Exogenous GSH Supplementation to Raw Semen Alters Sperm Kinematic Parameters in Infertile Patients.

Glutathione is an important antioxidant found in all mammalian cells. Sperm motility is positively correlated with seminal reduced glutathione (GSH) levels, and infertile men are known to have lower GSH levels. Studies on GSH supplementation in improving sperm functions in infertility patients are limited. Here, we re-investigate the effect of exogenous GSH supplementation on human sperm motility and kinematic parameters. Residual semen samples from 71 infertility patients who came for routine semen analysis for infertility assessment were studied. Liquefied raw semen was supplemented with GSH (0-10 mM) for 1 h. The untreated sample was the blank control. Only a 5 mM concentration was tested in all 71 samples. After two washes, the sperm was incubated and then analyzed for sperm motility and kinematic parameters by computer-assisted semen analysis (CASA), followed by adenosine triphosphate (ATP), reactive oxygen species (ROS) levels, free thiols, and DNA damage analyses. At 2 hrs post-treatment, GSH supplementation significantly altered many of the kinematics, compared to the control. Straight line velocity (VSL) (p = 0.0459), curvilinear velocity (VCL) (p < 0.0001), average path velocity (VAP) (p < 0.0001), and lateral head amplitude (ALH) (p < 0.0001) were decreased, whereas straightness (STR) (p = 0.0003), linearity (LIN) (p = 0.0008), and beat cross frequency (BCF) (p = 0.0291) were increased in 5 mM group. Wobble (WOB) (p = 0.4917), motility (MOT) (p = 0.9574), and progressive motility (PROG) (p = 0.5657) were unchanged. ATP level was significantly increased in the 5 mM group (p < 0.05). It is concluded that exogenous GSH supplementation does alter sperm kinematics in humans. These altered kinematic parameters together with increased energy (ATP) may have a positive role in influencing the success rates of ART procedures.

Mitochondria Transfer from Adipose Stem Cells Improves the Developmental Potential of Cryopreserved Oocytes.

Although it is not a well-established technology, oocyte cryopreservation is becoming prevalent in assisted reproductive technologies in response to the growing demands of patients' sociological and pathological conditions. Oocyte cryopreservation can adversely affect the developmental potential of oocytes by causing an increase in intracellular oxidative stresses and damage to the mitochondrial structure. In this study, we studied whether autologous adipose stem cell (ASC) mitochondria supplementation with vitrified and warmed oocytes could restore post-fertilization development that decreased due to mitochondrial damage following cryopreservation. ASC mitochondria showed similar morphology to oocytes' mitochondria and had a higher ATP production capacity. The vitrified-warmed oocytes from juvenile mice were supplemented with ASC mitochondria at the same time as intracellular sperm injection (ICSI), after which we compared their developmental capacity and the mitochondria quality of 2-cell embryos. We found that, compared to their counterpart, mitochondria supplementation significantly improved development from 2-cell embryos to blastocysts (56.8% vs. 38.2%) and ATP production in 2-cell embryos (905.6 & 561.1 pmol), while reactive oxygen species levels were comparable. With these results, we propose that ASC mitochondria supplementation could restore the quality of cryopreserved oocytes and enhance the embryo developmental capacity, signifying another possible approach for mitochondrial transplantation therapy.

A Global Survey of Reproductive Specialists to Determine the Clinical Utility of Oxidative Stress Testing and Antioxidant Use in Male Infertility.

PURPOSE: The use of antioxidants is common practice in the management of infertile patients. However, there are no established guidelines by professional societies on antioxidant use for male infertility. MATERIALS AND METHODS: Using an online survey, this study aimed to evaluate the practice pattern of reproductive specialists to determine the clinical utility of oxidative stress (OS) testing and antioxidant prescriptions to treat male infertility. RESULTS: Responses from 1,327 participants representing 6 continents, showed the largest participant representation being from Asia (46.8%). The majority of participants were attending physicians (59.6%), with 61.3% having more than 10 years of experience in the field of male infertility. Approximately two-thirds of clinicians (65.7%) participated in this survey did not order any diagnostic tests for OS. Sperm DNA fragmentation was the most common infertility test beyond a semen analysis that was prescribed to study oxidative stress-related dysfunctions (53.4%). OS was mainly tested in the presence of lifestyle risk factors (24.6%) or sperm abnormalities (16.3%). Interestingly, antioxidants were prescribed by 85.6% of clinicians, for a duration of 3 (43.7%) or 3-6 months (38.6%). A large variety of antioxidants and dietary supplements were prescribed, and scientific evidence were mostly considered to be modest to support their clinical use. Results were not influenced by the physician's age, geographic origin, experience or training in male infertility. CONCLUSIONS: This study is the largest online survey performed to date on this topic and demonstrates 1) a worldwide understanding of the importance of this therapeutic option, and 2) a widely prevalent use of antioxidants to treat male infertility. Finally, the necessity of evidence-based clinical practice guidelines from professional societies is highlighted.

Treatment with Laevo (L)-carnitine reverses the mitochondrial function of human embryos.

PURPOSE: Laevo (l)-carnitine plays important roles in reducing the cytotoxic effects of free fatty acids by forming acyl-carnitine and promoting beta-oxidation, leading to alleviation of cell damage. Recently, the mitochondrial functions in morula has been shown to decrease with the maternal age. Here, we assessed the effect of l-carnitine on mitochondrial function in human embryos and embryo development. METHODS: To examine the effect of L-carnitine on mitochondrial function in morulae, 38 vitrified-thawed embryos at the 6-11-cell stage on day 3 after ICSI were donated from 19 couples. Each couple donated two embryos. Two siblings from each couple were divided randomly into two groups and were cultured in medium with or without 1 mM L-carnitine. The oxygen consumption rates (OCRs) were measured at morula stage. The development of 1029 zygotes cultured in medium with or without L-carnitine was prospectively analyzed. RESULTS: Addition of L-carnitine to the culture medium significantly increased the OCRs of morulae and improved the morphologically-good blastocyst formation rate per zygote compared with sibling embryos. Twenty healthy babies were born from embryos cultured in L-carnitine-supplemented medium after single embryo transfers. CONCLUSION(S): L-carnitine is a promising culture medium supplement that might be able to counteract the decreased mitochondrial function in human morula stage embryos.

Oral administration of l-carnitine improves the clinical outcome of fertility in patients with IVF treatment.

Age-dependent decline of mitochondrial function has been proposed to be a main cause of decline of embryo quality. Then, l-carnitine plays important roles in reducing the membranous toxicity of free-fatty acids by forming acyl-carnitine and promoting ß-oxidation, preventing cell damage. Recent research revealed that l-carnitine played important roles in vitro in oocyte growth, oocyte maturation and embryo development. However, such beneficial effects of l-carnitine in vivo have yet to be verified. The effect of oral l-carnitine supplementation on embryo quality and implantation potential was examined. A total of 214 patients were included in this study. They all previously received in vitro fertilization-embryo transfer (IVF-ET) and failed to conceive. Then they were administered l-carnitine for 82 days on average and underwent IVF-ET again. There were no significant differences in the total number of retrieved oocytes, and their maturation and fertilization rates between before and after l-carnitine administration. The quality of embryos on Days 3 and 5 after insemination was improved following l-carnitine administration (p < .05) in cycles after l-carnitine administration compared with previous cycles. Healthy neonates were born after IVF-ET following l-carnitine administration. Our data suggested that oral administration of l-carnitine to fertility patients improved the developmental competence of their oocytes after insemination.

Oral melatonin supplementation improves oocyte and embryo quality in women undergoing in vitro fertilization-embryo transfer.

The aim of this study was to evaluate the efficacy of oral melatonin supplementation on oocyte and embryo quality in patients in an assisted reproductive technologies program. All patients were treated for at least 2 weeks with melatonin (3 mg/day). To evaluate the cumulative effect of melatonin supplementation, we compared cycle outcomes between the first (no supplementation) and second cycles (melatonin supplementation) of patients who completed two treatment cycles. There were no significant differences in maturation rates (p = 0.50), blastocyst rates (p = 0.75), and the rate of good quality blastocysts (p = 0.59) between the first and second cycles. The fertilization rate of ICSI was higher in the second cycle than that in the first cycle (69.3 versus 77.5%). Being limited to patients with a low fertilization rate in the first cycle (<60%), the fertilization rate dramatically increased after melatonin treatment (35.1 versus 68.2%). The rate of good quality embryos also increased (48.0 versus 65.6%). An important finding in our study was that oral melatonin supplementation can have a beneficial effect on the improvement of fertilization and embryo quality and this may have occurred due to a reduction in oxidative damage.

Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.

Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation-in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.