Impact of Melatonin Supplementation in Women with Unexplained Infertility Undergoing Fertility Treatment.

Unexplained infertility occurs when common causes for a couple's inability to conceive have been excluded. Although origins of idiopathic infertility are still unclear, factors, such as an altered oxidative balance, are believed to be involved. Melatonin is an outstanding antioxidant reportedly present in the follicular fluid (FF), which has been suggested as a useful tool in the management of human fertility. Herein, we observed that intrafollicular concentrations of melatonin were blunted in women with unexplained infertility (UI), which was associated with a marked oxidative imbalance in UI patients' FF. Based on these findings, this randomized pilot study was aimed at assessing whether exogenous melatonin ameliorated oxidative stress and improved in vitro fertilization (IVF) success rates in UI. Thus, 3 mg/day or 6 mg/day of melatonin were given to UI patients for a period spanning from the first appointment to control ovarian stimulation until the day of follicular puncture. Our results indicate that melatonin supplementation, irrespective of the two doses tested, ameliorated intrafollicular oxidative balance and oocyte quality in UI patients, and that this translated into a slight increase in the rate of pregnancies/live births. Therefore, although the indoleamine has shown therapeutic potential in this clinical setting, larger clinical trials in populations with different backgrounds are encouraged to corroborate the usefulness of melatonin.

Melatonin diminishes oxidative damage in sperm cells, improving assisted reproductive techniques.

Sperm preparation procedures are a potential generator of oxidative stress-induced DNA damage, which leads to a dramatic drop in fertility. An increasing number of studies suggest that melatonin reduces the oxidative stress induced by manipulation. However, very little is known about the preservative role of melatonin in sperm preparation medium during assisted reproduction procedures. For this aim to be achieved, semen was divided into two fractions and preincubated with and without 1 mM melatonin. Afterwards, both fractions were divided into two subfractions to perform swim-up in the presence and absence of 1 mM melatonin. Labeling with anti-CD46 and antiactive caspase-3 allowed the monitoring of acrosome reaction and apoptosis by flow cytometry. Sperm DNA fragmentation and compaction were analyzed through propidium iodide staining. The normozoospermic and oligozoospermic samples that were preincubated with melatonin underwent a significant increase in the ratio of adequate spermatozoa and a reduction of caspase-3 activation. Additionally, preincubation with melatonin enhanced the migration of sperm cells with compacted DNA in oligozoospermic samples (P < 0.05) and prevented DNA fragmentation in normozoospermic samples (P < 0.05). In light of the current results, the cytoprotective capacity and innocuousness of melatonin make it a great candidate to be applied in assisted reproduction techniques in order to prevent triaogenic oxidative damage.

Exogenous melatonin supplementation prevents oxidative stress-evoked DNA damage in human spermatozoa.

Reactive oxygen species (ROS) are essential for sperm physiological functions such as capacitation, hyperactivation, and acrosome reaction, on the one hand, and for stimulating the apoptotic processes involved in the regulation of spermatogenesis, on the other hand. However, the imbalance between production and removal of ROS leads to oxidative stress, which is referred to as one of the main factors involved in male infertility. The pineal hormone melatonin, given its low toxicity and well-known antioxidant capacity, could be an excellent candidate to improve sperm quality. For this reason, the objective of the present work was to analyze whether long-term supplementation with melatonin to infertile men affects human sperm quality and the quality of the embryos retrieved from their couples. Our findings showed that the daily supplementation of 6 mg melatonin, as early as after 45 days of treatment, produced an increase in melatonin endogenous levels, indirectly measured as urinary 6-sulfatoxymelatonin (aMT6-s), an enhancement of both urinary and seminal total antioxidant capacity, and a consequent reduction in oxidative damage caused in sperm DNA. Moreover, couples whose men were given melatonin showed a statistically significant increase in the percentage of grade A (embryo with blastomeres of equal size; no cytoplasmic fragmentation), B (embryo with blastomeres of equal size; minor cytoplasmic fragmentation), and C (embryo with blastomeres of distinctly unequal size; significant cytoplasmic fragmentation) embryos at the expense of grade D (embryo with blastomeres of equal or unequal size; severe or complete fragmentation.) embryos which were clearly reduced. In summary, melatonin supplementation improves human sperm quality, which is essential to achieve successful natural and/or assisted reproduction outcome.

A multicenter prospective study to assess the effect of early cleavage on embryo quality, implantation, and live-birth rate.

OBJECTIVE: To investigate the impact of early cleavage (EC) on embryo quality, implantation, and live-birth rates. DESIGN: Prospective cross-sectional study. SETTING: Multicenter study. PATIENT(S): Seven hundred embryo transfers and 1,028 early-stage human embryos. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation according to the presence of EC and embryo quality. RESULT(S): The presence of EC is associated with embryo quality, especially in cycles with autologous oocytes. However, the use of EC as an additional criterion for selecting an embryo for transfer does not appear to significantly improve likelihood of implantation. Furthermore, embryos that presented EC had live-birth rates per implanted embryo similar to those that did not show any sign of cleavage. CONCLUSION(S): At least for conventional embryo culture and morphologic evaluations, the additional evaluation of EC in embryos may not be valuable to improve embryo implantation.

Melatonin protects human spermatozoa from apoptosis via melatonin receptor- and extracellular signal-regulated kinase-mediated pathways.

OBJECTIVE: To evaluate whether the protective effect of melatonin on H2O2-induced caspase activation and DNA fragmentation depends on the interaction between melatonin and its surface receptors. DESIGN: Laboratory study. SETTING: Center for assisted human reproduction at a Spanish hospital. PATIENT(S): Twenty-one healthy donors. INTERVENTION(S): Human spermatozoa were treated with increasing concentrations of hydrogen peroxide (H2O2; 1 µM, 10 µM, 100 µM, 1 mM) and preincubated with 1 mM melatonin. MAIN OUTCOMES MEASURE(S): Activation of caspase-3 and -9 as well as DNA fragmentation were examined by fluorescence methods. RESULT(S): Our findings showed that H2O2 induced a significant increase in caspase-9 and caspase-3, which was dose independent. Conversely, pretreatment with melatonin reduced H2O2-mediated caspase activation in a dose-dependent way. Moreover, the antiapoptotic effects of melatonin in ejaculated human spermatozoa may involve membrane melatonin receptor MT1. In addition, we found that the survival-promoting pathway extracellular signal-regulated kinase (ERK) is likely to have a role in the protective actions of melatonin in ejaculated human spermatozoa. Finally, we confirmed these results further by demonstrating that melatonin prevention of H2O2-induced DNA fragmentation is dependent on both MT1 receptor and ERK signaling. CONCLUSION(S): These results indicate that the stimulation with melatonin triggers a set of events culminating in cell death prevention in ejaculated human spermatozoa.

The correlation between urinary 5-hydroxyindoleacetic acid and sperm quality in infertile men and rotating shift workers.

BACKGROUND: Serotonin is a neurotransmitter that modulates a wide range of neuroendocrine functions. However, excessive circulating serotonin levels may induce harmful effects in the male reproductive system. The objective of this study was to evaluate whether the levels of urinary 5-hydroxyindoleacetic acid (5-HIIA), a major serotonin metabolite, correlate with different classical seminal parameters. METHODS: Human ejaculates were obtained from 40 men attending infertility counselling and rotating shift workers by masturbation after 4-5 days of abstinence. Urinary 5- HIIA concentration was quantified by using a commercial ELISA kit. Forward motility was assessed by a computer-aided semen analysis (CASA) system. Sperm concentration was determined using the haemocytometer method. Sperm morphology was evaluated after Diff-Quik staining, while sperm vitality was estimated after Eosin-Nigrosin vital staining. RESULTS: Our results show that urinary 5-HIIA levels obtained from a set of 20 volunteers negatively correlated with sperm concentration, forward motility, morphology normal range and sperm vitality. On the other hand, we checked the relationship between male infertility and urinary 5-HIIA levels in 20 night shift workers. Thus, urinary 5-HIIA levels obtained from 10 recently-proven fathers were significantly lower than those found in 10 infertile males. Additionally, samples from recent fathers exhibited higher sperm concentration, as well as better forward motility and normal morphology rate. CONCLUSIONS: In the light of our findings, we concluded that high serotonin levels, indirectly measured as urinary 5-HIIA levels, appear to play a role as an infertility determinant in male subjects.

Melatonin as a potential tool against oxidative damage and apoptosis in ejaculated human spermatozoa.

It is assumed somatic cells can die in the apoptotic, the autophagic, or the necrotic way; however, the mechanisms of sperm death are not clear. Here, ejaculated human spermatozoa were evaluated for apoptosis and reactive oxygen species production in the absence or presence of melatonin, and we concluded that melatonin reverses sperm apoptosis due to its free radical scavenging actions.

Relationship between caspase activity and apoptotic markers in human sperm in response to hydrogen peroxide and progesterone.

Apoptosis plays an essential role in normal spermatogenesis, but deregulations of this biological process, which is closely associated with male infertility, have been found. Whereas calcium homeostasis is a key regulator of cell survival, sustained elevation of intracellular calcium plays a role in apoptosis. The aim of this research was to determine the role of two different calcium mobilizing agents, hydrogen peroxide (H(2)O(2)) and the physiological agonist progesterone, on the apoptosis process of human ejaculated spermatozoa. Translocation of membrane phosphatidylserine was examined with an annexin V binding assay, DNA damage was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL assay) and caspase-3 activity was assessed using a fluorometric assay. After incubation of spermatozoa for 1 h with either 10 microM H(2)O(2) or 20 microM of progesterone, there was a significant increase in both caspase-3 activity and the percentage of annexin V-positive cells. Similarly, the TUNEL results were significantly higher 1 h after incubation with either 10 microM H(2)O(2) or 20 microM of progesterone. In fact, progesterone-treated cells showed a three-fold increase (from 17.6 to 52.9%) of TUNEL-positive cells compared to untreated cells, while H(2)O(2)-treated cells exhibited a two-fold increase (from 17.6 to 37.9%). In sum, our results suggest that spermatozoa treated with calcium mobilizing agents, such as H(2)O(2) and progesterone, seem to undergo an apoptosis process that is dependent on caspase-3 activation.

Reduced levels of intracellular calcium releasing in spermatozoa from asthenozoospermic patients.

BACKGROUND: Asthenozoospermia is one of the most common findings present in infertile males characterized by reduced or absent sperm motility, but its aetiology remains unknown in most cases. In addition, calcium is one of the most important ions regulating sperm motility. In this study we have investigated the progesterone-evoked intracellular calcium signal in ejaculated spermatozoa from men with normospermia or asthenozoospermia. METHODS: Human ejaculates were obtained from healthy volunteers and asthenospermic men by masturbation after 4-5 days of abstinence. For determination of cytosolic free calcium concentration, spermatozoa were loaded with the fluorescent ratiometric calcium indicator Fura-2. RESULTS: Treatment of spermatozoa from normospermic men with 20 micromolar progesterone plus 1 micromolar thapsigargin in a calcium free medium induced a typical transient increase in cytosolic free calcium concentration due to calcium release from internal stores. Similar results were obtained when spermatozoa were stimulated with progesterone alone. Subsequent addition of calcium to the external medium evoked a sustained elevation in cytosolic free calcium concentration indicative of capacitative calcium entry. However, when progesterone plus thapsigargin were administered to spermatozoa from patients with asthenozoospermia, calcium signal and subsequent calcium entry was much smaller compared to normospermic patients. As expected, pretreatment of normospermic spermatozoa with both the anti-progesterone receptor c262 antibody and with progesterone receptor antagonist RU-38486 decreased the calcium release induced by progesterone. Treatment of spermatozoa with cytochalasin D or jasplakinolide decreased the calcium entry evoked by depletion of internal calcium stores in normospermic patients, whereas these treatments proved to be ineffective at modifying the calcium entry in patients with asthenozoospermia. CONCLUSION: Our results suggest that spermatozoa from asthenozoospermic patients present a reduced responsiveness to progesterone.

Caspase 3 activation in human spermatozoa in response to hydrogen peroxide and progesterone.

OBJECTIVE: To determine the role of calcium signaling on apoptosis evoked by the reactive oxygen species H2O2 and by the physiological agonist P in human ejaculated spermatozoa. DESIGN: Laboratory study. SETTING: Center for assisted human reproduction in a hospital in Spain. PATIENT(S): Forty-five healthy volunteers. INTERVENTION(S): Spermatozoa were treated with increasing concentrations of hydrogen peroxide (H2O2; 10 microM, 100 microM, and 1 mM) or with 20 microM of P for 5-120 minutes. MAIN OUTCOME MEASURE(S): Activation of caspase-3 and -9 as well as phosphatidylserine externalization were examined in human ejaculated spermatozoa by fluorescence methods. RESULT(S): Hydrogen peroxide and P induced activation of caspase-3 and -9. In addition, the effect of H2O2 and P was time dependent. Dimethyl-1,2-bis (aminophenoxy) ethane-N,N,N',N'-tetraacetic acid loading was able to inhibit H2O2- and P-induced caspase-3 activation and phosphatidylserine externalization. Pretreatment of spermatozoa with Ru360, to block the calcium uptake into mitochondria, also was able to decrease the activation of caspase-3 and phosphatidylserine exposure that was stimulated by either H2O2 or P. CONCLUSION(S): These findings suggest that H2O2- and P-induced mitochondrial apoptosis is dependent on calcium signaling.

Expression of transcription factors in endometrium during natural cycles.

PURPOSE: The sex steroid control of the endometrial cycle is mediated by transcription factors, four of which are the estrogen and progesterone receptors, c-jun and c-fos, all expressed by the endometrium. The aim of this study was to analyze the distribution of the transcription factors in the different endometrial compartments during natural cycles. METHODS: We studied 53 reproductively-normal women, of whom 26 were in the proliferative phase and 27 in the secretory phase. An endometrial biopsy was performed and serum values of LH, FSH, estradiol, and progesterone were determined. We studied the expression of transcription factors using monoclonal antibodies. RESULTS: A correlation between estrogen receptor and c-jun and c-fos expression was observed in stroma and epithelia, and progesterone receptor expression correlated with c-jun expression in epithelia. C-jun and c-fos presented greater expression in the proliferative phase than in the secretory phase, in the stroma and in both epithelia. No relation was found between estradiol serum levels and any transcription factor, but progesterone serum levels correlated significantly with most such factors. CONCLUSION: The two proto-oncogenes could play a decisive role in regulating the endometrial cycle; they could mediate the effects induced by sex steroid, and could be related to other transcription factors.