Oocyte polarized light microscopy, assay of specific follicular fluid metabolites, and gene expression in cumulus cells as different approaches to predict fertilization efficiency after ICSI.

BACKGROUND: The complex relationship between oocyte morphology, specific follicular fluid metabolites, gene expression in cumulus granulosa cells, and oocyte competence toward fertilization and embryo development still needs further clarification. METHODS: Forty-six oocytes retrieved from the largest pre-ovulatory follicle of patients undergoing intra-cytoplasmic sperm injection (ICSI) were considered assessing: (a) oocyte morphological characteristics at polarized light microscopy (PLM), (b) specific follicular fluid (FF) metabolites previously suggested to influence oocyte competence (AMH, markers of redox status and of cytotoxicity), (c) transcription of AMH and AMH type II receptor genes in cumulus cells. Data were analyzed using mono-parametric tests and multivariable logistic analysis in order to correlate morphological and biochemical data with fertilization. RESULTS: Comparing normally fertilized oocytes (n = 29, F group) with unfertilized (n = 17, nF group) we observed that: (a) the meiotic spindle area and major axis were significantly higher in nF group and in fertilized oocytes undergoing an early embryo development arrest; (b) AMH level in FF was comparable in F and nF groups; (c) the FF of nF group contained significantly higher levels of cytotoxicity (lactate dehydrogenase) and oxidative stress (Cu,Zn-superoxide dismutase, catalase, 4-hydroxynonenal-protein conjugates) markers; (d) cumulus cells of nF group showed significantly higher AMH receptor type II gene expression. CONCLUSIONS: Taken together, these observations suggest that an excessive cytotoxicity level can alter AMH signal transduction within cumulus cells, in turn leading to partial inhibition of aromatase activity, altered cytoplasmic maturation and increased oxidative stress, factors able to impair oocyte fertilization competence and embryo growth.

Effects of oncological treatments on semen quality in patients with testicular neoplasia or lymphoproliferative disorders.

Pretherapy sperm cryopreservation in young men is currently included in good clinical practice guidelines for cancer patients. The aim of this paper is to outline the effects of different oncological treatments on semen quality in patients with testicular neoplasia or lymphoproliferative disorders, based on an 8-year experience of the Cryopreservation Centre of a large public hospital. Two hundred and sixty-one patients with testicular neoplasia and 219 patients with lymphoproliferative disorders who underwent chemotherapy and/or radiotherapy and pretherapy semen cryopreservation were evaluated. Sperm and hormonal parameters (follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, inhibin B levels) were assessed prior to and 6, 12, 18, 24 and 36 months after the end of cancer treatment. At the time of sperm collection, baseline FSH level and sperm concentration were impaired to a greater extent in patients with malignant testicular neoplasias than in patients with lymphoproliferative disorders. Toxic effects on spermatogenesis were still evident at 6 and 12 months after the end of cancer therapies, while an improvement of seminal parameters was observed after 18 months. In conclusion, an overall increase in sperm concentration was recorded about 18 months after the end of cancer treatments in the majority of patients, even if it was not possible to predict the evolution of each single case 'a priori'. For this reason, pretherapy semen cryopreservation should be considered in all young cancer patients.

Ovarian sensitivity index is strongly related to circulating AMH and may be used to predict ovarian response to exogenous gonadotropins in IVF.

BACKGROUND: Serum anti-Mullerian hormone (AMH) is currently considered the best marker of ovarian reserve and of ovarian responsiveness to gonadotropins in in-vitro fertilization (IVF). AMH assay, however, is not available in all IVF Units and is quite expensive, a reason that limits its use in developing countries. The aim of this study is to assess whether the "ovarian sensitivity index" precisely reflects AMH so that this index may be used as a surrogate for AMH in prediction of ovarian response during an IVF cycle. METHODS: AMH serum levels were measured in 61 patients undergoing IVF with a "long" stimulation protocol including the GnRH agonist buserelin and recombinant follicle-stimulating hormone (rFSH). Patients were divided into four subgroups according to the percentile of serum AMH and their ovarian stimulation was prospectively followed. Ovarian sensitivity index (OSI) was calculated dividing the total administered FSH dose by the number of retrieved oocytes. RESULTS: AMH and OSI show a highly significant negative correlation (r = -0.67; p = 0.0001) that is stronger than the one between AMH and the total number of retrieved oocytes and than the one between AMH and the total FSH dose. CONCLUSIONS: OSI reflects quite satisfactory the AMH level and may be proposed as a surrogate of AMH assay in predicting ovarian responsiveness to FSH in IVF. Being very easy to calculate and costless, its use could be proposed where AMH measurement is not available or in developing countries where limiting costs is of primary importance.

Nitric oxide stimulates human sperm motility via activation of the cyclic GMP/protein kinase G signaling pathway.

Nitric oxide (NO), a modulator of several physiological processes, is involved in different human sperm functions. We have investigated whether NO may stimulate the motility of human spermatozoa via activation of the soluble guanylate cyclase (sGC)/cGMP pathway. Sperm samples obtained by masturbation from 70 normozoospermic patients were processed by the swim-up technique. The kinetic parameters of the motile sperm-rich fractions were assessed by computer-assisted sperm analysis. After a 30-90  min incubation, the NO donor S-nitrosoglutathione (GSNO) exerted a significant enhancing effect on progressive motility (77, 78, and 78% vs 66, 65, and 62% of the control at the corresponding time), straight linear velocity (44, 49, and 48 µm/s vs 34, 35, and 35.5 µm/s), curvilinear velocity (81, 83, and 84 µm/s vs 68 µm/s), and average path velocity (52, 57, and 54 µm/s vs 40, 42, and 42 µm/s) at 5 µM but not at lower concentrations, and in parallel increased the synthesis of cGMP. A similar effect was obtained with the NO donor spermine NONOate after 30 and 60  min. The GSNO-induced effects on sperm motility were abolished by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (a specific sGC inhibitor) and mimicked by 8-bromo-cGMP (8-Br-cGMP; a cell-permeating cGMP analog); the treatment with Rp-8-Br-cGMPS (an inhibitor of cGMP-dependent protein kinases) prevented both the GSNO- and the 8-Br-cGMP-induced responses. On the contrary, we did not observe any effect of the cGMP/PRKG1 (PKG) pathway modulators on the onset of hyperactivated sperm motility. Our results suggest that NO stimulates human sperm motility via the activation of sGC, the subsequent synthesis of cGMP, and the activation of cGMP-dependent protein kinases.

The effects of conformational heterogeneity on the binding of the Notch intracellular domain to effector proteins: a case of biologically tuned disorder.

Cell-fate decisions in metazoans are frequently guided by the Notch signalling pathway. Notch signalling is orchestrated by a type-1 transmembrane protein, which, upon interacting with extracellular ligands, is proteolytically cleaved to liberate a large intracellular domain [NICD (Notch intracellular domain)]. NICD enters the nucleus where it binds the transcription factor CSL (CBF1/suppressor of Hairless/Lag-1) and activates transcription of Notch-responsive genes. In the present paper, the interaction between the Drosophila NICD and CSL will be examined. This interaction involves two separate binding regions on NICD: the N-terminal tip of NICD {the RAM [RBP-Jkappa (recombination signal-binding protein 1 for Jkappa)-associated molecule] region} and an ankyrin domain approximately 100 residues away. CD studies show that the RAM region of NICD lacks alpha-helical and beta-sheet secondary structure, and also lacks rigid tertiary structure. Fluorescence studies show that the tryptophan residues in RAM are highly solvated and are quenched by solvent. To assess the impact of this apparent disorder on the bivalent binding of NICD to CSL, we modelled the region between the RAM and ANK (ankyrin repeat)-binding regions using polymer statistics. A WLC (wormlike chain) model shows that the most probable sequence separation between the two binding regions is approximately 50 A (1 A=0.1 nm), matching the separation between these two sites in the complex. The WLC model predicts a substantial enhancement of ANK occupancy via effective concentration, and suggests that the linker length between the two binding regions is optimal for bivalent interaction.

Varicocele sclerotherapy improves serum inhibin B levels and seminal parameters.

The usefulness of treating varicocele in order to improve fertility is still a matter of debate. The aim of this study was to evaluate variations in seminal parameters and inhibin B concentrations in a group of males affected by varicocele and treated by percutaneous retrograde sclerotherapy in comparison with a group of patients who did not undergo varicocele treatment. Thirty-eight patients with left varicocele underwent spermatic vein phlebography and percutaneous retrograde sclerotherapy with hydroxy-polyaethoxy-dodecanol. Serum inhibin B, follicle-stimulating hormone (FSH), testosterone levels and seminal parameters (sperm concentration, motility and morphology) were performed before and 6 months after sclerotherapy. Forty patients with left varicocele who did not undergo sclerotherapy were studied as controls. A significant increase (p < 0.01) in serum inhibin B levels and a significant decrease (p < 0.05) in FSH levels were observed 6 months after treatment. Semen analysis showed a significant improvement in sperm concentration (p < 0.05) and progressive motility (p < 0.01) after treatment. In control group no significant variations in hormonal and seminal parameters were observed 6 months after the basal examination. Six months after the basal evaluation, inhibin B levels were significantly higher in treated subjects than in controls (p < 0.05) whereas FSH levels were significantly lower (p < 0.05). Sperm concentration and progressive motility were significantly increased (p < 0.05 and p < 0.001, respectively) in treated subjects in comparison with controls. In conclusion, varicocele sclerotherapy improves inhibin B levels and seminal parameters, confirming the positive effect of this treatment on spermatogenesis and Sertoli cell function.

Alterations of seminal and hormonal parameters: An extrahepatic manifestation of HCV infection?

AIM: To evaluate the possible influences of HCV infection and relative antiviral treatment on seminal parameters and reproductive hormonal serum levels. METHODS: Ten male patients with HCV-related chronic hepatitis and 16 healthy male volunteers were studied. In all subjects seminal parameters (nemaspermic concentration, progressive motility, morphology) and hormonal levels were determined. Seminal parameters and inhibin B, follicle-stimulating hormone, luteinizing hormone, total and free testosterone, estradiol, prolactine in patients were measured after six and twelve months of antiviral combined (interferon+ribavirin) treatment. RESULTS: Patients before treatment showed a significantly lower nemaspermic motility and morphology as well as lower inhibin B and free testosterone levels than controls. Inhibin B levels in cases were improved six and 12 mo after treatment in five responders (161.9+/-52.8 pg/mL versus 101.7+/-47.0 pg/mL and 143.4+/-46.1 pg/mL versus 95.4+/-55.6 pg/mL, respectively). Hormonal pattern of patients did not significantly change after treatment, with the exception of estradiol levels with an initial reduction and an overall subsequent increment (19.7+/-6.4 pg/mL versus 13.6+/-5.0 pg/mL versus 17.3+/-5.7 pg/mL). However in 1-year responders a significant increment of free testosterone (14.2+/-2.54 pg/mL versus 17.1+/-2.58 pg/mL) occurred. An impairment of nemaspermic morphology occurred, while other seminal parameters did not change significantly during antiviral treatment. CONCLUSION: Patients with HCV infection show worse spermatic parameters than controls, suggesting a possible negative influence of virus on spermatogenesis, with further mild impairment during antiviral treatment. However therapy could improve the spermatic function, as suggested by the increased inhibin B levels and improved hormonal pattern in responders. Further studies are needed to confirm these preliminary intriguing results.

Nonspecific hydrophobic interactions stabilize an equilibrium intermediate of apomyoglobin at a key position within the AGH region.

Acid-induced unfolding of apomyoglobin (apoMb) proceeds in a multistate process involving at least one equilibrium intermediate (I) at pH 4.2. The structure of the I form has been investigated thoroughly, with significant effort devoted to identifying potentially stabilizing native contacts. Here, we test whether rigid side-chain packing interactions like those in holomyoglobin persist at a buried position, Met-131, within the low-pH apoMb intermediate. We have measured the urea-induced unfolding transitions of overpacking, underpacking, and polar substitutions of Met-131 to determine the effect on the stability of the native and intermediate states of apoMb. Whereas underpacking substitutions should destabilize the I form irrespective of the degree of native side-chain-packing interactions, we anticipate that overpacking replacements might show opposite effects in a tightly packed environment, compared with a region lacking native side-chain packing interactions. We observe that, whereas underpacking and polar substitutions destabilize the I form, overpacking substitutions are stabilizing, implying that I is structurally plastic. We also report a strong correlation between the I state unfolding free energies and side-chain transfer free energies from water to octanol. Our results suggest that, whereas side-chain hydrophobicity is important for the stability of the I form, specific side-chain packing interactions are not.