Direct visualization of pre-protamine 2 detects protamine assembly failures and predicts ICSI success.

Histone-to-protamine transition is an essential step in the generation of fully functional spermatozoa in various mammalian species. In human and mouse, one of the two protamine-encoding genes produces a precursor pre-protamine 2 (pre-PRM2) protein, which is then processed and assembled. Here, we design an original approach based on the generation of pre-PRM2-specific antibodies to visualize the unprocessed pre-PRM2 by microscopy, flow cytometry and immunoblotting. Using mouse models with characterized failures in histone-to-protamine replacement, we show that pre-PRM2 retention is tightly linked to impaired nucleosome disassembly. Additionally, in elongating/condensing spermatids, we observe that pre-PRM2 and transition protein are co-expressed spatiotemporally, and their physical interaction suggests that these proteins act simultaneously rather than successively during histone replacement. By using our anti-human pre-PRM2 antibody, we also measured pre-PRM2 retention rates in the spermatozoa from 49 men of a series of infertile couples undergoing ICSI, which shed new light on the debated relation between pre-PRM2 retention and sperm parameters. Finally, by monitoring 2-pronuclei embryo formation following ICSI, we evaluated the fertilization ability of the sperm in these 49 patients. Our results suggest that the extent of pre-PRM2 retention in sperm, rather than pre-PRM2 accumulation per se, is associated with fertilization failure. Hence, anti-pre-PRM2 antibodies are valuable tools that could be used in routine monitoring of sperm parameters in fertility clinics, as well as in experimental research programmes to better understand the obscure process of histone-to-protamine transition.

Proteomics study reveals the molecular mechanisms underlying cryotolerance induced by mild sublethal stress in human sperm.

The preconditioning of human sperm with sublethal nitrosative stress before cryopreservation can potentially improve the thawed sperm quality. However, the underlying mechanisms behind this protective strategy are not entirely understood. We compared the cryosurvival of human sperm exposed to 0.01 µM nitric oxide (NO) throughout the cryopreservation and used multiplexed quantitative proteomics approach to identify changes in the proteome profile of preconditioned sperm cells. Semen samples were obtained from 30 normospermia donors and then each sample was divided into three equal parts: fresh (F), frozen-control (C), and frozen exposed to nitric oxide (NO). The sperm undergoing mild sublethal stress showed higher values for motility and viability compared to the frozen control sperm. Moreover, out of 2912 identified proteins, 248 proteins were detected as differentially abundant proteins (DAPs) between cryopreserved groups and fresh group (F) (p < 0.05). Gene ontology (GO) analysis of differentially abundant proteins indicated that the abundance of proteins associated with glycolysis, gluconeogenesis, and fertilization processes was reduced while oxidative phosphorylation pathway was increased in abundance in cryopreserved sperm compared to the fresh sperm. Moreover, redox protein such as thioredoxin 17 was increased in abundance in the NO group compared to the control freezing group. Therefore, the pre-conditioning of sperm prior to cryopreservation may play an important role in maintaining the redox balance in mitochondria of sperm after freezing. Overall, our results indicate that arylsulfatase A (ARSA), serine protease 37 (PRSS37), and sperm surface protein (SP17) may potentially serve as protein biomarkers associated with screening the fertilization potential of the thawed sperm.

Human uterine fluid lavage-derived extracellular vesicle isolation: a comparative study for minimally invasive endometrial receptivity assessment.

RESEARCH QUESTION: Does pre-implantation uterine fluid lavage (UFL) of patients undergoing IVF and frozen embryo transfer (FET) affect implantation and clinical pregnancy rates? Which methods among ultracentrifugation, sucrose cushion and qEV column are suitable for isolating UFL extracellular vesicles? DESIGN: First, UFL was collected from 20 patients undergoing IVF and FET 2 days before embryo transfer as the case group. The control group consisted of 20 patients undergoing IVF and FET patients without lavage. All patients were monitored for 6 weeks. In the next step, the UFLs (n = 30) were collected and pooled. The UFL-derived extracellular vesicles were extracted by ultracentrifugation, sucrose cushion and qEV column methods and characterized. RESULTS: Preimplantation uterine lavage sampling did not affect implantation and clinical pregnancy rates. Extracellular vesicles were successfully isolated from UFL by all three methods. Scanning electron microscopy and dynamic light scattering analysis showed that the isolated vesicles were morphologically spherical. The qEV technique showed that they were smaller and homogenized in size. SDS-PAGE of extracellular vesicles showed a weaker albumin band in the qEV column. Western blot analysis indicated that the isolated extracellular vesicles by the qEV column were more immunoreactive for all the common extracellular vesicle markers (CD81, CD9, CD63, and TSG101). Six reference genes were compared by real-time polymerase chain reaction in the isolated extracellular vesicle subpopulations, and lowest cycle threshold value was observed for the 18SrRNA gene. CONCLUSIONS: The isolation of endometrial secretome extracellular vesicles is a minimally invasive procedure for individual assessment of endometrial receptivity and can be carried out during conception cycles along with transvaginal ultrasonography. Molecular analysis of UFL-derived extracellular vesicle components could suggest biomarkers to determine precise extracellular vesicle timing.

Epigenetic modifications at DMRs of imprinting genes in sperm of type 2 diabetic men.

High rates of infertility in type 2 diabetic (T2DM) men have led to attempts to understand the mechanisms involved in this process. This condition can be investigated from at least two aspects, namely sperm quality indices and epigenetic alterations. Epigenetics science encompasses the phenomena that can lead to inherited changes independently of the genetics. This study has been performed to test the hypothesis of the relationship between T2DM and the epigenetic profile of the sperm, as well as sperm quality indices. This research included 42 individuals referred to the infertility clinic of Royan Institute, Iran in 2019-2021. The study subjects were assigned to three groups: normozoospermic non-diabetic (control), normozoospermic diabetic (DN) and non-normozoospermic diabetic (D.Non-N). Sperm DNA fragmentation was evaluated using the sperm chromatin structure assay technique. The global methylation level was examined using 5-methyl cytosine antibody and the methylation status in differentially methylated regions of H19, MEST, and SNRPN was assessed using the methylation-sensitive high-resolution melting technique. The results showed that the sperm global methylation in spermatozoa of D.Non-N group was significantly reduced compared with the other two groups (P < 0.05). The MEST and H19 genes were hypomethylated in the spermatozoa of D.Non-N individuals, but the difference level was not significant for MEST. The SNRPN gene was significantly hypermethylated in these individuals (P < 0.05). The results of this study suggest that T2DM alters the methylation profile and epigenetic programming in spermatozoa of humans and that these methylation changes may ultimately influence the fertility status of men with diabetes.

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

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.

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

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.

Effect of oocyte dysmorphisms on intracytoplasmic sperm injection cycle outcomes in normal ovarian responders.

AIM: The aim of this study was to identify the influence of oocyte dysmorphisms on clinical outcomes after intracytoplasmic sperm injection cycle in normal responders. MATERIAL AND METHODS: In the prospective study, morphology of 1999 metaphase II oocytes retrieved from 316 intracytoplasmic sperm injection cycles was evaluated from March 2011 to March 2013 at Royan Institute. Controlled ovarian stimulation was performed by long standard agonist protocol. Oocyte morphology was assessed before sperm injection by one embryologist. The associations between fertilization rate, embryo quality and the independent variables were analyzed using odds ratio (OR) calculated with unconditional logistic regression test. RESULTS: From all retrieved oocytes, 1543 (77.1%) showed at least one morphologic aberration. Presence of cytoplasmic vacuoles and high cytoplasmic viscosity were associated with a significant decrease in the fertilization rate (OR: 0.5; P = 0.004 and OR: 0.6; P = 0.03, respectively). The results showed that oocyte morphology did not affect embryo quality. The number of gonadotrophin injections used showed a direct relation with presence of large perivitelline space. No significant difference was observed among four groups (women with total normal morphologic oocytes [group 1], women with total extracytoplasmic dysmorphic oocytes [group 2], women with total cytoplasmic dysmorphic oocytes [group 3] and women with total oocytes containing multiple dysmorphic features [group 4]) in terms of implantation and clinical pregnancy rates. CONCLUSIONS: Metaphase II oocyte morphology had minor impacts on fertilization rate, pronuclear morphology and embryo quality in women with normal ovarian response.

Can We Harvest More Mature Oocytes by Repeating Gonadotropin-Releasing Hormone Agonist Doses in Polycystic Ovarian Syndrome Patients at Risk of OHSS in Antagonist Cycles? A Randomised Clinical Trial.

BACKGROUND: There is an ongoing debate about the optimal dosage of gonadotropin-releasing hormone (GnRH) agonist for oocyte triggering in polycystic ovarian syndrome (PCOS) patients at risk for ovarian hyperstimulation syndrome (OHSS). In this study, we intend to ascertain whether the use of repeated doses of a GnRH agonist for oocyte triggering in these patients can enhance the outcomes of controlled ovarian stimulation (COS) for in vitro fertilization/ intracytoplasmic sperm injection (IVF/ICSI) cycles. MATERIALS AND METHODS: This randomised clinical trial enrolled 70 PCOS women candidates for IVF/ICSI with the standard antagonist protocol at Royan Institute (Tehran, Iran) from May 2020 to June 2022. Patients at risk of OHSS with oestradiol (E2) levels >3000 pg/ml on the day of trigger were randomly assigned to a control or experimental group. Group A (control group) patients received 0.2 mg triptorelin (Decapeptyl®) for final oocyte maturation. Group B (experimental group) patients received a second dose of 0.1 mg Decapeptyl®12 hours after their first dose, for a total dose of 0.3 mg. IVF/ICSI outcomes were compared between the groups. RESULTS: Ultimately, 35 women from the study group and 33 from the control group completed the treatment cycle. Both groups were comparable in terms of demographic characteristics, baseline hormonal profiles, and PCOS phenotypes. The dosage of gonadotropin, stimulation duration, number of retrieved oocytes, oocyte maturation rate, and oocyte recovery ratio did not significantly differ between the groups. No significant differences were found in terms of the number of blastocyst and cleavage embryos, nor the quality of obtained embryos between the groups. The mild to moderate OHSS rate was significantly lower in the study group (P=0.038). CONCLUSION: A second dose of GnRH agonist 12 hours after the first dose did not improve the number and maturity of oocytes, or pregnancy outcomes in PCOS patients (registration number: NCT04600986).

Does The Culture of Post-Thawed Cleavage-Stage Embryos to Blastocysts Improve Infertility Treatment Outcomes of Frozen-Thawed Embryo Transfer Cycles? A Randomised Clinical Trial.

BACKGROUND: There is a definite shift in assisted reproductive centres from cleavage-stage embryo transfer (ET) to blastocyst transfer that is attributed to improvements in laboratory environments and advances in the development of embryo culture media. The aim of the study was to investigate the reproductive outcomes of thawed cleavage-stage ET versus blastocysts derived from an extended culture of these embryos. MATERIALS AND METHODS: This open-label, randomised, parallel group clinical trial study enrolled 182 women aged ≤37 years who underwent frozen-thawed ET from November 2015 to June 2020 at Royan Institute Research Centre, Tehran, Iran. The women were randomly assigned to either the thawed cleavage ET group (n=110) or the post-thaw extended culture blastocysts group (n=72). The primary outcome measure was the clinical pregnancy rate. Secondary outcome measures were implantation rate, live birth rate (LBR), and miscarriage rate. A P<0.05 indicated statistical significance. RESULTS: There were no significant differences between the two groups in terms of demographic characteristics. Both the mean numbers of embryos transferred and good quality embryos transferred were significantly lower in the postthaw extended culture blastocysts group compared to thawed cleavage-stage ET cycles. However, the post-thaw extended culture blastocysts group had higher clinical pregnancy (56.94 vs. 40.91%, P=0.034), implantation (34.43 vs. 19.84%, P=0.001) and live birth (49.3 vs. 33.63%, P=0.036) rates compared to the thawed cleavage-stage ET group. Miscarriage and multiple gestations rates were comparable between the groups. CONCLUSION: These results allow us to take a position in favour of post-thaw extended culture blastocysts; thus, it is important to improve the post-thawing extended culture technique (registration number: NCT02681029).

Upregulation of Oxidative Phosphorylation Genes in Cumulus Cells of The Polycystic Ovary Syndrome Patients with or without Insulin Resistance.

OBJECTIVE: The relationship between oxidative stress (OS), insulin resistance (IR), and polycystic ovary syndrome (PCOS) is an important medical issue in human reproduction. Some of the oxidative phosphorylation (OXPHOS) genes have been previously studied in granulosa and muscle cells of PCOS patients. Cumulus cells (CCs) remain close to the oocyte even after ovulation. This research has been designed to compare the expression of OXPHOS genes in CCs of PCOS, with or without insulin resistance. MATERIALS AND METHODS: In this experimental study, patients were included in PCOS insulin-resistant, PCOS insulinsensitive (IS), and control (fertile women with male infertility history) groups. The expression of NCF2, TXNIP, UCP2, NDUFB6, ATP5H, COX7C, NDUFA3, SDHA, and SDHB was studied by real-time polymerase chain reaction (PCR), and normalization was performed considering HPRT1 and CYC1 as reference genes. One-way ANOVA and Tukey test were used for data analysis. RESULTS: The results showed that the expression of NCF2, TXNIP, UCP2, and ATP5H was significantly higher in the IR group than IS and control groups (P<0.01). NDUFB6 showed the highest expression in the IS group, which was significantly different from other groups (P<0.01). The other genes of interest, except COX7C, were observed with the most transcriptional levels in the IS group, although there was no significant difference for those genes. CONCLUSION: Altered expression of genes involved in mitochondrial function compared to the control group in CCs of both IR and IS categories of the PCOS patients suggests that alteration in OXPHOS genes can contribute to the pathophysiology of PCOS.

Enhanced generation of human embryonic stem cells from single blastomeres of fair and poor-quality cleavage embryos via inhibition of glycogen synthase kinase ß and Rho-associated kinase signaling.

STUDY QUESTION: Could selected pluripotency-enhancing small molecules (SMs) lead to efficient derivation of human embryonic stem cells (hESCs) from cleavage embryos-derived single blastomeres (SBs)? SUMMARY ANSWER: Inhibition of glycogen synthase kinase ß (GSK3ß) and Rho-associated kinase (ROCK) signaling can enhance the derivation of hESCs from cleavage embryo-derived SBs. WHAT IS KNOWN ALREADY: Parameters involved in sustaining the pluripotency of biopsied blastomeres for generating hESCs without causing injury to a viable embryo have remained obscure. This research seeks to improve the culture conditions for increasing the efficiency of deriving hESCs from SBs from cleavage-stage embryos by using SMs. STUDY DESIGN, SIZE, DURATION: In order to identify SMs which may enhance hESC generation from SBs, 11 pluripotency-enhancing SMs were screened and CHIR99021 (CH), a GSK3ß inhibitor, was selected. To optimize culture condition in hESC generation from SMs, we used ROCK inhibitor Y27632 (Y) and basic fibroblast growth factor in combination with CH or its alternative, Kenpaullone, in different time courses over 12 days. We also assessed a critical time point for CH + Y treatment of cleavage embryos from 4- to 8-cell embryo. In total, 224 embryos and 1607 SBs were used in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blastomeres of fair and poor-quality from 6- to 8-cell stage human embryos were mechanically dispersed and individually seeded into a 96-well plate that was precoated with mitotically inactivated feeder cells. Derivation of hESC line from each SB was carried out in hESC defined medium supplemented with SMs. Randomly selected hESC lines were evaluated by immunostaining for pluripotency markers, karyotype analysis and differentiation potential into the three embryonic germ layer derivatives. MAIN RESULTS AND THE ROLE OF CHANCE: We found that 3 µM CH was the only SM that was capable of directing SBs from fair and poor-quality 6-8-cell embryos into hESC lines. The application of hESC-conditioned medium had no additive effect on hESC establishment from SBs. Also, we indicated that CH combined with Y improved hESC generation efficiency by up to 31%. By using of Kenpaullone as an alternative to CH, we confirmed the involvement of GSK3 inhibition in hESC derivation from SBs. Interestingly, by treatment of 4-cell embryos, these SMs could enhance the derivation efficiency of SB-derived hESC lines up to 73% and the maximum number of hESC lines from SBs of one embryo was achieved in this state. LIMITATIONS, REASONS FOR CAUTION: The low quality of the embryos used in this study most likely had an effect on hESC generation. Furthermore, although we attempted to minimize any differences in inter-embryo quality, we cannot exclude the possibility that small differences in starting quality between embryos may have contributed to the differences observed, other than the addition of SMs. WIDER IMPLICATIONS OF THE FINDINGS: This approach would allow the establishment of autogeneic or allogeneic matched cells from embryos fertilized in vitro without destroying them. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the National Elite Foundation and the Royan Institute for Stem Cell Biology and Technology. The authors have no conflict of interest to declare.

Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells.

There are growing numbers of recombinant proteins that have been expressed in milk. Thus one can consider the placement of any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Among the transgene introducing techniques, only nuclear transfer (NT) allows 100 % efficiency and bypasses the mosaicism associated with counterpart techniques. In this study, in an attempt to produce a transgenic goat carrying the human coagulation factor IX (hFIX) transgene, goat fetal fibroblasts were electroporated with a linearized marker-free construct in which the transgene was juxtaposed to ß-casein promoter designed to secret the recombinant protein in goat milk. Two different lines of transfected cells were used as donors for NT to enucleated oocytes. Two transgenic goats were liveborn. DNA sequencing of the corresponding transgene locus confirmed authenticity of the cloning procedure and the complementary experiments on the whey demonstrated expression of human factor IX in the milk of transgenic goats. In conclusion, our study has provided the groundwork for a prosperous and promising approach for large-scale production and therapeutic application of hFIX expressed in transgenic goats.

The effect of intra-peritoneal administration of Papaver bracteatum Lindl. extract on development of NMRI mice oocytes treated with Doxorubicin.

PURPOSE: The effect of water-alcohol Papaver bracteatum Lindl. extract on development of mice oocytes treated with Doxorubicin (dox) was examined in this study. METHODS: The mice were classified into four groups. Control group, mice injected intraperitoneally (IP) with saline. Extract group alone, mice treated with 200 mg/kg of body weight (bw), IP, twelve consecutive days. Dox group alone, mice were given dox, IP, 10 mg/kg bw. Experimental group treated with extract and dox together. Effect of the extract on the development of mice oocytes treated with dox were evaluated through assisted reproductive technology techniques (ARTs). RESULTS: Developmental rate and blastocyst formation was improved by using the extract. A significant increase in in vitro developmental competence in comparison with dox group (P < 0.05) was observed. CONCLUSIONS: The results of this study indicated that P. bracteatum Lindl. extract could prevent dox toxicity of dox affecting both follicle or oocytes, and therefore it can result in improved embryo development which was observed in mice treated with dox plus P. bracteatum Lindl. compared to mice treated with dox alone.

Abdominal Obesity May Play a Significant Role in inflammation Exacerbation in Polycystic Ovary Syndrome Patients.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is an endocrine disorder that seems to be pro-inflammatory at many levels, abdominal obesity (AO) is a prevalent pro-inflammatory phenotype in PCOS patients, and it seems to contribute to the initiation or worsening of inflammation in PCOS patients. In this study, we investigated the role of the AO phenotype in the occurrence of other obesity indicators (neck and arm) and augmentation of inflammation in the follicular fluid (FF) of PCOS patients. METHODS: 40 patients under the age of 35 were divided into four groups: PCOS with AO, PCOS without AO, non-PCOS with AO, and non-PCOS without AO. The FF samples were collected from each patient. Clinical and anthropometric characteristics of the participants, as well as tumor necrosis factor-α (TNF-α) concentration in the FF samples, were quantitatively assessed using enzyme-linked immunosorbent assays. The number of retrieved cumulus-oocyte complexes (COC) and their quality were scored. RESULTS: The PCOS+AO+ group had significantly increased neck circumference, compared to the other groups (p<0.001). The concentration of TNF-α was significantly higher in the PCOS+AO+ group than in the other groups (p<0.001). There were no significant differences in the number of retrieved COC per patient and the quality of oocytes between the groups (p>0.05). CONCLUSIONS: Given the significant role of inflammation in the development of PCOS, managing AO in PCOS patients may aid in reducing inflammation and could potentially help in the design of customized treatment approaches.

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.

Embryo Condition Media Collected from Polycystic Ovary Syndrome Patients with Abdominal Obesity Can Increase The Decidualization Potential of Healthy Endometrial Stromal Cells.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrinological disorder associated with abdominal obesity (AO) and some reproductive complications including low pregnancy rate. Embryo-endometrium cross-talk has a key role in successful embryo implantation and subsequent normal pregnancy rate. The primary objective of this study is to evaluate the decidualization potential of endometrial stromal cells (ESCs) using the embryo condition media (ECM) collected from PCOS patients with AO, compared to ECM of those patients without AO. MATERIALS AND METHODS: In this experimental study, we measured the capacity of ECM collected from PCOS patients with or without AO for decidualization induction in healthy ESCs after coculture. A total number of 53 embryos from 40 couples belonging to PCOS with AO, PCOS without AO, nonPCOS with AO, and nonPCOS without AO patients, were included in our study. The embryosof four groups were single-cultured up to the blastocyst stage. Their ECM (45λ/well) were pooled and added to healthy ESCs monolayer culture media to investigate their effects on decidualization potential via gene (PRL, IGFBP1, IL1-ß, HOXA10, IL-6 and TNF-α) and protein (PRL, IGFBP1, IL1-ß) expression analysis and ESCs migration assay. RESULTS: The morphological analysis, migration assay (P≤0.0321), protein (P≤0.0139) and gene expression analysis showed PCOS with AO accounted for the highest gene (PRL, IGFBP1, IL1-ß, HOXA10, IL-6, TNF-α) and protein markers (PRL, IGFBP1, IL1-ß) (P≤0.05). NonPCOS individuals without AO had the lowest level of both gene and protein decidualization markers (P≤0.05). CONCLUSION: Considering decidualization as an inflammatory process, a higher level of decidualization markers was associated with a higher inflammatory status created by AO and PCOS, separately. Inflammation may disrupt the process of inflammatory to anti-inflammatory phase required for prevention of pregnancy loss, this could explain the high rate of abortion in these cases.

Ultrastructural changes of sheep cumulus-oocyte complexes following different methods of vitrification.

To determine the ultrastructural changes of sheep cumulus-oocyte complexes (COCs) following different methods of vitrification, good quality isolated COCs (GV stage) were randomly divided into the non-vitrified control, conventional straw, cryotop and solid surface vitrification groups. In both conventional and cryotop methods, vitrified COCs were respectively loaded by conventional straws and cryotops, and then plunged directly into liquid nitrogen (LN2); whereas in the solid surface group, vitrified COCs were first loaded by cryotops and then cooled before plunging into LN2. Post-warming survivability and ultrastructural changes of healthy COCs in the cryotop group especially in comparison with the conventional group revealed better viability rate and good preservation of the ooplasm organization. However in all vitrification groups except the cryotop group, mitochondria were clumped. Solely in the conventional straw group, the mitochondria showed different densities and were extremely distended. Moreover in the latter group, plenty of large irregular connected vesicles in the ooplasm were observed and in some parts their membrane ruptured. Also, in the conventional and solid surface vitrification groups, cumulus cells projections became retracted from the zona pellucida in some parts. In conclusion, the cryotop vitrification method as compared with other methods seems to have a good post-warming survivability and shows less deleterious effects on the ultrastructure of healthy vitrified-warmed sheep COCs.

Altered Expression of GDF9 and BMP15 Genes in Granulosa Cells of Diminished Ovarian Reserve Patients: A Case-Control Study.

OBJECTIVE: Diminished ovarian reserve (DOR) is a challenging issue encountered during assisted reproductive technology. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) belong to the transforming growth factor-beta (TGF-ß) superfamily which are essential for folliculogenesis. We aimed to the evaluation of the GDF9 and BMP15 expression in the granulosa cells (GCs) of DOR patients. MATERIALS AND METHODS: This case-control study included 14 women with DOR and 12 controls, who were between 28- 40 years of age undergoing controlled ovarian stimulation with a gonadotropin releasing hormone (GnRH) antagonist protocol. DOR patients were selected by the Bologna criteria. The GCs were extracted from the aspirated follicular fluids and RNA isolated from this. The fold change of gene expressions was assessed by real-time polymerase chain reaction (PCR). RESULTS: GDF9 expression in patients was 0.23 times lower than the control group, which was significant (P<0.0001). BMP15 expression in patients was 0.32 times lower than the control group, which was significant (P<0.0001). The number of archived oocytes, MII, and two pronuclei (PN) embryos was higher in the control group and these differences were statistically significant (P<0.05). CONCLUSION: Given that GDF9 and BMP15 are specifically involved during follicular recruitmen., we expect expression of these two genes in DOR patients which is greatly reduced by reducing follicular reserve.

Underestimated Aspects in Male Infertility: Epigenetics is A New Approach in Men with Obesity or Diabetes: A Review.

Infertility is a complex multifactorial problem that affects about 7% of men and 15% of couples worldwide. Many molecular mechanisms involved in male infertility. Destructive effects of infertility on the next generations are not well understood. Approximately 60-75% of male infertility cases have idiopathic causes, and there is a need for additional investigations other than routine examinations. Molecular factors that surround DNA, which are mitotically stable and independently regulate genome activity of DNA sequences, are known as epigenetics. The known epigenetic mechanisms are DNA methylation, histone modifications and non-coding RNAs. Prevalence of metabolic diseases has been increased dramatically because of changes in lifestyle and the current levels of inactivity. Metabolic disorders, such
as obesity and diabetes, are prevalent reasons for male infertility; despite the association between metabolic diseases and male infertility, few studies have been conducted on the effects of epigenetic alterations associated with these diseases and sperm abnormalities. Diabetes can affect the reproductive system and testicular function at multiple levels;
however, there are very few molecular and epigenetic studies related to sperm from males with diabetes. On the other hand, obesity has similar conditions, while male obesity is linked to notable alterations in the sperm molecular architecture affecting both function and embryo quality. Therefore, in this review article, we presented new and developed technologies to study different patterns of epigenetic changes, and explained the exact mechanisms of epigenetic changes linked to metabolic diseases and their relationship with male infertility.

Chemical modification of hyaluronic acid improves its supportive action on embryo implantation.

Hyaluronic acid (HA) is a supplement of the embryo transfer medium that improves embryo implantation. We have suggested that the supportive action of HA can be promoted by introducing additional artificial binding sites on the HA structure. HA was modified at carboxyl sites separately with thiol (SH) and N-hydroxysuccinimide (NHS), as mucoadhesive and amine-reactive groups, respectively. The mouse blastocysts were incubated with HA derivatives for 15 min. The HA coatings maintained their potential for enzymatic degradation and showed no detrimental effect on embryonic viability and developmental potential. After in vivo transfer, a significantly higher implantation rate was attained by HA-NHS treatment (80 %) compared with the HA-SH (53 %) and the commercial transfer medium, EmbryoGlue® (56 %). The HA-NHS was produced by a slight modification on the native structure of HA using a simple, fast, non-expensive and scalable chemistry which all promise applicability of this new HA derivative in assisted reproductive technologies.