What is the maximal timeframe between sperm acquisition to sperm cryopreservation, in different "culture" conditions?

OBJECTIVE: Most of the literature about postmortem sperm retrieval (PMSR) deals with the controversies surrounding ethical and legal aspects, while the optimal time interval between the death and viable sperm acquisition is indefinite. In an attempt to aid fertility specialists, while counseling whether to pursue and adopt PMSR, we aim to explore the maximal time frame from ejaculated sperm acquisition to sperm cryopreservation in different "culture" conditions, observations that might be extrapolated to PMSR requests. PATIENTS AND METHODS: Five healthy men with normal semen analysis were enrolled. The sperm specimen from each man was diluted to 6.5 mL. After extracting 0.5 mL for cryopreservation, the remaining 6 mL were divided into three tubes: one was maintained in room temperature (23-25 °C), the second in an incubator (37 °C), and the third in a refrigerator (4 °C). Thereafter, every day, a 0.5 mL of each sample was extracted, examined, and cryopreserved. A week later, all the cryopreserved samples were thawed and tested for sperm motility and viability. RESULTS: While at room temperature, frozen/thawed sperm were still motile (6.5%) and viable (9.9%) up to 96 h; those maintained in the refrigerator, following freezing/thawing were immotile already at 48 h in culture, but still viable (6.0%) up to 72 h in culture. Those maintained in the incubator demonstrated the worse results with negligible motility (1.5%) and viability (3.7%) following freezing/thawing, already after 48 h in culture. CONCLUSIONS: The timeframe cut-off between ejaculated sperm acquisition and cryopreservation should be 72 h, unless sperm was maintained at room temperature, where it might be longer. It would be prudent to check for sperm vitality prior to freezing in cases where only immotile sperms are present.

Cleavage-stage human embryo arrest, is it embryo genetic composition or others?

Embryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle.

Embryos derived from delayed mature oocyte should be cryopreserved and are favourable to transfer in a following endometrium synchronize frozen-thawed cycle.

Oocytes eligible for intracytoplasmic sperm injection (ICSI) are those that have progressed through meiosis to metaphase 2 (MII). The remaining delayed mature oocytes can be injected, aiming to achieve more embryos and a better chance to conceive. We aimed to assess the outcome of delayed matured oocytes, derived from either germinal vesicles or metaphase 1 (MI), that reached maturity (MII) 24 h following retrieval. The study population consisted of 362 women who underwent 476 IVF cycles. While fertilization rates were comparable between the sibling delayed mature oocyte group compared with injection on day 0 group (58.4% vs 62%, respectively, P = 0.07), the top-quality embryo rate per injected MII day 0 oocyte was significantly higher compared with day 1 injected oocyte (57.5% vs 43.9% respectively, P < 0.001). Moreover, following fresh transfer of embryos derived from delayed mature oocytes, implantation rate and the clinical pregnancy (CPR) and live-birth rates (LBR) per transfer were 3.9%, 3.3% and 1.6% respectively. When considering the following thawed embryo transfer cycles, implantation, pregnancy and LBR were non-significantly higher (10%, 8.3% and 8.3%, respectively). Although clinical outcomes are significantly lower when using embryos derived from delayed mature oocyte to mature day 0 oocytes, the additional embryos derived from delayed mature oocytes might contribute to the embryo cohort and increase the cumulative live-birth rate per retrieval. Moreover, the embryos derived from delayed mature oocyte favour a transfer in a frozen-thawed cycle rather than in a fresh cycle.

Cumulative IVF outcomes after retrieval of testicular spermatozoa: should we use immotile spermatozoa for ICSI?

RESEARCH QUESTION: What are the cumulative clinical pregnancy rates (CPR) and live births rates (LBR) in intracytoplasmic sperm injection (ICSI) cycles using testicular motile compared with immotile spermatozoa, obtained from testicular sperm aspiration (TESA) or extraction (TESE)? DESIGN: A retrospective analysis of ICSI cycles using TESA or TESE over a period of 7 years. Cycles were divided into two groups according to the motility of the retrieved spermatozoa: Group A consisted of couples with motile spermatozoa; Group B of couples with immotile spermatozoa. Group B was subdivided into two groups: B1 consisted of couples with motile spermatozoa and B2 with immotile spermatozoa after the addition of pentoxifylline. RESULTS: No differences in CPR and LBR per transfer was found between the study groups after fresh embryo transfer. No pregnancies were achieved by vitrified-warmed embryo transfer in group B2. Fertilization rates decreased when using immotile spermatozoa (64.4%, 56%, 37.9%, for groups A, B1 and B2, respectively, P < 0.001). Top-quality embryo rates were higher in groups A and B1 compared with B2 (40.7% and 40.1% versus 19.1%, respectively, P = 0.015). Cumulative CPR (53%, 41.7%, 13.6% for groups A, B1 and B2, respectively, P = 0.005) and LBR (42.4%, 30%, 13.6% for groups A, B1 and B2, respectively P = 0.03) per oocyte retrieval was significantly higher when using motile spermatozoa compared with motile or immotile spermatozoa after adding pentoxifylline. CONCLUSIONS: Although fertilization, top-quality embryo rates, cumulative CPR and LBR decreased when using immotile spermatozoa, ICSI is still valid; therefore, it should be considered and offered to couples before embarking on a donor sperm insemination cycle, or cryopreserving oocytes for future additional testicular sperm retrieval.

Does the number of embryos loaded on a single cryo-carrier affect post-vitrification survival rate?

BACKGROUND: We aimed to assess whether the survival rate of embryos is influenced by the number of embryos/oocytes loaded on a single cryo-carrier during vitrification. METHODS: This was a retrospective study that included 974 patients who underwent thawing of 1896 embryo-warming cycles between September 2016 and January 2020. A distinct analysis was made for cleavage stage embryos (2-10-cell stage) and blastocysts. For vitrification, embryos were placed in a Cryotop™ open device using a SAGE vitrification kit following the manufacturer's instructions. Warming was carried using a SAGE warming vitrification kit according the manufacturer's instructions. RESULTS: Total post-vitrification survival rates of embryos at the cleavage stage or blastocyst stage was 94.8%. At the cleavage stage, cryo-preserving three embryos per single cryo-carrier gave the highest full intact embryo survival rate (91.5%) compared with one or two embryo(s) per single cryo-carrier (85.7%, P < 0.0002 and 87.3%, P < 0.004). Conversely, post warmed full intact blastocyst survival rate for two blastocysts was significantly lower compared with one blastocyst (76.7% vs. 87.9%, P < 0.0193) per single cryo-carrier. CONCLUSION: Post-thawing survival rate following vitrification is affected by the number of embryos per single cryo-carrier undergoing the vitrification equilibration phase, with the optimum number of three cleaved embryos or one blastocyst per single cryo-carrier. Further studies are required to determine the optimum number of cleaved embryos or blastocysts that should be loaded onto a single cryo-carrier vitrification device.

Testicular sperm retrieval: What should we expect from the fresh and subsequent cryopreserved sperm injection?

We sought to compare ICSI outcomes of cycle using fresh versus thawed TESE spermatozoa obtained during the previous fresh TESE. All consecutive couples undergoing ICSI cycles using fresh TESE spermatozoa, followed by ICSI cycle using cryopreserved sperm remaining from the previous fresh TESE procedure were included. Ovarian stimulation (OS)/laboratory variables and cycle outcome were assessed and compared between those utilising fresh versus thawed TESE spermatozoa. Seventy-five couples were evaluated, with no in-between groups differences in OS nor embryological variables. While implantation and LBR per embryo transfer were nonsignificantly higher in the frozen as compared to the fresh TESE, there was a trend towards higher LBRs per patient in the frozen TESE group. The cumulative miscarriage rate (4% versus 14.7%, p < .022 respectively) was significantly lower and the cumulative LBR (34.7% versus 16%, p < .007 respectively) was significantly higher using frozen TESE spermatozoa. Moreover, significantly higher proportion of frozen TESE sperm samples used pentoxifylline to enhance sperm motility. In conclusion, the results of ICSI cycles using frozen TESE spermatozoa are as good, or even better than using fresh TESE spermatozoa. Further studies are required to explore the factors responsible for the improved ICSI outcome, while using frozen versus fresh TESE sperm samples.

Spermatozoa retrieved by electroejaculation: Should we prefer fresh or cryopreserved spermatozoa for intracytoplasmic sperm injection?

We aim to evaluate our experience, comparing intracytoplasmic sperm injection (ICSI) outcomes of cycle using fresh versus thawed electroejaculated spermatozoa. All consecutive couples undergoing ICSI cycles using electroejaculated spermatozoa, during a 16-year period, were evaluated. Embryological/laboratory variables of the ICSI cycles were assessed and compared between those utilising fresh (fresh group) versus thawed (thawed group) electroejaculated spermatozoa. Fifty-seven couples were evaluated, 30 used a fresh electroejaculated spermatozoa in 55 ICSI cycles, while 27 used a thawed sperm sample in 41 ICSI cycles. There were no in-between group differences in the mean numbers of oocytes retrieved per oocyte retrieval nor the percentage of MII oocytes. The fresh group demonstrated significantly higher fertilisation (71.5% vs. 64.1%, respectively, p < .05), top-quality embryos (66.5% vs. 54.9%, respectively, p < .02), clinical pregnancy per transfer (41.3% and 21.2%, respectively, p < .05) and cumulative clinical pregnancy (58.2% vs. 26.8%, respectively, p < .001) rates, as compared to the thawed group. Independent of the source of spermatozoa used, no pregnancy was achieved following ICSI utilising immotile spermatozoa. In conclusion, ICSI cycles using ejaculated spermatozoa of patients suffering from neurologic or psychogenic anejaculation are reassuring. The use of fresh ejaculated spermatozoa retrieved on the day of the female spouse oocyte retrieval might improve outcome. Whenever a thawed electroejaculated spermatozoa yield no motile spermatozoa, emergency electroejaculation is mandatory.

Timing day-3 vitrification for PGT-M embryos: pre- or post-blastomere biopsy?

PURPOSE: To assess the efficacy and clinical outcomes of preimplantation genetic testing for monogenic diseases (PGT-M), following blastomere biopsy prior or following vitrification. METHODS: A cohort-historical study of all consecutive patients admitted to IVF in a large tertiary center for PGT-M and PCR cycle from September 2016 to March 2020. Patients were divided into 4 groups: Group A1 consisted of patients undergoing day-3 embryos biopsy followed by a fresh transfer of unaffected embryos. Group A2 consisted of Group A1 patients that their surplus unaffected embryos were vitrified, thawed, and transferred in a subsequent FET cycle. Group B1 consisted of patients that their day-3 embryos were vitrified intact (without biopsy) for a subsequent FET cycle. Later embryos were thawed and underwent blastomere biopsies, and the unaffected embryos were transferred, while the surplus unaffected embryos were re-vitrified for a subsequent FET cycle. Group B2 consisted of Group B1 patients that their surplus unaffected embryos were re-vitrified, thawed, and transferred in a subsequent FET cycle. The laboratory data and clinical results were collected and compared between groups. RESULTS: A total of 368 patients underwent 529 PGT-M cycles in our center: 347 with day-3 embryos biopsied before undergoing vitrification (Group A1) and 182 following vitrification and thawing (Group B1). There were no between group differences in embryo survival rate post-thawing, nor the ongoing implantation and pregnancy rates. CONCLUSION: In PGT-M cycles, the timing of embryos vitrification, whether prior or following blastomere biopsy, has no detrimental effect on post-thawing embryo survival rate, nor their potential ongoing implantation and pregnancy rates.

FMR6 may play a role in the pathogenesis of fragile X-associated premature ovarian insufficiency.

The aim of this study was to evaluate whether long noncoding RNA accumulation play a role in the pathophysiology of fragile X-associated premature ovarian insufficiency (FXPOI). The study population consisted of 22 consecutive fragile X mental retardation 1 (FMR1) premutation carriers (CGGn 55-199 repeats) undergoing in vitro fertilization and pre-implantation genetic diagnosis (IVF-PGD) treatment. The control group consists of 11 patients, with <55 CGG repeats, undergoing IVF-ICSI for male factor infertility, matched by age, treated in the same period. After oocyte retrieval, granulosa cells from follicular fluid were washed and stored at -80 °C. RNA was transcribed to generate cDNA and the RNA levels were measured using RT-PCR. Transcripts levels in granulosa cells of long noncoding RNA's FMR4 and FMR6 were measured. In FMR1 premutation carriers there was a significant nonlinear association between the number of CGG repeats and the levels of FMR6 (p = 0.03), but not FMR4. The highest level of FMR6 was seen in women with mid-size CGG repeats (80-120). In addition, a significant negative linear correlation was observed between the number of oocytes retrieved and the RNA levels in granulosa cells of FMR6 (r = -0.53, p = 0.01) but not FMR4. Our study supports previous findings suggesting RNA toxic gain-of-function as one of the possible pathophysiologic mechanisms underlying FXPOI.

Peptide-binding GRP78 protects neurons from hypoxia-induced apoptosis.

Brain ischemia has major consequences leading to the apoptosis of astrocytes and neurons. Glucose-regulated protein 78 (GRP78) known for its role in endoplasmic reticulum stress alleviation was discovered on several cell surfaces acting as a receptor for signaling pathways. We have previously described peptides that bind cell surface GRP78 on endothelial cells to induce angiogenesis. We have also reported that ADoPep1 binds cardiomyocytes to prevent apoptosis of ischemic heart cells. In this study we describe the effect of hypoxia on astrocytes and neurons cell surface GRP78. Under hypoxic conditions, there was an increase of more than fivefold in GRP78 on cell surface of neurons while astrocytes were not affected. The addition of the GRP78 binding peptide, ADoPep1, to neurons decreased the percentage of GRP78 positive cells and did not change the percent of astrocytes. However, a significant increase in early and late apoptosis of both astrocytes and neurons under hypoxia was attenuated in the presence of ADoPep1. Intravitreal administration of ADoPep1 to mice in a model of optic nerve crush significantly reduced retinal cell loss after 21 days compared to the crush-damaged eyes without treatment or by control saline vehicle injection. Histological staining demonstrated reduced GRP78 after ADoPep1 treatment. The mechanism of peptide neuroprotection was demonstrated by the inhibition of hypoxia induced caspase 3/7 activity, cytochrome c release and p38 phosphorylation. This study is the first report on hypoxic neuronal and astrocyte cell surface GRP78 and suggests a potential therapeutic target for neuroprotection.

The effect of coronavirus disease 2019 immunity on frozen-thawed embryo transfer cycles outcome.

OBJECTIVE: To study the effect of patients' immunization after coronavirus disease 2019 (COVID-19) infection or messenger ribonucleic acid (mRNA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine on frozen-thawed embryo transfer (FET). DESIGN: Cohort retrospective study. SETTING: Tertiary university affiliated medical center. PATIENT(S): All consecutive patients undergoing FET cycles in our center. The study group (immune group) consisted of patients treated during the COVID-19 pandemic (between January 2021 and August 2021) who either recovered from COVID-19 infection or received the mRNA SARS-CoV-2 vaccine. The control groups consisted of patients treated during the COVID-19 pandemic (between January 2021 and August 2021) but were not infected or did not receive the mRNA SARS-CoV-2 vaccine (not-immune2021 group) and those treated between January 2019 and August 2019 (before the pandemic) (not-immune2019 group). INTERVENTION(S): Frozen-thawed embryo transfer cycles. MAIN OUTCOME MEASURE(S): Ongoing pregnancy rates and FET cycles' characteristics. Data on patient age and variables related to infertility treatment were collected from the patient records. RESULT(S): During the study periods, 428 patients underwent 672 FET cycles. The immune group consisted of 141 patients who underwent 264 FET cycles (44 in postinfection and 220 in postvaccination), whereas the not-immune2021 and not-immune2019 groups consisted of 93 and 194 patients undergoing 125 and 283 FET cycles, respectively. Patients' characteristics and the types of endometrial preparations were comparable between the study groups. The implantation rate and clinical and ongoing pregnancy rates per transfer were similar between the study groups (immune group, postinfection and postvaccination; not-immune2021 group; not-immune2019 group). CONCLUSION(S): Coronavirus disease 2019 infection or vaccination did not affect patients' performance or implantation in their subsequent FET cycle.

Does haptoglobin genotype affect early onset of diabetic retinopathy in patients with type 2 diabetes?

PURPOSE: There are 2 common alleles for Hp (Hp-1 and Hp-2) and 3 common Hp genotypes: Hp1-1, Hp2-1, and Hp2-2. The haptoglobin genotype may play a dual role in morbidities of diabetes: Hp1-1, protective and Hp2-2, provocative. This study investigated the possible association of haptoglobin genotypes with onset of retinopathy in Type 2 diabetes (DM2). METHODS: The sample included 98 consecutive adults with DM2 under routine outpatient follow-up from 2007 to 2009 who met the criteria for either no retinopathy at ≥10 years after diagnosis (Group 1) or proliferative retinopathy at ≤10 years after diagnosis (Group 2). Blood samples were collected for haptoglobin genotyping by polymerase chain reaction. Findings were compared between and within groups. RESULTS: Eighty-four patients had no retinopathy and 14 had early proliferative retinopathy. The distributions of the Hp genotypes were as follows: no-retinopathy group: 28.6% Hp1-1, 35.7% Hp2-1, and 35.7% Hp2-2 and proliferative retinopathy group: 22.6% Hp1-1, 27.4% Hp2-1, and 50% Hp2-2 (NS). On statistical analysis (limited to the larger no-retinopathy group), a predominance of Hp1-1 was noted in older patients; Hp2-2 was associated with an increased rate of stroke. CONCLUSION: The Hp genotype apparently plays no role in the development or worsening of proliferative retinopathy in DM2. Hp1-1 may be involved in delaying the onset of diabetes. Hp2-2 may pose a microvascular risk.

Protective effect of Bax ablation against cell loss in the retinal ganglion layer induced by optic nerve crush in transgenic mice.

BACKGROUND: Bax expression is a prerequisite for retinal ganglion cell (RGC) apoptosis. Experimental studies have reported Bax protein upregulation following optic nerve transection. The stimuli that trigger apoptosis share a common executioner proteolysis cascade, including caspase-3 and poly-(adenosine diphosphate ribose) polymerase cleavage. This study sought to elucidate the role of the mitochondrial apoptotic pathway in RGCs using a Bax transgenic knockout mouse model. METHODS: The right optic nerves of 26 C57BL mice, 7 Bax, 7 Bax, and 12 Bax, were subjected to crush injury and analyzed for apoptosis and neuronal cell loss on days 1, 3, and 21. Levels of Bax, Bcl-2, and caspase-3 messenger RNA expression were determined with real-time polymerase chain reaction. RESULTS: Multiple apoptotic cells were detected in the retinas of the Bax and Bax mice at days 1 and 3, but not in the Bax mice. The Bax/Bcl-2 ratio was higher in the Bax than in the Bax mice on day 1 (1.33 and 0.83, respectively), with a trend toward an increase on day 3 (1.47 and 1.66, respectively); Bax/Bcl-X showed the same elevation on day 1 in the wild-type mice (1.34) but decreased on day 3 (0.8). Bax gene expression was undetectable in the Bax mice. Caspase-3 gene expression was higher in the Bax than in the Bax mice on day 1 and dropped toward baseline on day 3. The opposite trend was noted in the Bax mice. CONCLUSION: The lack of apoptosis combined with the reduction in proapoptotic genes in the Bax mice after injury compared to the Bax and Bax mice suggests that Bax plays a crucial role in the induction of apoptosis. Suppression of Bax expression may reduce retinal cell loss.

Effect of subconjuctival and intraocular bevacizumab injection on angiogenic gene expression levels in a mouse model of corneal neovascularization.

PURPOSE: This study sought to characterize the expression of angiogenesis-related genes in a mouse model of corneal neovascularization, either untreated or after treatment with a single injection of bevacizumab by three different routes. In addition, the effectiveness of the treatment was compared to a rabbit model. METHODS: A chemical burn was induced in the mid-cornea of the right eye in 119 mice; 56 of them were untreated and 63 were bevacizumab-treated. Neovascularization was evaluated 2, 4, 8, 10, and 14 days later using digital photos, angiography and India ink perfusion. The relative area of new blood vessels was analyzed using slit-lamp examination in vivo and on histological and flat-mount sections. The levels of gene expression involved in the angiogenic process vascular endothelial growth factor [VEGF], insulin-like growth factor-1 [IGF-1], pigment epithelium derived factor [PEDF], and macrophage-inflammatory protein-2 [MIP-2]) were measured by a real-time polymerase chain reaction. Six rabbits underwent the same injury and treatment, and the response was compared to the mouse model. RESULTS: Neovascularization was first observed two days after injury. The affected section increased from 11.24% (+/-7.0) of the corneal area to 47.42% (+/-25.4) on day 8 and 50.62% (+/-24.7) on day 10. In the mice treated with bevacizumab, the relative area of neovascularization was significantly lower at the peak time points (p<0.005): 24.90% (+/-21.8) on day 8 and 28.29% (+/-20.9) on day 10. Spontaneous regression was observed on day 14 in both groups, to 26.98% (+/-19.9) in the untreated mice and 10.97% (+/-10.8) in the bevacizumab-treated mice (p<0.005). Rabbits also showed peak corneal neovascularization on days 8-10, with significant regression of the vessels following intracameral bevacizumab injection. In the mice, intraocular (intravitreal, intracameral) injection was more effective than subconjuctival injection. VEGF gene expression was upregulated in both the untreated and treated mice, but was slightly less in the treated mice. PEDF gene expression decreased in both the treated and untreated mice. In the untreated group, gene expression peaked (above baseline) at 14 days, and in the untreated mice, it had already peaked by day 8. IGF-1 was upregulated early in the model; at 8 days, there was only a slight change in the untreated group compared to a significant increase in the treated group. MIP-2 was upregulated in both groups in the early stage and returned to baseline on day 14. CONCLUSIONS: Bevacizumab treatment partially inhibits the progressive corneal neovascularization induced by chemical damage in a mouse model. Treatment is more effective when administered via the intraocular than the subconjunctival route. The clinical findings are compatible with the angiographic and histologic data and are supported by molecular analysis showing a partial change in expression of proangiogenic genes. The molecular mechanisms involved in corneal neovascularization and inflammation warrant further exploration. These findings may have important therapeutic implications in the clinical setting.

Possible neuroprotective effect of brimonidine in a mouse model of ischaemic optic neuropathy.

PURPOSE: To investigate the neuroprotective effect of brimonidine following induction of ischaemic optic neuropathy in rodents (rAION). METHODS: Mice were treated with an intraperitoneal injection of brimonidine 48, 24 or 0 h before rAION induction or eye drops for 5 days after rAION induction. Retinal ganglion cell (RGC) loss and expression of genes involved in the angiogenesis (vascular endothelial growth factor [VEGF], pigment epithelium-derived factor [PEDF], The epidermal growth factor homology domains-2 [Tie-2]), ischaemia (haem oxygense-1 [HO-1], hypoxia-inducible factor 1alpha[HIF-1alpha], endothelial nitric oxide synthase [eNOS]) and oxidative stress (superoxide dismutase-1 [SOD-1], glutathione peroxidase-1 [GPX-1]) response to ischaemic damage were compared with sham or rAION-untreated mice. RESULTS: No RGC loss was detected in the brimonidine-treated mice. Effect of post-rAION eye drops: day 1--no decrease in retinal mRNA levels of angiogenesis-related genes, increase in ischaemia- and oxidative stress-related genes except HIF-1alpha; day 3--baseline or higher levels of oxidative and ischaemia-related genes except HIF-1alpha, increase in VEGF, decrease in PEDF; day 21--no change in angiogenesis-related genes. Effect of pre-rAION injection: baseline levels of angiogenesis-related genes with all injection schedules; increase in ischaemia-related genes with 48-h and 0-h pretreatment; decrease in HO-1 and eNOS with 24-h pretreatment; increase in oxidative-related genes except GPX-1. In optic nerve tissue, HO-1, HIF-1alpha and SOD-1 decreased on day 1 after topical administration and were still below baseline on day 3. CONCLUSIONS: The increase in HO-1 associated with rAION is mitigated with brimonidine treatment, especially when administered intraperitoneally. Topical brimonidine apparently reduces VEGF, Tie-2, HIF-1alpha and GPX-1 expression on day 21. These results agree with published data and may have therapeutic implications for patients diagnosed with AION in the acute phase.

Pathophysiology Mechanisms in Fragile-X Primary Ovarian Insufficiency.

Women who carry the FMR1 premutation may suffer from ongoing deterioration of ovarian function. The lucidity of the molecular mechanism of FXTAS is emerging and findings from research in the field of FXTAS could elucidate the pathogenesis of FXPOI. To date there are three possible mechanisms for ovarian dysfunction in FMR1 permutation carriers. The first is the RNA toxic gain-of-function mechanism initiating loss of function of over 30 specific RNA-binding proteins. The second is associated to the formation of an abnormal polyglycine-containing protein (FMRpolyG), and the third is related to novel lncRNAs, named FMR4 and FMR6. Herein we describe our laboratory methodology, focusing on the culturing and manipulation of granulosa cells from human female premutation carriers, trying to reveal the actual possible mechanisms liable to FXPOI. Detecting the precise pathways in premutation carrier might facilitate in offering these women the opportunity to make an informed decision regarding their reproductive and family planning.

Interleukin-2 Production by Cultured Human Granulosa Cells.

PROBLEM: Ovarian hyperstimulation syndrome (OHSS) is similar to vascular leak syndrome (VLS), which may be attributable to the massive increase in systemic inflammatory cytokines. The hyperstimulated human ovaries were demonstrated to contain interleukin (IL)-2, which, in turn, was suggested to activate the systemic inflammatory response characteristic of OHSS. As the source of follicular fluid IL-2 is still unclear, in the present study, we sought to validate the presence of IL-2 and IL-2 mRNA expression in human luteinized granulosa cells. METHOD OF STUDY: IL-2 nuclear expression was detected using real-time PCR and immunofluorescence staining of human luteinized granulosa cell from 6 patients undergoing in vitro fertilization treatment. Calretinin immunofluorescence staining was used as a marker of granulosa cells. RESULTS: IL-2-positive immunofluorescence staining was detected within nuclei of granulosa cells, together with positive stain for calretinin, confirming the presence of granulosa cell. Moreover, IL-2 gene expression was demonstrated in luteinized granulosa cells by real-time PCR. CONCLUSION: In the present study, we provided firm evidence for the IL-2 production by human luteinized granulosa cells, as demonstrated by the presence of IL-2 and IL-2 mRNA expression in luteinized granulosa cells. Further studies are justified in an attempt to clarify the regulation and the cause-and-effect relationship between IL-2 production by the hyperstimulated ovaries and OHSS.

Interleukin-2 and SOCS-1 proteins involvement in the pathophysiology of severe ovarian hyperstimulation syndrome--a preliminary proof of concept.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS), is characterized by marked ovarian enlargement and acute third space fluid sequestration that almost always develops after hCG administration or in early pregnancy. OHSS is similar to vascular leak syndrome (VLS), which may be attributable to the massive increase in systemic inflammatory cytokines. In the present pilot exploratory case series, we sought to evaluate interleukin (IL)-2 and suppressor of cytokine signaling (SOCS)-1 expressions in the peripheral blood mononuclear cells (PBMCs) of patients suffering from severe ovarian hypertimulation syndrome (OHSS), and to examine whether their expressions differ when compared to PBMCs originated from normal early pregnant women (without OHSS). METHODS: Interleukin-2 and SOCS-1 mRNA expressions were examined in PBMCs of 5 women who were hospitalized due to severe OHSS (OHSS group) and 5 women with early IVF pregnancies and without OHSS (control group). RESULTS: Interleukin-2 mRNA levels in PBMCs were significantly higher in the OHSS as compared to the control groups. Moreover, while SOCS-1 mRNA levels were non-significantly lower, the ratio between IL-2 and SOCS-1 mRNA levels was significantly higher in the OHSS, as compared to the control group. CONCLUSIONS: The inflammatory response to hCG, leading to dysregulation of Il-2 expression and SOCS activation, might be the culprit of OHSS. Additional large prospective studies are required to elucidate the effect of hCG on patients' inherited inflammatory cascades, which may help discriminating those at risk to develop severe OHSS from those who are not.

Elevated levels of FMR1 mRNA in granulosa cells are associated with low ovarian reserve in FMR1 premutation carriers.

AIM: To assess the role of mRNA accumulation in granulosa cells as the cause of low ovarian response among FMR1 premutation carriers undergoing pre-implantation genetic diagnosis (PGD). DESIGN: Case control study in an academic IVF unit. Twenty-one consecutive FMR1 premutation carriers and 15 control women were included. After oocyte retrieval the granulosa cells mRNA levels of FMR1 was measured using RT-PCR. RESULTS: In FMR1 premutation carriers, there was a significant non-linear association between the number of CGG repeats and the number of retrieved oocytes (p<0.0001) and a trend to granulosa cells FMR1 mRNA levels (p = 0.07). The lowest number of retrieved oocytes and the highest level of mRNA were seen in women with mid-size CGG repeats (80-120). A significant negative linear correlation was observed between the granulosa cells FMR1 mRNA levels and the number of retrieved oocytes (R2 linear = 0.231, P = 0.02). CONCLUSION: We suggest that there is a no-linear association between the number of CGG repeats and ovarian function, resulting from an increased granulosa cells FMR1 mRNA accumulation in FMR1 carriers in the mid-range (80-120 repeats).

BRAF, GNAQ, and GNA11 mutations and copy number in pediatric low-grade glioma.

Fifty-two samples of pediatric low-grade glioma (48 primary, 4 recurrent) were analyzed for BRAF copy number variation (digital PCR analysis, CopyCaller) and point mutations of BRAF V600E, and exon 5 Q209 in GNAQ, and GNA11, using the MALDI-TOF mass spectrometer with validation by direct sequencing. An increased BRAF copy number was found in 18/47 primary samples tested; 15 of them (83.3%) were pilocytic astrocytomas. A BRAF mutation was found in 3/48 primary tumors, all with a normal BRAF copy number and no GNAQ mutation. One sample had a GNAQ209 mutation (Q209P626) with a normal BRAF gene; none of the tumors had a GNA11Q209 mutation. Recurrent or progressive tumors, analyzed in four patients, had the same molecular genotype as their primary. Increased BRAF copy number and activating BRAF mutations may be involved in the development of low-grade glioma via overactivation of the Ras/Raf pathway. This is the first report of a mutation in GNAQ209 in pediatric low-grade glioma. Understanding the molecular mechanisms underlying glioma initiation and growth may assist in the development of targeted therapies.