Publications by authors named "Abdolhossein Shahverdi"

75 Publications

Gene Expression Alteration of Sperm-Associated Antigens in Human Cryopreserved Sperm.

Biopreserv Biobank 2021 May 18. Epub 2021 May 18.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Sperm-associated antigens (SPAGs) are 18 types of proteins, some of which play important roles in various biological functions associated with assisted reproductive technology outcomes, and are consequently important to the success of fertility programs. Despite the favorable outcomes of fecundity rates among male patients with cancer using cryopreserved sperm, the detrimental impact of freezing on cells has been noted in many studies. Cryopreservation has been thought to have adverse effects on sperm quality through disruptions in the expressions of genes. This study aimed to evaluate the effects of cryopreservation on the expressions of genes and their transcriptome alterations in human sperm. A total of 12 normal ejaculations were prepared using the density gradient centrifugation procedure, and the motile sperm fractions were divided into fresh and frozen groups. In the latter, sperm samples were mixed with SpermFreeze solution as the cryoprotectant. The cryovial of sperm suspension was first held just over nitrogen vapor and then dipped inside liquid nitrogen. After 3 days, the specimens were thawed in tap water and incubated for 2 hours for recovery. Then, RNA from sperm was extracted for gene expression analysis, using real-time polymerase chain reaction. Our findings showed a decrease in expression of (-value = 0.009), (-value = 0.004), and ; -value = 0.039) genes during cryopreservation. The results indicate that the freezing procedure could negatively affect gene expression and to some extent proteins in human spermatozoa. The alteration of expression could provide new information on the molecular correlation between cryopreservation and increased failure in intracytoplasmic sperm injection and fertilization.
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http://dx.doi.org/10.1089/bio.2020.0165DOI Listing
May 2021

Sperm chromatin integrity in a man with macrocephaly syndrome.

Andrologia 2021 May 7:e14100. Epub 2021 May 7.

Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.

This study reports chromatin status and ICSI outcomes in a case of sperm macrocephaly syndrome(SMS), showing 100% of spermatozoa with abnormal morphology. Percentages of sperm DNA fragmentation for TUNEL (31.7% versus 6.5%), SCSA (33% versus 25%) assays, chromatin maturity tests, CMA3 (58% versus 29%) and aniline blue (63% versus 35%) staining were higher in case sample compared to the fertile sample. Artificial oocyte activation resulted in a similar fertilisation rate between case and control samples (71% versus 66.7%), but the case showed delayed embryo development on day 3 post-insemination. Unlike fertile case, no embryos reached the blastocyst stage. The result of this case study shows that macrocephaly is associated with reduced chromatin maturity and DNA integrity. Although both cases showed a similar chance for fertilisation through artificial chemical activation for only macrocephalic man, the developmental competency is jeopardised in such cases.
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http://dx.doi.org/10.1111/and.14100DOI Listing
May 2021

The effect of methyl-beta-cyclodextrin on DNA absorption and quality of posttransfected sperm.

Poult Sci 2021 May 17;100(5):101058. Epub 2021 Feb 17.

Prestage Department of Poultry Science, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, 27695-7608 USA. Electronic address:

Sperm can be selected as a natural vector for the production of transgenic animals. Methyl-beta-cyclodextrin (MBCD) removes cholesterol from the phospholipid membrane of sperm and improves the efficiency of DNA uptake by sperm. In experiment 1, fresh sperm was treated with various concentrations of MBCD. The direct effects of MBCD on sperm parameters were monitored. In experiment 2, different concentrations of MBCD (0, 1, 2, and 4 mmol) were assessed for the transfection of genetically exogenous construction to rooster sperm. Washed semen was divided into 5 equal groups for the incubation and transfection with a pcDNA3.1+/hG-CSF vector (exogenous DNA) as follows; Treatment I-Control (washed semen without DNA); Treatment II-Control (washed semen with DNA); Treatment III-(washed semen incubated with DNA and 1 mmol MBCD); Treatment IV-(washed semen incubated with DNA and 2 mmol MBCD); and Treatment V-(washed semen incubated with DNA and 4 mmol MBCD). We demonstrated that rooster spermatozoa spontaneously can uptake exogenous DNA; this was assessed using exogenous DNA amplification (sperm genomic DNA used as a template for PCR reaction) after DNase I treatment. In addition, total motility (TM), progressive motility (PM), velocity parameters [curvilinear velocity (VCL), straight linear velocity (VSL), sperm track straightness (STR), linearity (LIN)], membrane integrity (MI), and membrane functionality were posttransfectionally evaluated. The concentrations of 1 and 2 mmol MBCD significantly (P < 0.05) improved the motion characteristics and membrane integrity of fresh sperm. The presence of hG-CSF in rooster sperm was detected by PCR and based on sperm analyses MBCD (1 mmol) improved the percentage of motility (98.9 ± 0.81), membrane functionality (64 ± 1.64), and MI (76.2 ± 1.65) after transfection when compared with the other groups (P < 0.05). For the production of transgenic chicken, hens were inseminated (AI) by transfected sperm treated with 1 and 0 mmol MBCD. A PCR analysis of the blood samples and dead embryo tissues of chicks did not reveal the transgene integration.
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http://dx.doi.org/10.1016/j.psj.2021.101058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010517PMC
May 2021

Oxidation of Sperm DNA and Male Infertility.

Antioxidants (Basel) 2021 Jan 12;10(1). Epub 2021 Jan 12.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran 16635-148, Iran.

One important reason for male infertility is oxidative stress and its destructive effects on sperm structures and functions. The particular composition of the sperm membrane, rich in polyunsaturated fatty acids, and the easy access of sperm DNA to oxidative damage due to sperm cell specific cytologic and metabolic features (no cytoplasm left and cells unable to mount stress responses) make it the cell type in metazoans most susceptible to oxidative damage. In particular, oxidative damage to the spermatozoa genome is an important issue and a cause of male infertility, usually associated with single- or double-strand paternal DNA breaks. Various methods of detecting sperm DNA fragmentation have become important diagnostic tools in the prognosis of male infertility and such assays are available in research laboratories and andrology clinics. However, to date, there is not a clear consensus in the community as to their respective prognostic value. Nevertheless, it is important to understand that the effects of oxidative stress on the sperm genome go well beyond DNA fragmentation alone. Oxidation of paternal DNA bases, particularly guanine and adenosine residues, the most sensitive residues to oxidative alteration, is the starting point for DNA damage in spermatozoa but is also a danger for the integrity of the embryo genetic material independently of sperm DNA fragmentation. Due to the lack of a spermatozoa DNA repair system and, if the egg is unable to correct the sperm oxidized bases, the risk of de novo mutation transmission to the embryo exists. These will be carried on to every cell of the future individual and its progeny. Thus, in addition to affecting the viability of the pregnancy itself, oxidation of the DNA bases in sperm could be associated with the development of conditions in young and future adults. Despite these important issues, sperm DNA base oxidation has not attracted much interest among clinicians due to the lack of simple, reliable, rapid and consensual methods of assessing this type of damage to the paternal genome. In addition to these technical issues, another reason explaining why the measurement of sperm DNA oxidation is not included in male fertility is likely to be due to the lack of strong evidence for its role in pregnancy outcome. It is, however, becoming clear that the assessment of DNA base oxidation could improve the efficiency of assisted reproductive technologies and provide important information on embryonic developmental failures and pathologies encountered in the offspring. The objective of this work is to review relevant research that has been carried out in the field of sperm DNA base oxidation and its associated genetic and epigenetic consequences.
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http://dx.doi.org/10.3390/antiox10010097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827380PMC
January 2021

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.

Theriogenology 2021 Feb 7;161:8-15. Epub 2020 Oct 7.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. Electronic address:

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.
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http://dx.doi.org/10.1016/j.theriogenology.2020.10.005DOI Listing
February 2021

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

Biopreserv Biobank 2021 Feb 4;19(1):33-40. Epub 2020 Nov 4.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

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. 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. The addition of 10% BSA to the extender significantly improved sperm viability at 24 and 120 hours of refrigerated liquid storage ( < 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% ( > 0.05). Sperm motion characteristics were higher in the BSA-free group at 120 hours of preservation ( < 0.05). No statistical difference was found for nuclear protamination between experimental groups ( > 0.05). BSA supplementation in TSL-based extender can preserve the viability of epididymal ram spermatozoa during liquid storage at 4°C.
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http://dx.doi.org/10.1089/bio.2020.0041DOI Listing
February 2021

The Relationship of Mitochondrial Membrane Potential, Reactive Oxygen Species, Adenosine Triphosphate Content, Sperm Plasma Membrane Integrity, and Kinematic Properties in Warmblood Stallions.

J Equine Vet Sci 2020 11 17;94:103267. Epub 2020 Sep 17.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Equine sperm possesses a unique physiology because its energy supply is mostly dependent on oxidative phosphorylation of mitochondria as an aerobic source of adenosine triphosphate (ATP) generation. The present study was, therefore, conducted to investigate the relationship between sperm kinematic and functional variables in stallions. Semen samples were collected from five warmblood stallions (three ejaculates from each stallion), diluted with INRA96 and transferred to the laboratory. Next, sperm motility, mitochondrial membrane potential (MMP), production of superoxide anion (as a reactive oxygen species; ROS), ATP content, and plasma membrane integrity were assessed. Motion and functional characteristics differed among investigated stallions (P < .05). In addition, it was revealed MMP was positively correlated with the level of ROS and ATP content and progressive motility (P < .05). The level of ROS was positively correlated with ATP content and negatively correlated with plasma membrane integrity and straightness (P < .05). Adenosine triphosphate content was positively correlated with progressive motility, curvilinear velocity, average path velocity, and beat cross frequency and reversely correlated with plasma membrane integrity and straightness (P < .05). Plasma membrane integrity was positively correlated with straight line velocity, linearity, and straightness and negatively correlated with curvilinear velocity (P < .01). In conclusion, the present study substantiated that kinematic and functional characteristics varied among various warmblood stallions. Furthermore, the present study implicated although higher mitochondrial activity increases ATP synthesis, it leads to elevated superoxide anion production, which could culminate in disintegration of the sperm plasma membrane, thereby altering motion characteristics and swimming pattern of sperm.
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http://dx.doi.org/10.1016/j.jevs.2020.103267DOI Listing
November 2020

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

Theriogenology 2020 Dec 15;158:331-338. Epub 2020 Sep 15.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, 148-16635, Iran.

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.
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http://dx.doi.org/10.1016/j.theriogenology.2020.09.020DOI Listing
December 2020

Effect of Maternal Age on Hippo Pathway Related Gene Expressions and Protein Localization Pattern in Human Embryos.

Cell J 2020 Jul 18;22(Suppl 1):74-80. Epub 2020 Jul 18.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. Electronic Address:

Objective: The Hippo pathway plays an important role in embryo development, and separation of trophectoderm (TE) and inner cell mass (ICM) cell lines. Therefore, this study investigated effect of maternal age on activity of Hippo pathway in human embryos.

Materials And Methods: In this experimental study, the developed up embryos to the blastocyst stage and the embryos whose growth stopped at the morula stage were collected from women aged 20-30 years old (young group, 94 embryos) and >37 years (old group, 89 embryos). Expression of genes and the relevant proteins, in the both groups were evaluated using respectively quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence methods.

Results: There was no significant difference in the expression level of and genes in blastocyst and morula stages, between the two groups. However, and gene expressions in morula stage embryos of the old group was statistically lower than that of the young group (P=0.007 and P=0.008, respectively). Additionally, in the embryos collected from women with >37 years of age, at the blastocyst stage, phospho-YAP (p-YAP) protein was found to be accumulated in the TE, but it was almost disappeared from the ICM. Additionally, in the old group, contrary to the expectation, YAP protein was expressed in the ICM, rather than TE.

Conclusion: The results of this study showed that YAP and P-YAP among the Hippo signalling pathway may be altered by increasing age.
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http://dx.doi.org/10.22074/cellj.2020.6860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481894PMC
July 2020

A novel approach for human sperm cryopreservation with AFPIII.

Reprod Biol 2020 Jun 10;20(2):169-174. Epub 2020 Apr 10.

Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. Electronic address:

Sperm cryopreservation causes different stresses including thermal shock, osmotic damage, and ice crystal formation, thereby reducing sperm quality. Few studies have evaluated the application of AFPs in cryopreservation. The effects of antifreeze protein III (AFP III) on human sperm cryopreservation is not fully understood therefore, we conducted this study to investigate the effects of AFPIII treatment on human sperm parameters following cryopreservation. First, for 20 semen samples the effects of various concentrations of AFPIII (0, 0.01, 0.1, 1, 5, 10 μg/ml) were evaluated. Sperm parameters, such as motility and viability were assessed in order to identify an optimal dose. Next, liquefied 20 semen samples were divided into three aliquots and diluted in glycerol-egg-yolk-citrate (GEYC) cryopreserved without AFPIII (control), with optimal dose of AFPIII, as well as fresh groups. After thawing, samples were evaluated for plasma membrane integrity (PMI), DNA fragmentation index (DFI), reactive oxygen species (ROS), and total antioxidant capacity (TAC) levels. Spermatozoa treatment with 0.01, 0.1 and 1 μg/ml AFPIII increased the sperm motility and viability compared to the control group, but the highest concentrations were ineffective. In conclusion, the results showed that the addition of AFPIII to GEYC at 1 μg/ml improved motility, PMI, viability and TAC, and decreased ROS and DNA fragmentation of cryopreserved human semen compared to the control group.
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http://dx.doi.org/10.1016/j.repbio.2020.03.006DOI Listing
June 2020

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.

Andrologia 2020 May 13;52(4):e13541. Epub 2020 Feb 13.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

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.
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http://dx.doi.org/10.1111/and.13541DOI Listing
May 2020

Knockout serum replacement is an efficient serum substitute for cryopreservation of human spermatozoa.

Cryobiology 2020 02 28;92:208-214. Epub 2020 Jan 28.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran; Reproductive Epidemiology Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. Electronic address:

The freeze-thaw procedure causes irreversible structural and functional changes in human spermatozoa. In order to decrease the detrimental effects of cryopreservation and improve the quality of post-thawed spermatozoa, the constituents of the freezing solution attracted considerable attention. In this study, for the first time, we evaluated the efficacy of knockout serum replacement (KSR) as a substitute for human serum albumin (HSA) for cryopreservation of human spermatozoa. Twenty semen samples were collected from normozoospermic men and divided them into five equal groups. One of the aliquots was diluted with glycerol-based medium as a control group (CON). The other four aliquots were diluted with the sucrose solution containing 5% HSA (H5), 10% HSA (H10), 5% KSR (K5), and 10% KSR (K10). The diluted samples were frozen and preserved in liquid nitrogen. Post thawed sperm parameters including motion characteristics, viability, membrane integrity, mitochondrial activity, acrosome integrity and DNA intactness in all of the sucrose-based groups were comparable with glycerol-based medium. The replacement of HSA by 10% KSR in the freezing medium resulted in significantly higher post-thawed viability, acrosome integrity and DNA intactness compared with other sucrose-based groups. In conclusion, the addition of 10% KSR to the sucrose-based freezing solution improves the quality of post-thawed human spermatozoa and may have potential to develop chemically defined freezing medium.
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http://dx.doi.org/10.1016/j.cryobiol.2020.01.013DOI Listing
February 2020

Suppression of transforming growth factor-beta signaling enhances spermatogonial proliferation and spermatogenesis recovery following chemotherapy.

Hum Reprod 2019 12;34(12):2430-2442

Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

Study Question: Could small molecules (SM) which target (or modify) signaling pathways lead to increased proliferation of undifferentiated spermatogonia following chemotherapy?

Summary Answer: Inhibition of transforming growth factor-beta (TGFb) signaling by SM can enhance the proliferation of undifferentiated spermatogonia and spermatogenesis recovery following chemotherapy.

What Is Known Already: Spermatogonial stem cells (SSCs) hold great promise for fertility preservation in prepubertal boys diagnosed with cancer. However, the low number of SSCs limits their clinical applications. SM are chemically synthesized molecules that diffuse across the cell membrane to specifically target proteins involved in signaling pathways, and studies have reported their ability to increase the proliferation or differentiation of germ cells.

Study Design, Size, Duration: In our experimental study, spermatogonia were collected from four brain-dead individuals and used for SM screening in vitro. For in vivo assessments, busulfan-treated mice were treated with the selected SM (or vehicle, the control) and assayed after 2 (three mice per group) and 5 weeks (two mice per group).

Participants/materials, Setting, Methods: We investigated the effect of six SM on the proliferation of human undifferentiated spermatogonia in vitro using a top-bottom approach for screening. We used histological, hormonal and gene-expression analyses to assess the effect of selected SM on mouse spermatogenesis. All experiments were performed at least in triplicate and were statistically evaluated by Student's t-test and/or one-way ANOVA followed by Scheffe's or Tukey's post-hoc.

Main Results And The Role Of Chance: We found that administration of SB431542, as a specific inhibitor of the TGFb1 receptor (TGFbR1), leads to a two-fold increase in mouse and human undifferentiated spermatogonia proliferation. Furthermore, injection of SB to busulfan-treated mice accelerated spermatogenesis recovery as revealed by increased testicular size, weight and serum level of inhibin B. Moreover, SB administration accelerated both the onset and completion of spermatogenesis. We demonstrated that SB promotes proliferation in testicular tissue by regulating the cyclin-dependent kinase (CDK) inhibitors 4Ebp1 and P57 (proliferation inhibitor genes) and up-regulating Cdc25a and Cdk4 (cell cycle promoting genes).

Limitations, Reasons For Caution: The availability of human testis was the main limitation in this study.

Wider Implications Of The Findings: This is the first study to report acceleration of spermatogenesis recovery following chemotherapy by administration of a single SM. Our findings suggest that SB is a promising SM and should be assessed in future clinical trials for preservation of fertility in men diagnosed with cancer or in certain infertility cases (e.g. oligospermia).

Study Funding/competing Interest(s): This study was supported by Royan Institute and National Institute for Medical Research Development (NIMAD, grant no 963337) granted to H.B. The authors have no conflict of interest to report.
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http://dx.doi.org/10.1093/humrep/dez196DOI Listing
December 2019

The effect of chick embryo extract on mice pre-antral follicles.

Vet Res Forum 2019 15;10(3):213-219. Epub 2019 Sep 15.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.

Chick embryo extract (CEE) contains a variety of growth factors which may improve follicle growth. Therefore, the effect of CEE on mouse pre-antral follicle culture was evaluated. Different percentages of CEE (0, 0.50%, 1.00%, 5.00% and 10.00%) were added to culture medium. Hence, the osmolarity of media was measured. Pre-antral follicles with diameter of 120-150 μm were isolated from 12-14 days old mouse ovary and cultured for 12 days. After culture, the maturation rate was assessed. Granulosa cells viability was evaluated using MTT test and estradiol levels were evaluated using related radio-immunoassay (RIA). Genes expression (BMP15 and ALK6) was also evaluated. The osmolarity of media and granulosa cells viability were the same in all groups. Estradiol level in group with 10.00% CEE was significantly decreased compared to the control group. After 12 days culture, the percentage of antral follicles development was significantly higher in the group with 5.00% CEE compared to control group. The percentage of metaphase II and germinal vesicle breakdown oocytes was significantly higher in group 5.00% CEE compared to control group. The expression of BMP15 gene in antral follicles in 5.00% CEE and control groups was significantly lower compared to pre-antral follicles. However, the expression of ALK6 gene in antral follicles in 5.00% CEE and control groups was not significantly different compared to pre-antral follicles. The increasing effect of CEE on follicle viability with keeping normal gene expression indicates that addition of proper percentage of CEE to culture media improves culture conditions, making it a possible choice to be used as a follicular growth enhancer in infertility clinics.
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http://dx.doi.org/10.30466/vrf.2019.79305.2054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828170PMC
September 2019

The alteration of PLCζ protein expression in unexplained infertile and asthenoteratozoospermic patients: A potential effect on sperm fertilization ability.

Mol Reprod Dev 2020 01 17;87(1):115-123. Epub 2019 Nov 17.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Failed oocyte activation has been observed in unexplained infertile (UI) and asthenoteratozoospermic (AT) men. The deficiency of phospholipase C-zeta (PLCζ) could be a possible reason for such failures and has not been studied yet. We investigated the expression and localization of PLCζ protein in the sperms of patients with UI and AT conditions. The relationships between PLCζ-related parameters with male age, sperm characteristics, DNA integrity, and cellular maturity were assessed. Semen samples were collected from fertile (n = 40), UI (n = 40), and AT (n = 40) men. Subsequently, semen analysis, DNA fragmentation, hyaluronic acid-binding ability, and PLCζ level along with its distribution were evaluated using computer-assisted sperm analyzer, sperm chromatin structure assay (SCSA), hyaluronic acid-binding assay (HBA), western blot analysis and immunofluorescence microscopy, respectively. Unlike SCSA, the values of HBA, and PLCζ expression were significantly reduced in UI and AT patients compared to fertile men, whereas no significant differences were observed among the experimental groups in terms of PLCζ localization patterns. The regression analysis also showed that HBA is the only variable associated with PLCζ levels. Furthermore, the correlation of male age with PLCζ localization in postacrosomal, equatorial, and acrosomal+postacrosomal+equatorial (A+PA+E) patterns, as well as the relation of normal morphology, with the (A+PA+E) pattern, remained in the regression model. Our findings indicated that reduced PLCζ level along with the increased DNA fragmentation and impaired maturation may be possible etiologies of decreased fertilization in the studied subjects.
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http://dx.doi.org/10.1002/mrd.23293DOI Listing
January 2020

Peroxisome Proliferator-Activated Receptors (PPARs) levels in spermatozoa of normozoospermic and asthenozoospermic men.

Syst Biol Reprod Med 2019 Dec 1;65(6):409-419. Epub 2019 Nov 1.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Interest in the role of male factor in infertility continues to mount with defects related to sperm movement considered as one of the more severe forms of subfertility. The peroxisome proliferator-activated receptor gamma (PPARγ) primarily regulates the expression of target genes involved in energy control as well as lipid and glucose metabolism. Although the pivotal roles of these receptors on female fertility have been reported, there are limited studies addressing PPARs role(s) in the male. This study was designed to determine and compare and mRNA expression in sperm cells of normozoospermic and asthenozoospermic men. In addition, flow cytometric analyses, immunofluorescence and western blot were used to evaluate PPARγ protein levels in spermatozoa. We have compared the sperm mRNA relative expression in 27 normozoospermic and 28 asthenozoospermic samples and monitored sperm PPARγ protein levels in 39 normozoospermic and 40 asthenozoospermic samples using flow cytometry. We have also assessed in a sub-group of seven normozoospermic and eight asthenozoospermic samples, PPARγ protein levels by western blotting. Relative expression of PPARγ mRNA in normozoospermic men was found to be significantly higher (P = 0.004) than in asthenozoospermic men while and relative expression was similar in the two groups. Likewise, showed a positive correlation with motility (r = 0.34; P < 0.05), sperm concentration (r = 0.33) and the percentage of progressive motile spermatozoa (r = 0.31). In agreement with the mRNA behavior, sperm PPARγ protein levels as measured by flow cytometry (P = 0.066) and western blot (P = 0.089) showed a tendency to be higher in normozoospermic than asthenozoospermic men. The present study proposes a link between PPARγ gene expression level and motility in human sperm. PPARs: Peroxisome Proliferator-Activated Receptors; CASA: Computer Assisted Semen Analysis; TFA: Trans Fatty Acids; HTF: Human Tubal Fluid; PBS: Phosphate-Buffered Saline; PPP: Pentose Phosphate Pathway; PI3K: Phosphoinositide 3-Kinase; G6PDH: Glucose 6-Phosphate Dehydrogenase.
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http://dx.doi.org/10.1080/19396368.2019.1677801DOI Listing
December 2019

DGC/Zeta as A New Strategy to Improve Clinical Outcome in Male Factor Infertility Patients following Intracytoplasmic Sperm Injection: A Randomized, Single-Blind, Clinical Trial.

Cell J 2020 Apr 8;22(1):55-59. Epub 2019 Sep 8.

School of Allied Medical Science, Tehran University of Medical Sciences, Tehran, Iran.

Objective: The aim of this blind randomised clinical trial study was to assess the clinical efficiency of combined density gradient centrifugation/Zeta (DGC/Zeta) sperm selection procedure compared to conventional DGC in infertile men candidates for intracytoplasmic sperm injection (ICSI). The literature shows that DGC/Zeta is more effective compared to DGC alone in selection of sperms with normal chromatin and improves the clinical outcome of the ICSI procedure. Therefore, this study re-evaluates the efficiency of DGC/Zeta in improving the clinical outcomes of ICSI in an independent clinical setting.

Materials And Methods: In this randomized, single-blind, clinical trial, a total of 240 couples with male factor infertility and at least one abnormal sperm parameter were informed regarding the study and 220 participated. Based on inclusion and exclusion criteria, 103 and 102 couples were randomly allocated into the DGC/Zeta and DGC groups, respectively. ICSI outcomes were followed and compared between the two groups.

Results: Although there was no significant difference in fertilization rate (P=0.67) between the DGC/Zeta and DGC groups, mean percentage of good embryo quality (P=0.04), good blastocysts quality (P=0.049), expanded blastocysts (P=0.007), chemical pregnancies (P=0.005) and clinical pregnancies (P=0.007) were significantly higher in the DGC/ Zeta group compared to DGC. In addition, implantation rate was insignificantly higher in DGC/Zeta compared to DGC (P=0.17).

Conclusion: This is the second independent study showing combined DGC/Zeta procedure improves ICSI outcomes, especially the pregnancy rate, compared to the classical DGC procedure and this is likely related to the improved quality of sperm selected by the DGC/Zeta procedure (Registration number: IRCT20180628040270N1).
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http://dx.doi.org/10.22074/cellj.2020.6525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791063PMC
April 2020

Cryopreservation of rooster semen: Evidence for the epigenetic modifications of thawed sperm.

Theriogenology 2020 Jan 19;142:15-25. Epub 2019 Sep 19.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Although semen cryopreservation is an important, widely used technique for long-term sperm storage, it not only induces partially irreversible damages to sperm but might also deteriorate anatomical, biochemical, and structural organelles. These cellular and epigenetic modifications are the main reasons underlying the decline in sperm motility and fertility during the freeze-thaw process. Using the two Lake and Beltsville semen extenders, the present study aims to evaluate the epigenetic patterns (DNA methylation and histone modification), cellular parameters (e.g., membrane integrity, viability, DNA stability, mitochondria activity, and apoptosis status), and fertility potential of rooster semen collected from six mature roosters before and after cryopreservation according to a standard protocol. The results show that cryopreservation leads to significantly (P < 0.05) reduced values of the parameters examined when compared with those of fresh sperms. While the extenders used exhibit no difference with respect to DNA methylation (DNMT), the Lake extender leads to significant reductions (P < 0.05) in H3K9 acetylation (17.4 ± 1.8) and H3K4 methylation (42 ± 2.3) compared to the Beltsville (9.2 ± 1.8 and 23 ± 2.3, respectively). Compared to the Beltsville extender, the Lake one is also observed to yield a significantly (P < 0.05) superior sperm quality in terms of total motility (TM; 77.2 ± 1.6 vs. 68.3 ± 1.6), average path velocity (VAP; 71 ± 1.4 vs. 53 ± 1.4), and straight-line velocity (VSL; 52 ± 1.5 vs. 34 ± 1.5) as well as significantly (P < 0.05) higher viability (60 ± 1.69 vs. 51 ± 1.69) and membrane functionality (55 ± 3.2 vs. 46 ± 3.2). The Lake extender is also found to outperform the Beltsville one due to its significantly (P < 0.05) higher fertility rate (59.5% vs. 47.2%). The two extenders, however, exhibit no differences in DNA fragmentation, mitochondrial activity, or hatchability rate. The Beltsville extender showed to be superior to the Lake one due to its significantly greater reactive oxygen species percentage (ROS; 45.9 ±.3.2 vs. 28.5 ± 3.2) and apoptosis (29 ± 2.3 vs. 27 ± 2.3). It may be concluded that the Lake extender is capable of improving the cellular and epigenetic parameters of rooster sperms during cryopreservation due to the crucial roles it plays in the protection of sperms against cryo-damages.
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http://dx.doi.org/10.1016/j.theriogenology.2019.09.030DOI Listing
January 2020

Electromagnetic field in human sperm cryopreservation improves fertilizing potential of thawed sperm through physicochemical modification of water molecules in freezing medium.

PLoS One 2019 5;14(9):e0221976. Epub 2019 Sep 5.

Department of Embryology at Reproduction Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACER, Tehran, Iran.

Physicochemical properties of water molecules as the main compositions of the freezing media can be affected by the electromagnetic fled. The purpose of this study was to apply extremely low repetition rate electromagnetic fields (ELEFs) to change the molecular network of water molecules existing in freezing media used for human sperm cryopreservation. First, different time periods and pulsed electromagnetic fields were used to evaluate the physiochemical properties of water. The lowest rate of cluster size, surface tension, viscosity, and density was observed for water samples exposed to 1000 Hz ELEF for 60 min (P < 0.05) that could be results in small ice crystal formation. Therefore, this treatment was selected for further evaluations in human sperm freezing because there was minimal probability of amorphous ice crystallization in this group. To assess fertilizing potential, human semen samples were subjected to ELEF (1000 Hz) water-made freezing medium and cryopreserved. The highest percentage of total motility, progressive motility, viability, membrane integrity, mitochondrial membrane potential, DNA integrity, and TAC were obtained in frozen ELEF as compared to other groups. The percentage of viable spermatozoa (Annexin V-/PI-) in frozen ELEF was significantly higher than in frozen control. The level of ROS was significantly lower in frozen ELEF when compared to frozen control. It can be concluded that the modification of physicochemical properties of water existing in cryopreservation media by ELEF is a suitable strategy to improve the outcome of cryopreservation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221976PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728042PMC
March 2020

Comparison of polymerization and structural behavior of microtubules in rat brain and sperm affected by the extremely low-frequency electromagnetic field.

BMC Mol Cell Biol 2019 08 29;20(1):41. Epub 2019 Aug 29.

Department of Embryology at Reproduction Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACER, Tehran, Iran.

Background: Microtubule proteins are able to produce electromagnetic fields and have an important role in memory formation, and learning. Therefore, microtubules have the potential to be affected by exogenous electromagnetic fields. This study aimed to examine the comparison of microtubule polymerization and its structural behavior in brain and sperm affected by 50 Hz extremely low-frequency electromagnetic field (ELEF).

Results: Twenties adult male rats were randomly and equally divided into control and experimental groups, to evaluate the effect of 50 Hz ELEF on the sperm and brain functions. Plus-maze, serum testosterone and corticosterone, and sperm evaluation were performed. Next, the semen and brain samples were obtained, and they were divided into four experimental groups for investigation of microtubule polymerization. There was no significant difference in testosterone and, corticosterone levels, anxiety behaviors, and sperm morphology between control and ELEF-exposure groups. The sperm viability, total and progressive motility were significantly higher in the ELEF-exposed group than that of the control group. The microtubule polymerization in sperm ELEF was significantly higher than in other groups. The secondary and tertiary structures of tubulins were significantly affected in the brain, and sperm ELEF groups.

Conclusion: It seems that the polymerization of microtubules and conformational changes of tubulin dimers are improved by ELEF application.
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http://dx.doi.org/10.1186/s12860-019-0224-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716927PMC
August 2019

Pre-conditioning with Xanthine oxidase to improve post thawed quality of bull sperm.

Cryobiology 2019 08 17;89:1-5. Epub 2019 Jul 17.

Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.

The purpose of this study was to examine the effects of sub-lethal concentration of Xanthine oxidase (XO) on the post-thawed bull sperm quality. Semen samples were collected from four Holstein bulls, twice a week and during three consecutive weeks (n = 24 total ejaculates). After collection in each replicate, semen samples were pooled and then frozen by semen extender containing different concentrations [0 (XO-0), 0.05 (XO-0.05), 0.5 (XO-0.5), 5 (XO-5), 50 (XO-50) and 500 (XO-500) μM] of XO. After thawing, motion parameters (SCA), plasma membrane functionality (HOST), apoptosis status (Phosphatidylserine translocation assay), mitochondrial activity (Rhodamine 123), and acrosome integrity (PSA), were evaluated. The results showed that total motility, VAP, VSL, VCL, STR, and LIN were lower in XO-50 and XO-500 compared to other groups (P < 0.05). Progressive motility were higher in XO-0.05 and XO-0.5 compared to XO-0, XO-50, and XO-500 (P < 0.05). Mitochondrial activity was highest in XO-0.05 and XO-0.5 groups. Sperm plasma membrane functionality was significantly greater in XO-0, XO-0.05, XO-0.5, and XO-5 than that of XO-50 and XO-500. Xanthine oxidase had not significant effects on acrosome integrity and dead spermatozoa. Higher percentage of live spermatozoa was recorded for XO-0, XO-0.05, XO-0.5, and XO-5; however, the lower amount of apoptotic spermatozoa was detected in the aforementioned groups (P < 0.05). In conclusion, it seems that XO at lower doses may have beneficial effects on post-thawed bull sperm quality.
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http://dx.doi.org/10.1016/j.cryobiol.2019.07.003DOI Listing
August 2019

The Application of Ultrasonic Vibration in Human Sperm Cryopreservation as a Novel Method for the Modification of Physicochemical Characteristics of Freezing Media.

Sci Rep 2019 07 11;9(1):10066. Epub 2019 Jul 11.

Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran.

The application of ultrasonic vibration was performed to modify the water molecules as the main compositions of the freezing medium used for human sperm cryopreservation. Different time periods of ultrasonic vibration (ULV) at the frequency of 28 kHz were applied for the evaluation of physicochemical properties of the water molecules. The most significant bubble size, zeta potential, and pH were obtained for the water molecules exposed to ultrasonic vibrations for 18 minutes and this time period was selected for further experiments due to the optimum results. In the next stage, semen samples were diluted with freezing medium containing ULV-exposed water and then cryopreserved. All the semen parameters were significantly reduced in cryopreserved groups as compared with the fresh control group. The highest percentage of total and progressive motility, viability, membrane and DNA integrity, and mitochondrial membrane potential were observed in frozen ULV compared with the frozen control. The rate of apoptosis in frozen ULV was significantly lower than that of in the frozen control. Furthermore, the gene expression ratios of α- and β-tubulins were significantly increased during cryopreservation, while the expression ratio of the tubulin polymerization promoting protein (TPPP) gene was decreased. Similar results were also observed when the protein levels of the genes mentioned earlier were evaluated by the ELISA method. Therefore, the changes in physicochemical properties of the freezing medium of human sperm cryopreservation using ULV can improve the quality of frozen products.
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http://dx.doi.org/10.1038/s41598-019-46424-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624318PMC
July 2019

Changes in seminal parameters and hormonal profile with use of aromatase inhibitor in management of aging broiler breeder roosters.

Poult Sci 2019 Nov;98(11):6100-6107

Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran 16635-148, Iran.

An excessive amount of aromatase enzyme reduces reproductive performance in aging roosters. Testosterone metabolism by aromatase enzyme is one of the reasons for reduced testosterone and lower fertility of aging roosters. The purpose of this study was to determine the effects of Exemestane (EX), as a steroidal aromatase inhibitor, on the seminal parameters and reproductive hormones of aging roosters. A total of 20 roosters (45 wk of age) were housed in individual cages and received a standard basal diet and oral EX capsules for 60 D at the daily doses per rooster (mg/rooster) in the following experimental groups: 0 mg (CTRL), 0.25 mg (Ex-0.25), 0.5 mg (Ex-0.5), and 1.5 mg (Ex-1.5). Sperm samples were obtained on days 1, 20, 40, and 60 of experiment. Blood samples were taken on days 1 and 60. The results indicated that different EX dosages affected semen parameters (P < 0.05) other than semen volume, morphology, apoptosis, and acrosome integrity. Various semen characteristics were significant (P < 0.05) during different times of the experiment, with the exception of semen volume, total motility, membrane integrity, morphology, apoptosis, and acrosome integrity. Roosters that received 0.5 mg of EX had higher percentages of sperm concentration, total motility, progressive motility, membrane integrity, viability, and mitochondrial activity (P < 0.05). There were lower concentrations of malondialdehyde in the CTRL (0 mg) and Ex-0.25 groups (P < 0.05). Although there was no significant difference in hormones at day 0 of the experiment (P > 0.05), roosters in the Ex-0.5 had higher concentration of testosterone as well as lower of aromatase activity at day 60 (P < 0.05). It can be concluded that EX improved semen parameters and testosterone, which ultimately can increase fertility in the aging broiler breeder roosters.
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http://dx.doi.org/10.3382/ps/pez325DOI Listing
November 2019

Dietary trans and saturated fatty acids effects on semen quality, hormonal levels and expression of genes related to steroid metabolism in mouse adipose tissue.

Andrologia 2019 Jun 15;51(5):e13259. Epub 2019 Mar 15.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Our objectives were to assess sperm alteration and adipose tissue (AT) genes expression related to steroid metabolism subsequent to fatty acids consumption. Twenty-nine mature male mice were divided into: fat diet (FD; n = 15) and the control group (n = 14). FD group was fed with low level of trans and saturated fatty acids source for 60 days. Sperm parameters, levels of hormones and the mRNA abundance of the target genes in AT were assessed. The sperm concentration, total and progressive motilities were lower in FD group compared to that of control (p < 0.01). Blood estradiol levels increased in FD (p < 0.001), whereas no significant difference was observed in testosterone. The mRNA levels of StAR, CYP11A1, CYP17A1, 17βHSD7 and 17βHSD12 in AT of FD were higher than those of the control (p < 0.05). In contrast, mRNA level of Cyp19a1 in FD was significantly (p < 0.05) lower than that of control. 17βHSD12 and 17βHSD7 (as oestrogenic genes) increased, while 17βHSD5 and 17βHSD3 (as androgenic genes) remained unchanged, indicating that dietary trans/saturated fatty acids affect AT genes expression. Probably, sperm parameters were altered by increment of expression level of genes involved in oestrogenic metabolism rather than those engaged in androgenic metabolism after fatty acids consumption.
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http://dx.doi.org/10.1111/and.13259DOI Listing
June 2019

Preconditioning of sperm with sublethal nitrosative stress: a novel approach to improve frozen-thawed sperm function.

Reprod Biomed Online 2019 Mar 8;38(3):413-425. Epub 2019 Jan 8.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR Tehran, Iran.

Research Question: Can sublethal stress induced by nitric oxide on fresh human spermatozoa protect the functional properties of post-thaw human spermatozoa?

Design: Semen samples were obtained from 46 donors. Twenty semen samples were used to determine toxicity level of nitric oxide by incubation of semen with different concentrations of nitric oxide (0.01 to 400 μM). Then, 26 semen samples were cryopreserved with optimized ranges of nitric oxide: control (NO-0.00), 0.01 μM nitric oxide (NO-0.01), 0.1 μM nitric oxide (NO-0.1), 1 μM nitric oxide (NO-1), 10 μM nitric oxide (NO-10), 100 μM nitric oxide (NO-100). Frozen-thawed spermatozoa were assessed for motion characteristics, viability, morphology, apoptosis-like changes, caspase 3 activity, DNA fragmentation and intracellular reactive oxygen species levels. Fertilization potential was investigated by heterologous piezo-intracytoplasmic sperm injection (piezo-ICSI) of human spermatozoa into mouse oocytes.

Results: In fresh spermatozoa, nitric oxide did not induce a negative effect, except a significant reduction in motility and viability at 200 µM and 400 µM (P < 0.05). Cryopreservation significantly reduced sperm motility and increased reactive oxygen species, apoptosis-like changes, caspase 3 activity, and DNA damage (P < 0.05). NO-0.01 significantly increased total and progressive motility versus the other groups (P < 0.05). The lowest percentage of caspase 3 activity was in the NO-0.01 and NO-0.1 compared with the other freezing groups. In the fertilization trial, the rate of two-cell embryo formation after heterologous piezo-ICSI was higher (P < 0.05) in NO-0.01 (69%) versus controls (42%).

Conclusions: Sublethal oxidative stress induced by nitric oxide might improve human sperm function after cryopreservation.
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http://dx.doi.org/10.1016/j.rbmo.2018.11.029DOI Listing
March 2019

Micro-quantity straw as a carrier for cryopreservation of oligozoospermic semen samples: Effects of storage times and cryoprotectant.

Cryobiology 2019 02 8;86:65-70. Epub 2018 Dec 8.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. Electronic address:

Application of an appropriate freezing carrier is crucial for improving post-thaw recovery of oligozoospermic samples. In this study, our purpose is developing a user-friendly, easy handling and close micro-quantity (MQ) straw along with different freezing media, for cryopreservation of oligozoospermic samples. Twenty oligozoospermic semen samples were collected and mixed with glycerol egg yolk citrate (GEYC) or Spermfreeze (SPF) medium. The mixture was loaded into MQ straws, sealed and stored in liquid nitrogen (LN) vapor. After freezing, the straws were transferred into cryotube and plunged into LN. Post-thawed sperm parameters including motion characteristics, viability, membrane and DNA integrity were evaluated one and three months after cryopreservation. The post-thawed sperm parameters were significantly reduced in GEYC and SPF medium compared to fresh samples. No statistically significant differences were seen in sperm characteristics between the two storage times (i.e. month 1 vs. month 3). Furthermore, GEYC medium yielded higher motility, viability and membrane integrity compared to SPF at both storage time-points. Sperm DNA integrity was also improved in GEYC group compared to SPF after 1 month of storage. The findings of our study showed that application of MQ straw along with GEYC, as the cryoprotectant, was beneficial for cryopreservation of low count semen samples.
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http://dx.doi.org/10.1016/j.cryobiol.2018.12.003DOI Listing
February 2019

The effect of Verapamil on ischaemia/reperfusion injury in mouse ovarian tissue transplantation.

Biomed Pharmacother 2018 Dec 4;108:1313-1319. Epub 2018 Oct 4.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.

One of the challenges that must be overcome during ovarian tissue transplantation is Ischaemia/Reperfusion Injury (IRI). The most important hypothesis explaining the cellular events in I/R processes are calcium overload and oxygen free radicals constitute. Here, we study the effect of verapamil on IRI, and consequently on follicle survival during ovarian transplants in an autograft model. Female mice were randomly assigned into three groups in order to ovarian autotransplantation as follow: Group 1 (Control group), Group 2 (Transplanted group) and Groups 3 (Transplanted + Verapamil group). The grafted ovaries were collected at 3, 7 and 14 days after transplantation for evaluation of follicle content and morphology, apoptosis and Malondialdehyde (MDA) concentration. The results showed that verapamil treatment significantly preserved primordial follicular reserve and reduced the number of degenerated follicles compared to the transplanted group (P <  0.05). MDA levels were significantly higher on the 14th day after transplantation, in group 2 than in group 3. In conclusion, verapamil treatment is effective for the preservation of the follicular pool and reducing tissue damage induced by transplantation of ovarian tissue.
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http://dx.doi.org/10.1016/j.biopha.2018.09.130DOI Listing
December 2018

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

Anim Reprod Sci 2018 Nov 29;198:193-201. Epub 2018 Sep 29.

Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

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.
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http://dx.doi.org/10.1016/j.anireprosci.2018.09.019DOI Listing
November 2018

Optimizing methods for human testicular tissue cryopreservation and spermatogonial stem cell isolation.

J Cell Biochem 2019 01 22;120(1):613-621. Epub 2018 Sep 22.

Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

Cryopreservation of testicular tissue before cancer therapy for fertility preservation in prepubertal boys with cancer is of great interest in reproductive medicine. Isolation of spermatogonial stem cells (SSCs) from cryopreserved tissues would be a suitable cell source to re-establish spermatogenesis after cancer therapy. We herein establish optimized protocols for cryopreservation of human testicular tissue and isolation of SSCs from cryopreserved tissue. We developed a freezing protocol that provided high testicular cell viability and supported structural integrity and tubular epithelium coherence similar to fresh tissue. Then, we established a protocol that allowed efficient isolation of functional SSCs from cryopreserved tissues. Isolated cells were found on the testicular basement membrane after xenotransplantation. Our results demonstrated the preservation of testicular tissue structure and high cell viability with efficient isolation of SSCs after testicular cryopreservation, which is promising for future therapeutic applications in fertility preservation.
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http://dx.doi.org/10.1002/jcb.27419DOI Listing
January 2019

Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches.

Reprod Biomed Online 2018 09 22;37(3):327-339. Epub 2018 Aug 22.

Department of EmbryologyReproductive Biomedicine Research CentreRoyan Institute for Reproductive BiomedicineACECRTehranIran. Electronic address:

The cryopreservation of spermatozoa was introduced in the 1960s as a route to fertility preservation. Despite the extensive progress that has been made in this field, the biological and biochemical mechanisms involved in cryopreservation have not been thoroughly elucidated to date. Various factors during the freezing process, including sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for poor sperm quality post-thaw. Little is known regarding the new aspects of sperm cryobiology, such as epigenetic and proteomic modulation of sperm and trans-generational effects of sperm freezing. This article reviews recent reports on molecular and cellular modifications of spermatozoa during cryopreservation in order to collate the existing understanding in this field. The aim is to discuss current freezing techniques and novel strategies that have been developed for sperm protection against cryo-damage, as well as evaluating the probable effects of sperm freezing on offspring health.
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http://dx.doi.org/10.1016/j.rbmo.2018.05.012DOI Listing
September 2018