tet2) expression and DNA demethylation in blastocyst and melatonin supplementation rescued these processes. Innovation: This is the first report to show that embryos synthesize melatonin, and its synthetic enzyme Aanat was located in the mitochondria of embryos. An effect of melatonin is to maintain Tet2 expression and normal methylation status, and thereby promote embryonic development. Conclusion: Embryos can produce melatonin that reduces ROS production, preserves mitochondrial function, and maintains Tet2 expression and the normal DNA methylation.

Melatonin promotes development of haploid germ cells from early developing spermatogenic cells of Suffolk sheep under in vitro condition.

Promotion of spermatogonial stem cell (SSC) differentiation into functional sperms under in vitro conditions is a great challenge for reproductive physiologists. In this study, we observed that melatonin (10(-7) M) supplementation significantly enhanced the cultured SSCs differentiation into haploid germ cells. This was confirmed by the expression of sperm special protein, acrosin. The rate of SSCs differentiation into sperm with melatonin supplementation was 11.85 ± 0.93% which was twofold higher than that in the control. The level of testosterone, the transcriptions of luteinizing hormone receptor (LHR), and the steroidogenic acute regulatory protein (StAR) were upregulated with melatonin treatment. At the early stage of SSCs culture, melatonin suppressed the level of cAMP, while at the later stage, it promoted cAMP production. The similar pattern was observed in testosterone content. Expressions for marker genes of meiosis anaphase, Dnmt3a, and Bcl-2 were upregulated by melatonin. In contrast, Bax expression was downregulated. Importantly, the in vitro-generated sperms were functional and they were capable to fertilize oocytes. These fertilized oocytes have successfully developed to the blastula stage.