Alpha-Lipoic Acid Supplementation for Male Partner of Couples with Recurrent Pregnancy Loss: A Post hoc analysis in Clinical Trial.

BACKGROUND: Increased sperm DNA damage is known as one of the causes of recurrent pregnancy loss (RPL) which can be due to increased levels of oxidative stress. Therefore, the aim of this study was to assess the effect of alpha-lipoic acid (ALA) on sperm parameters and sperm functions in couples with a history of RPL. MATERIALS AND METHODS: In this post hoc analysis in clinical trial study, a total of 37 couples with RPL (n=12 and n=25 for placebo and ALA groups, respectively) were considered. Men were treated with ALA (600 mg/day) or placebo for 80 days. Semen samples were acquired from the participants before initiation and after completion of the medication course and assessed regarding conventional sperm parameters, chromatin damage/integrity, intracellular oxidative stress, lipid peroxidation, and seminal antioxidant characteristics. Individuals were further followed up for twelve months for pregnancy occurrence and outcomes. Finally, after excluding patients with no history of RPL, the data was analyzed. RESULTS: No significant differences were observed between the baseline measures of the aforementioned parameters except for seminal volume. After the intervention, the mean sperm DNA damage, protamine deficiency, and persisted histones were significantly lower in the ALA group than in placebo receivers (P<0.05). A decrease in the mean of seminal total antioxidant capacity (P=0.03), malondialdehyde (P=0.02), and sperm DNA damage (P=0.004) as well as an increase in sperm total motility (P=0.04) after treatment with ALA was noticed. In addition, the mean of protamine deficiency and persisted histones were declined post-ALA therapy (P=0.003 and 0.002, respectively). The percentage of spontaneous pregnancy in the ALA group (4 of 25 cases; 16%) was higher than in the placebo group (1 of 12, 8.3%). CONCLUSION: ALA-therapy attenuates sperm DNA damage and lipid peroxidation while enhancing sperm total motility and chromatin compaction in the male partner of couples with PRL (registration number: IRCT20190406043177N1).

Carob extract induces spermatogenesis in an infertile mouse model via upregulation of Prm1, Plzf, Bcl-6b, Dazl, Ngn3, Stra8, and Smc1b.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological studies for drug discovery from natural compounds play an important role for developing current therapeutical platforms. Plants are a group of natural sources which have been served as the basis in the treatment of many diseases for centuries. In this regard, Ceratonia siliqua (carob) is one of the herbal medicine which is traditionally used for male infertility treatments. But so far the main mechanisms for effects of carob are unknown. Here, we intend to investigate the ability of carob extract to induce spermatogenesis in an azoospermia mouse model and determine the mechanisms that underlie its function. AIM OF THE STUDY: This is a pre-clinical animal model study to evaluate the effect of carob extract in spermatogenesis recovery. METHODS: We established an infertile mouse model with the intent to examine the ability of carob extract as a potential herbal medicine for restoration of male fertility. Sperm parameters, as well as gene expression dynamics and levels of spermatogenesis hormones, were evaluated 35 days after carob administration. RESULTS: Significant enhanced sperm parameters (P < 0.05) showed that the carob extract could induce spermatogenesis in the infertile mouse model. Our data suggested an anti-apototic and inducer role in the expressions of cell cycle regulating genes. Carob extract improved the spermatogenesis niche by considerable affecting Sertoli and Leydig cells (P < 0.05). The carob-treated mice were fertile and contributed to healthy offspring that matured. Our data confirmed that this extract triggered the hormonal system, the spermatogenesis-related gene expression network, and signaling pathways to induce and promote sperm production with notable level (P < 0.05). We found that the aqueous extract consisted of a polar and mainly well water-soluble substance. Carob extract might upregulate spermatogenesis hormones via its amino acid components, which were detected in the extract by liquid chromatography-mass spectrometry (LC-MS). CONCLUSION: Our results strongly suggest that carob extract might be a promising future treatment option for male infertility. This finding could pave the way for clinical trials in infertile men. This is the first study that has provided reliable, strong pre-clinical evidence for carob extract as an effective candidate for fertility recovery in cancer-related azoospermia.

Manipulation of fatty acid profiles in roosters' testes, alteration in sexual hormones, improvements in testicular histology characteristics and elevation sperm quality factor by L-carnitine.

The present study was designed to investigate the effects of different levels of l-carnitine (LC) on sperm quality factor (SQF), alterations in testis fatty acid profiles, testicular histology and reproductive hormones in young roosters. Eighteen broiler breeders (Ross 308) weighed at 3 months of age. They were randomly classified while each group had six birds. There were three experimental groups based on the LC concentrations (i.e. LC-0, LC-250, LC-500 mg per kg of diet). After two weeks of adaptation, semen samples were collected and evaluated for seminal attributes every two weeks (from week 24 to week 34). At the end of the experiments, four roosters from each treatment group were sacrificed in order to analyze testicular histology, testis fatty acid profiles and reproductive hormones. Supplementing the diet with two of the LC levels for 22 weeks caused significant rise in sperm concentration, viability and SQF compared to that of the control group (P < 0.05). Quadratic analysis in terms of number of seminiferous tubules and spermatogenesis index were significant (P<0.05). Tubular differentiation index improved linearly by the increasing levels of LC supplementation (P<0.01). The analysis of fatty acid profiles showed that LC significantly (P < 0.05) reduced the percentages of C14:0, C21:0, total saturated fatty acids, total odd-chain fatty acids and n-6/n-3 ratio. Moreover, LC significantly increased the percentage of C20:5n-3 (Eicosapentaenoic acid; EPA) (P < 0.05). Analysis of the correlation coefficient revealed that the SQF is in consistency with EPA (r = 0.98; P < 0.04). In contrast, SQF negatively and significantly correlates with odd-chain fatty acids (r = - 0.99; P < 0.001). The desaturation index for C16 fatty acids (16:1cis/C16:0) negligibly increased linearly as LC was added to the diet (P < 0.05). Furthermore, LC caused the roosters to have significant (P < 0.05) high levels of total testosterone and FSH concentrations. The concentration of LH in different treatment groups, however, turned out to be similar in response to the different levels of LC. In conclusion, long-term supplementation of rooster diet with LC can have beneficial effects on SQF and testis histology. The benefits include alterations in testicular histology, reproductive hormones and testicular fatty acid profiles.

Effect of Bovine Serum Albumin Supplementation in Tris-Soybean Lecithin-Based Extender on Quality of Chilled Ram Epididymal Spermatozoa.

Objective: This study was designed to assess the effects of using tris-soybean lecithin (TSL)-based extender supplemented with bovine serum albumin (BSA) on the quality of ram epididymal spermatozoa during refrigerated storage. Method: Epididymal sperm were collected from 22 Zandi rams, diluted in TSL-based extender at different concentrations (0%, 2.5%, 5%, 7.5%, and 10%) of BSA, and stored for 5 days at 4°C. Sperm parameters including motility, viability, plasma membrane integrity, chromatin protamination, and malondialdehyde (MDA) content were evaluated at 0, 24, 72, and 120 hours of refrigeration. Results: The addition of 10% BSA to the extender significantly improved sperm viability at 24 and 120 hours of refrigerated liquid storage (p < 0.05). An enhancement in plasma membrane integrity was observed along with a decrease in MDA level by increasing the concentration of BSA from 0% to 10% (p > 0.05). Sperm motion characteristics were higher in the BSA-free group at 120 hours of preservation (p < 0.05). No statistical difference was found for nuclear protamination between experimental groups (p > 0.05). Conclusion: BSA supplementation in TSL-based extender can preserve the viability of epididymal ram spermatozoa during liquid storage at 4°C.

Peroxisome proliferator-activated receptors (PPARs) as a mediator of dietary fatty acids affects reproductive performance in broiler breeder roosters.

This study analyzed the effects of dietary sources of omega-3 and omega-6 fatty acids on semen parameters and fertility potential in broiler breeder roosters. The mRNA and protein profiles of peroxisome proliferator-activated receptors-γ (PPAR-γ) expression in sperm as potential mediator of FAs were considered. Roosters were categorized into three groups and received their diets for 24 weeks as follows: 1) control diet received a basal diet (CTRL); 2) Fish oil based diet (FO) received the basal diet supplemented with 15 g/kg of diet fish oil; and 3) sunflower oil based diet (SO) received the basal diet supplemented with 15 g/kg of diet sunflower oil. While the different diets had significant effects on semen parameters, the effect of sampling time was not significant. The effect of the diets on sperm parameters were significantly higher in the SO and FO groups in total motility, progressive motility, amplitude of lateral head displacement, linearity, straightness, wobble and viability (P ≤ 0.05). Fertility rate was significantly improved in the FO and SO groups (P = 0001). The highest value for PPAR-γ mRNA was observed in the SO group compared to other groups (P ≤ 0.05). Moreover, supplementation of the roosters' diets with FO and SO increased PPAR-γ protein expression compared to the control. It seems that PPAR-γ could be a strong potential mediator of the underlying mechanism of improvement in semen parameters and reproductive performance of roosters under the effects of both dietary omega-3 and omega-6 polyunsaturated fatty acids.

Inclusion of ovine enriched serum with vitamin E and polyunsaturated fatty acids in the freezing medium: a new strategy to improve human frozen-thawed sperm parameters.

The objective was to evaluate the effect of inclusion of 2.5% and 5% ovine serum, enriched with vitamin E (Vit E) and fish oil (FO), in human sperm freezing medium. Serum samples were prepared from sixteen rams (n = 4) feeding on a without supplemented diet, and diets supplemented with Vit E, FO and Vit E + FO. Semen samples, from 60 normozoospermic men, were frozen in: (I) a commercial freezing medium (SpermFreeze™; control medium), (II) the commercial freezing medium containing foetal bovine serum, (III) the commercial freezing medium + nonenriched serum (serum group), (IV) the commercial freezing medium + Vit E enriched serum (Vit E group), (V) the commercial freezing medium + FO enriched serum (FO group) and (VI) the commercial freezing medium + Vit E + FO enriched serum (Vit E + FO group). Sperm total and progressive motility, morphology, viability and plasma membrane integrity were significantly higher (p ≤ .05) in Vit E and Vit E + FO groups compared with the control group. Mitochondrial membrane potential did not differ between treatments (p > .05). It was concluded that ovine serum enriched with vitamin E and vitamin E + FO improved the quality of human spermatozoa but enriched serum containing FO could not improve the sperm cryo-injuries.

Supplementation of extender with coenzyme Q10 improves the function and fertility potential of rooster spermatozoa after cryopreservation.

The effects of coenzyme Q10 (CoQ10) has not yet been assessed for cryopreservation of rooster semen. The aim of this study was to evaluate the effect of different concentrations of CoQ10 in Lake extender for cryopreservation of rooster semen. The viability and apoptosis status, DNA fragmentation, abnormal morphology, motion parameters, membrane functionality, mitochondrial activity, acrosome integrity, lipid peroxidation, and fertility potential were evaluated after the freeze-thaw process. Semen samples were collected from ten roosters, twice a week, and then diluted in extender contained different concentrations of CoQ10 as follows: Lake without CoQ10 (control, Q 0), Lake containing 1 µM (Q 1), 2 µM (Q 2), 5 µM (Q 5), and 10 µM (Q 10) CoQ10. Supplementation of Lake with 1 and 2 µM CoQ10 resulted in greater sperm viability, total motility, progressive motility, membrane functionality, mitochondrial activity, acrosome integrity, and fertility rate. Furthermore, the extent of lipid peroxidation in thawed spermatozoa treated with 1 and 2 µM CoQ10 was less than with the other groups. Different concentrations of CoQ10 had no effect on DNA fragmentation and sperm morphology. Results of the present study indicate that supplementation of Lake extender with 1 and 2 µM CoQ10 enhances the quality of rooster sperm after the freeze-thaw process.

Dietary fish oil supplemented with vitamin E improves quality indicators of rooster cold-stored semen through reducing lipid peroxidation.

Cockerel semen is sensitive to cooling, which limits chilled storage of semen for more than 24 h. Results of artificial insemination with cold-stored semen are not desirable. This study was conducted to evaluate the effects of dietary fish oil and vitamin E (vitE) for cold-storage of rooster semen and its effects on parameters of semen during 48 h cooling preservation. Roosters were assigned into four dietary treatments; 1) control group received a basal diet, 2) vitE group received a basal diet supplemented with 200 mg/kg vitE, 3) fish oil group (FO) received a basal diet supplemented with 2% fish oil and 4) fish oil and vitE group received a basal diet supplemented with 2% fish oil and 200 mg/kg vitE (FO + vitE). Semen samples were collected after 40 days of feeding and then diluted and cooled to 5 °C for preservation up to 2 days. Several quality indicators of sperm such as motion characteristics, membrane integrity, and viability, and abnormal morphology, activity of mitochondria, lipid peroxidation and acrosome integrity of the sperm were assessed at different times of storage (0, 24 and 48 h). None of sperm were significantly affected by the diets at the start of storage (0 h, p > 0.05). FO and FO + vitE improved the percentage of total motility, viability, and mitochondria activity at 24 h (P ≤ 0.05). After 48 h, only FO + vitE group produced the higher percentage of total motility, viability and membrane integrity (P ≤ 0.05). Lipid peroxidation was significantly reduced in sperm obtained from roosters fed diets of FO + vitE and vitE compared to FO and control (P ≤ 0.05) at times of 24 and 48 h. There was no significant difference between control and vitE groups in none of parameters (P > 0.05). Integrity of acrosome and abnormal morphology were not significantly affected by the diets (P > 0.05). Supplementation of roosters' diet with 2% fish oil and 200 mg/kg vitamin E improved the quality of cold-stored semen by supporting several indicators of sperm quality through reducing lipid peroxidation.

Improvements in semen quality, sperm fatty acids, and reproductive performance in aged Ross breeder roosters fed a diet supplemented with a moderate ratio of n-3: n-6 fatty acids.

The purpose of this study was to evaluate the effects of dietary n-3: n-6 fatty acid (FA) ratios and vitamin E (vitE) on the semen quality, FA composition, hormonal responses, and reproductive performance of aged roosters. Thirty-six Ross broiler breeder roosters were assigned to a 3 × 2 factorial design with 3 n-3: n-6 FA ratios (0.09, 0.16, and 0.23) based on the inclusion of 3 oil sources (canola, canola/fish, and fish) and 2 vitE levels (0 and 200 mg/kg). During the 60 d of treatment, semen parameters, FAs composition of sperm, lipid peroxidation, and hormonal responses were monitored. Reproductive performance using artificial insemination was also evaluated at the end of experiment (on day 60). Results showed that the 0.16 and 0.23 dietary ratios increased docosahexaenoic acid and n-3 polyunsaturated FA and decreased docosatetraenoic acid and arachidonic acid in rooster sperm (P < 0.05). The 0.16 dietary ratio supplemented with 200 mg vitE improved total sperm motility (P < 0.05) than the 0.09 and 0.23 ratios, both with and without vitE. Progressive motility, membrane functionality, and viability were significantly (P < 0.05) improved in the 0.16 and 0.23 ratios supplemented with vitE. The highest concentration of testosterone was observed in the roosters fed a diet ratio of 0.16 (P < 0.05). Compared with no supplementation of vitE, 200 mg/kg vitE reduced the lipid peroxidation of rooster sperm (P < 0.05). In the artificial insemination, the higher significant percentage of fertility rate (P = 0.02) was observed in the 0.16 ratio group that was supplemented with vitE compared to other groups. It can be concluded that supplementation of aged roosters' diet with 0.16 ratio of n-3: n-6 can be a beneficial strategy for improvement of their fertility.

Can permeable super oxide dismutase mimetic agents improve the quality of frozen-thawed ram semen?

This study was carried out to assess the effects of MnTBAP, a cell permeable antioxidant, on motility, membrane integrity, capacitation status and in vitro fertilization ability of frozen-thawed ram semen. Fresh semen ejaculates were collected with artificial vagina from five rams, mixed and divided into five equal fractions, and diluted (1:20 v/v) with commercial extender, Bioxell®, containing 0 (control), 50, 100, 150 and 200 µM of MnTBAP. All diluted sperm suspensions were cooled to 5°C for 2h followed by transfer into 0.5 ml French straws before being stored in liquid nitrogen. The results showed that MnTBAP supplementation of extender improved ram semen quality in a dose-dependent manner. Accordingly, the extender supplemented with 150µM MnTBAP resulted in higher sperm motility and improved acrosomal membrane integrity compared to control. However, further supplementation (200µM) with MnTBAP not only did not improve the results but inversely affected motility and membrane integrity. The results of in vitro fertilization (IVF) indicated that the presence of MnTBAP in semen extender has a marginal beneficial effect on developmental competence of inseminated oocytes, though this improvement was not significant. In conclusion, this study demonstrated that semen extender supplemented with MnTBAP can reduce the oxidative stress provoked by freeze/thaw processes. Moreover, beneficial effect of 100 µM of MnTBAP on preservation of spermatozoa in a non-capacitated state post freezing, an important criterion for in vitro or in vivo fertilization, was observed. However, at 150 µM of MnTBAP, the harmful effects of cryopreservation on membrane integrity were decreased. Regarding to importance of non-capacitated spermatozoa during IVF or artificial insemination, the optimum MnTBAP concentration appears to be 100 µM for commercial ram semen extender tested here.

Fatty Acid Prof iles of Ram's Sperm after Removing Some Fatty Acid Sources from the Diets and Persistency of Fatty Acids in Sperm.

BACKGROUND: Mammalian spermatozoa are characterized by a high proportion of polyunsaturated fatty acids (PUFAs), but reliable data concerning dietary effects on fatty acid (FA) profile in ram's sperm and the persistency of FA in the ration to the FA in sperm has not been reported. Therefore, the aim of this study was to determine the stability of saturated and unsaturated FAs in ram's sperm despite removing FA sources from their diet. MATERIALS AND METHODS: Nine Kalkoohi rams were used in a completely randomized design and they were assigned to 3 groups. The treatments were diet supplemented (35 g/d/ram) by C16:0 (RP-10®), C18: 2 (Sunflower oil; SO) and n-3 (Fish oil; FO) with Vitamin E. Fifteen weeks after the start of the supplemented diet, rams were offered a basal diet without any supplementary FA source for 35 days when the sperm's FA ratio was determined. The data were analyzed by ANOVA (Analysis of variance) using the General Linear Model (GLM) procedure of SAS Institute. RESULTS: THIRTY FIVE DAYS AFTER REMOVING THE FAT SUPPLEMENT FROM THE DIET, MAJOR FA IN SPERM CONSISTED OF: C14:0, C16:0, C18:0, C18:1 cis, C18:2 cis and C22:6 n-3 docosahexaenoic acid (DHA). The percentage of C14:0 (p=0.8) and C18:1 cis (P =0.4) were similar among all the treatments. Interestingly, 35 days after the removal of fatty acid source, the percentage of C22:6 was highest in the FO treated group. CONCLUSION: The different sperm FA profile among various groups suggests that dietary FA had significant direct or indirect impacts on sperm FA profile after 35 days which might lead to physical and chemical changes in sperm characteristics.