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.

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).

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.

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.

Effect of Mechanical Micro-Vibrations on The Efficiency of Leopard Inter-Species Somatic Cell Nuclear Transfer.

OBJECTIVE: Scarcity of oocytes for assisted reproduction in endangered species can be bypassed by interspecies somatic cell nuclear transfer (iSCNT). In Felids, domestic cat (Felis catus) oocytes can serve as recipients for the nucleus of the endangered Persian leopard (Panthera pardus saxicolor). However, in vitro oocyte maturation is still suboptimal in cats, whereas it has been reported to benefit from micro-vibration in non-felid species. Therefore, the present study is aimed to determine whether micro-vibration, applied during in vitro maturation (IVM), improves the embryogenic potential of cat oocytes transplanted with fibroblast nuclei of the Persian leopard. MATERIALS AND METHODS: In the experimental study, cat cumulus-oocyte complexes (COCs) were randomly assigned to the treatment group (micro-vibration) or control group (static culture). Resultant metaphase II (MII) oocytes were enucleated and reconstructed with nucleus transplants from leopard fibroblasts, followed by artificial oocyte activation and embryo culture under the same condition (static) for 7 days. RESULTS: While cumulus cell expansion and oocyte maturation profited from micro-vibration (P<0.05), the quantity and quality of blastocysts were significantly lower in micro-vibration than in the control group (P<0.05). The total number of blastocyst cells tended to be lower in the micro-vibration than in the control group (P=0.075). Nevertheless, the proportion of ICM and TE cells did not differ between the micro-vibration and control groups (P>0.05). CONCLUSION: The present study indicated that micro-vibration at a frequency of 44 Hz for 5 secs per hour enhanced nuclear maturation and cumulus cell expansion of cat oocytes. However, exposure to micro-vibration during IVM impaired the survival rate of reconstructed oocytes during the iSCNT process and their developmental competence toward the blastocyst stage.

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.

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.

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.

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.

Epigenetic reprogramming in cloned mouse embryos following treatment with DNA methyltransferase and histone deacetylase inhibitors.

We examined the effects of DNA methyltransferase inhibitor - RG108, and histone deacetylase inhibitor - SAHA, on the reprogramming parameters of cloned mouse embryos produced by somatic cell nuclear transfer into oocytes. The programming parameters studied included dynamics of histone reacetylation, developmental rate, DNA methylation, and transcript levels of genes, all of which are pivotal to lineage specification and blastocyst formation. At the pronuclear stage, somatic nucleus-transplanted oocytes treated with 5 µM SAHA presented higher histone acetylation at H3K9, H3K14, H4K16 and H4K12, compared to untreated clones (p < 0.05). At the morula stage, cloned embryos treated with 5 µM RG108 or 5 µM SAHA presented lower DNA methylation intensity compared to untreated clones (p < 0.05), resembling the intensity levels of fertilized embryos. However, these effects were not observed when RG108 and SAHA were used in combination. The rate of morula formation was significantly higher in cloned embryos treated with 5 µM SAHA than in untreated clones, whereas treatment with RG108 resulted in no obvious effects on morula formation rates. On the other hand, the combined treatment with RG108 and SAHA resulted in inferior rates of cloned morula formation, compared to untreated clones. At the blastocyst stage, the aberrant expression levels of key developmental genes Oct4 and Cdx2, but not Nanog, were corrected in cloned embryos by the treatment with RG108. This is similar to the intensity levels seen in fertilized embryos. The expression of Rpl7l1 gene was significantly higher in embryos treated with both RG108 and SAHA than in untreated and in control groups. In summary, the present study showed that SAHA and RG108, when applied separately, improve the rate and quality of cloned mouse embryos.

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.

Frequency of Sperm Aneuploidy in Oligoasthenoteratozoospermic (OAT) Patients by Comprehensive Chromosome Screening: A Proof of Concept.

BACKGROUND: Embryonic aneuploidy usually results in implantation failure and miscarriage. Considering significantly high frequency of sperm aneuploidy reported in oligoasthenoteratozoospermia (OAT) using fluorescence in situ hybridization (FISH) in limited number of chromosomes and lack of comprehensive chromosome screening (CCS) in OAT, the aim of this study was applying CCS in OAT sperm and comparison of the results with FISH findings. METHODS: Five OAT patients with normal blood karyotypes and history of implantation failure were included. The successfully amplified samples, each containing two sperm, were analyzed by array comparative genomic hybridization (aCGH). FISH was utilized mainly depending on the aneuploidies found by aCGH to assess their frequencies in total sperm population. RESULTS: In aCGH for 30 sperm, aneuploidy was found in 66% of samples. Following the study of 4300 sperm by FISH, an average of 55.46% aneuploidy was observed. No pregnancy was resulted with normal partners. CONCLUSION: Using aCGH, some abnormalities were observed that are not typically considered in sperm FISH studies. Despite small sample size of the comprehensive study, like other similar studies, the frequency of aneuploidies was considerable and similar to FISH. Aneuploidies revealed by aCGH at single sperm resolution were different from sperm population detected by FISH. Considering high frequency of aneuploidy in OATs sperm, preimplantation genetic testing for aneuploidy (PGT-A) can be used for in transfer of chromosomally normal embryos.

Controlling Semi-Invasive Activity of Human Endometrial Stromal Cells by Inhibiting NF-kB Signaling Pathway Using Aloe-emodin and Aspirin.

BACKGROUND: Inflammation and its master regulator, Nuclear Factor-kB (NF-kB), have been implicated in the development of endometriosis. Inhibition of NF-kB pathway using small molecules ameliorated disease progression and reduced the lesion size; nevertheless, the underlying mechanism is not fully understood. Therefore, this study, is an attempt to assess whether inhibiting NF-kB signaling by aloe-emodin (AE) or aspirin (Asp), as anti-inflammatory compounds, can suppresses the invasive activity of human endometrial stromal cells at stage IV endometriosis. METHODS: The eutopic and healthy endometrial biopsies from a total of 8 infertile women with confirmed endometriosis and 8 women without endometriosis were digested and the single cells were cultured. Gene and protein markers of proliferation, migration, adhesion, and invasion of eutopic endometrial stromal cells (EuESCs) with and without treatment with AE or Asp, as well as control endometrial stromal cells (CESCs) was analyzed using q-PCR and immunofluorescence staining, respectively. Comparison between groups was performed using one-way ANOVA and the Bonferroni post hoc and p≤0.5 was considered statistically significant. RESULTS: There was an association between NF-kB overexpression and higher proliferation/adhesion capacity in EuESCs. EuESCs (at stage IV endometriosis) displayed no invasive and migratory behaviors. Pre-treatment of EuESCs with AE or Asp significantly attenuated NF-kB expression and reduced proliferative, adhesive, invasive, and migratory activity of endometrial cells (p≤0.5). CONCLUSION: Eutopic endometrial stromal cells seem to have a semi-invasive activity which is largely suppressed by AE or Asp. It can be suggested that both Asp and AE (as potent NF-kB inhibitors) can be used as a supplement in conventional endometriosis treatments.

Preimplantation Genetic Screening and The Success Rate of In Vitro Fertilization: A Three-Years Study on Iranian Population.

OBJECTIVE: In vitro fertilization (IVF) is one of the most efficient approaches within the context of assisted reproductive technology (ART) to treat infertility. High pregnancy rates have become the major index of successful IVF in clinical studies. It is not clear yet which factors are certainly responsible for IVF success, as various outcomes were obtained in different IVF centers with different settings. In this study, we aimed to address controversies in the interpretation of promising results of IVF with respect to preimplantation genetic screening (PGS). MATERIALS AND METHODS: In this retrospective case series study, we built a dataset containing data from 213 IVF patient candidates for PGS (654 embryos) with blastomere biopsy at day 3 and trophectoderm biopsy in day 5, referred to Royan Institute, Tehran, Iran from 2015 to 2018. Next, the data were analyzed to find influential factors affecting success rate of ART cycles. RESULTS: Data analyses showed that regardless of PGS indications (ART failures, recurrent miscarriage, chromosomal abnormalities, etc.), the pregnancy rate is influenced by maternal and embryonic factors such as the age of mother as well as quantity and quality of transferred embryos. Furthermore, genotyping of embryos using array comparative genomic hybridization (aCGH) depicted the highest rate of chromosomal aberrations for chromosomes 1, 16 and 19 while the lowest frequency for chromosomes 11 and 17. Similarly, we detected 463 genetically abnormal embryos by aCGH, among which only 41.9% could be detected by classical fluorescent in situ hybridization (FISH) method. CONCLUSION: This study not only highlighted the advantages of aCGH over the FISH method in detection of chromosomal abnormalities, but also emphasized the importance of genetic abnormality as an indication for determination of IVF success rate.

Wharton's jelly derived mesenchymal stem cells differentiate into oocyte like cells in vitro by follicular fluid and cumulus cells conditioned medium.

Wharton's jelly derived-mesenchymal stem cells (WJ-MSCs) have a same developmental origin with primordial germ cells. WJ-MSCs perhaps differentiate into oocyte and germ like-cells (OLCs/GLCs) in the presence of appropriate inducers. Human follicular fluid (FF) and cumulus cells conditioned medium (CCM) are naturally rich sources for oocyte development. The aim of this study was to evaluate WJ-MSCs potential for differentiating into OLCs and GLCs exposed to FF and CCM. WJ-MSCs were cultured in two different induction media (10% FF, 10% CCM) for 21 days. Morphological changes and expression of developmental genes were evaluated on days 0, 7, 14 and 21 of culture. Also, on 21st day of culture, the expression of oocyte and germ cell proteins investigated using immunofluorescence staining. Appearance of round shaped cells from 7th day onwards indicated that WJ-MSCs can differentiate into OLCs when exposed to FF and CCM. The size of produced OLCs and expression of oocyte specific genes and proteins were increased more positively in FF group rather than CCM group. Although, WJ-MSCs could differentiate into OLCs by FF and CCM, however, the induction potential of FF for producing OLCs was better than CCM.

Establishment and characterization of a PCOS and a normal human granulosa cell line.

Oocyte maturation is an important phase in fertility and any disorder in this process could lead to infertility. The most common disorder during folliculogenesis is polycystic ovary syndrome (PCOS). Due to the secretive activity of granulosa cells (GCs), they play a vital role in folliculogenesis. Although scientists use various cellular and molecular methods to have a better understanding of the mechanism of these cells, some limitations still exist in GC culture such as low primary cell yield and proliferation capability. Therefore, immortalization of primary cells is an approach to overcome these limitations. In the current study, GCs were obtained from two females, one with PCOS and one with normal folliculogenesis. In the first stage, we established two human GC (hGC) lines by immortalizing them through retrovirus-mediated transfer of the human telomerase reverse transcriptase (hTERT) and c-Myc genes. Subsequently, the normal and PCOS cell lines were characterized and were investigated for their growth features. The cell lines were also examined in terms of immortal markers of hTERT, follicle stimulating hormone receptor (FSHR), aromatase, anti-Müllerian hormone (AMH), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), estrogen, and progesterone. Our results indicated that the normal and PCOS cell lines both showed similar characteristics to GCs during the follicular stage in normal and PCOS women. The normal and PCOS cell lines demonstrate molecular mechanisms similar to that of GCs such as folliculogenesis, oogenesis, and steroidogenesis, which enable researchers to perform further investigations in future.

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

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 OCT4, SOX2, CDX2, GATA3, YAP 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 OCT4, SOX2, CDX2, GATA3 and YAP genes in blastocyst and morula stages, between the two groups. However, SOX2 and CDX2 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.