Value of microsurgical varicocelectomy for severe oligo-asthenospermia patients failed in fertilization assisted by in vitro fertilization.

OBJECTIVE: To investigate the clinical effect of microsurgical varicocelectomy on severe oligo-asthenospermia patients failing in fertilization assisted by intracytoplasmic sperm injection. PATIENTS AND METHODS: From January 2013 to August 2014, forty-nine patients with severe oligo-asthenospermia and serious varicoceles were treated by microsurgical varicocelectomy after failing in fertilization assisted by intracytoplasmic sperm injection (ICSI), eleven of whom had varicoceles on the left side and thirty-eight had bilateral varicoceles. Patients were followed up for the natural pregnancy condition, changes of routine semen parameters and reproductive hormone level and the embryonic development and outcome of next IVF-ET (ICSI) cycles within 6 months. RESULTS: After surgery, 61.2% (30/49) of spouses obtained clinical pregnancy. Among whom 22.4% (11/49) were naturally pregnant, 32.65% (16/49) were conceived after second IVF-ET assistance, and 6.1% (3/49) were conceived with the third or further assistance of ICSI-ET. The overall miscarriage rate was 16.7% (5/30). All of the patients had improvement in the sperm concentration and forward motility. The sperm concentration increased from (10.53 ± 8.76) × 106/ml to (20.23 ± 11.76) × 106/ml. The ratio of forward motile sperm was increased to (30.52 ± 18.78) % from (8.75.52 ± 6.36) % (p < 0.01). The serum total testosterone (T) improved from (2.19 ± 1.03) ng/ml to (4.05 ± 0.64) ng/ml (p < 0.05). Serum follicle-stimulating hormone (FSH) changed from (5.23 ± 1.26) mIU/ml to (3.76 ± 2.22) mIU/ml after the procedure. Luteinizing hormone (LH) changed from (4.38 ± 1.36) to (3.98 ± 1.38) mIU/ml. Estrogen (E2) changed from 40.28 ± 7.26 pg/ml to 35.24 ± 5.75 pg/ml. Prolactin (PRL) level elevated from (18.24 ± 4.28) to (17.16 ± 2.16) ng/ml (p > 0.05). The fertility rate of in vitro fertilization significantly improved to (83.36 ± 19.36) % from (72.36 ± 17.88) % (p < 0.05). The rate of 2PN ratio increased from (66.73 ± 17.93) % to (75.96 ± 20.39) %. The cleavage rate increased from (83.26 ± 32.33) % to (90.35 ± 23.66). The abnormal fertility rate were (5.36 ± 12.58) % and (7.26 ± 13.89) % before and after the procedure (p > 0.05), while the rate of high-quality embryos increased significantly from (34.36 ± 33.27) % to (55.67 ± 23.36) % (p < 0.05). The rate of transferable embryos remained without significant change (70.67 ± 30.6% before and 60.53 ± 30.27% after the procedure). The anabiosis rate of frozen embryo increased from (66.32 ± 30.69) % to (89.72 ± 29.69) %. The further blastocyst rate improved from (10.98 ± 9.7) % to (30.27 ± 15.33) % (p < 0.01). CONCLUSIONS: The microsurgical varicocelectomy effectively improved sperm parameters, the fertility rate of oocyte fertilized in vitro and the anabiosis rate and blastocyst rate of the frozen embryo for on patients with severe oligo-asthenospermic, and further increased the odds of natural pregnancy, the rate of high-quality embryos and the success rate of in vitro fertilization.

Cryoprotectant-free ultra-rapid freezing of human spermatozoa in cryogenic vials.

The objective of this study was to investigate the effect of ultra-rapid freezing (direct immersion in liquid nitrogen) on human spermatozoa in cryogenic vials (≥0.5 ml) at different concentrations of sucrose. After swim-up, the sperm suspensions (N = 58) were diluted with sperm preparation medium and divided into six aliquots: swim-up (fresh), conventional freezing group (slow freezing) and four ultra-rapid freezing groups containing sucrose at different concentrations (0.15 m, 0.20 m, 0.25 m and 0.30 m). Sperm motility, progressive motility, plasma membrane integrity, DNA stability and acrosome integrity of fresh and cooled-warmed spermatozoa were analysed. The progressive motility, plasma membrane and acrosome integrity of spermatozoa in the 0.20 m sucrose group were significantly higher than those of the slow freezing group (47.5 ± 6.8% versus 36.4 ± 8.7%, 73.2 ± 6.9% versus 63.9 ± 6.3%, 53.7 ± 10.0% versus 35.9 ± 9.7% respectively, P < 0.05). However, no differences were found in sperm motility or DNA stability (58.5 ± 6.3% versus 54.2 ± 5.3%, 90.1 ± 2.8% versus 87.2 ± 4.7%, P > 0.05 respectively) between the 0.20 m sucrose and the slow freezing group. No differences were found between the ultra-rapid and slow freezing group at the other concentrations of sucrose. Our findings suggest that the method of ultra-rapid freezing of human spermatozoa in cryogenic vials with a solution containing 0.20 m sucrose results in recovery of spermatozoon of superior qualities. In contrast to slow freezing, the ultra-rapid freezing technique of human spermatozoa seems to reduce cryoinjuries and maintain important physiological characteristics of the spermatozoa after warming.

Skeletal development and abnormalities of the vertebral column and of the fins in hatchery-reared turbot Scophthalmus maximus.

To describe the skeletal development and abnormalities in turbot Scophthalmus maximus, samples were collected every day from hatching to 60 days after hatching (DAH). A whole-mount cartilage and bone-staining technique was used. Vertebral ontogeny started with the formation of anterior haemal arches at 5·1 mm standard length (L(S) ) c. 11 DAH, and was completed by the full attainment of parapophyses at 16·9 mm L(S) c. 31 DAH. Vertebral centra started to develop at 6·3 mm L(S) c. 16 DAH and ossification in all centra was visible at 11·0 mm L(S) c. 25 DAH. The caudal fin appeared at 5·1 mm L(S) c. 11 DAH and ossification was visible at 20·6 mm L(S) c. 37 DAH. The onset of dorsal and anal fin elements appeared at 5·8 mm L(S) c. 15 DAH and 6·3 mm L(S) c. 16 DAH, respectively. Ossifications of both dorsal fin and anal fin were visible at 20·6 mm L(S) c. 37 DAH. The pectorals were the only fins present before first feeding, their ossifications were completed at 23·5 mm L(S) c. 48 DAH. Pelvic fins began forming at 7·2 mm L(S) c. 19 DAH and calcification of the whole structure was visible at 19·8 mm L(S) c. 36 DAH. In the present study, 24 types of skeletal abnormalities were observed. About 51% of individuals presented skeletal abnormalities, and the highest occurrence was found in the haemal region of the vertebral column. As for each developmental stage, the most common abnormalities were in the dorsal fin during early metamorphic period (stage 2), vertebral fusion during climax metamorphosis (stage 3) and caudal fin abnormality during both late-metamorphic period (stage 4) and post-metamorphic period (stage 5). Such research will be useful for early detection of skeletal malformations during different growth periods of reared S. maximus.

Digestive enzyme activities of turbot (Scophthalmus maximus L.) during early developmental stages under culture condition.

Digestive enzyme activities were analysed in turbot (Scophthalmus maximus) from hatching until 60 days after hatching (DAH). Trypsin sharply increased to the climax at 17 DAH and decreased until 31 DAH followed by a stable level thereafter. Amylase was determined at 4 DAH, reached the maximum value at 19 DAH and declined sharply to 39 DAH and remained at a low level thereafter, suggesting the carbohydrate component should remain at a low level in formulated diets. Pepsin was detected at 9 DAH and increased to 34 DAH and then remained at a stable level. The above results revealed pancreatic enzymes are no longer main enzymes for food digestion after the formation of functional stomach. Leucine-alanine peptidase (Leu-ala) and alkaline phosphatase (AP) and leucine aminopeptidase N (LAP) were found in newly hatched larvae. Both AP and LAP activities markedly increased to 23 DAH, decreased abruptly to 50 DAH and increased gradually to 60 DAH. Leu-ala reached the plateau from 23 to 39 DAH, followed by a decline to 46 DAH and an increase until 60 DAH. The brush border membrane (BBM)-bound enzyme activities increased from 30% at 31 DAH to 81% at 38 DAH of the total activities, indicating the maturation of intestinal tract.

Changes in RNA, DNA, protein contents and growth of turbot Scophthalmus maximus larvae and juveniles.

The growth potential of turbot Scophthalmus maximus larvae and juveniles was studied using nucleic acid-based indices and protein variables. The experiment was carried out from 4 to 60 days post hatching (dph). A significant increase in instantaneous growth rate during metamorphosis and retarded growth rate during post-metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein all showed developmental stage-specific traits. The RNA:DNA ratio decreased up to 12 dph, then increased rapidly till 19 dph and fluctuated until 35 dph followed by a decline to the end. The RNA:DNA ratio was positively correlated with growth rate of juveniles during the post-metamorphic phase, whereas this ratio was not a sensitive indicator of growth during the pre-metamorphic phase and metamorphosis. The protein:DNA ratio showed a similar tendency to the RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of S. maximus performed mainly by hyperplasia from 4 to 12 dph and hypertrophy until 21 dph during the pre-metamorphic larval phase. Growth was dominantly hypertrophical from the early- to mid-metamorphosing phase and hyperplastic thereafter. The results show that the DNA content and protein:DNA ratio can evaluate growth rates of larval and juvenile S. maximus on a cellular level.

Cadmium-induced ultramorphological and physiological changes in leaves of two transgenic cotton cultivars and their wild relative.

The present study describes cadmium-induced alterations in the leaves as well as at the whole plant level in two transgenic cotton cultivars (BR001 and GK30) and their wild relative (Coker 312) using both ultramorphological and physiological indices. With elevated levels of Cd (i.e. 10, 100, 1000 microM), the mean lengths of root, stem and leaf and leaf width as well as their fresh and dry biomasses linearly decreased over their respective controls. Moreover, root, stem and leaf water absorption capacities progressively stimulated, which were high in leaves followed by roots and stems. BR001 accumulated more cadmium followed by GK30 and Coker 312. Root and shoot cadmium uptakes were significantly and directly correlated with each other as well as with leaf, stem and root water absorption capacities. The ultrastructural modifications in leaf mesophyll cells were triggered with increase in Cd stress regime. They were more obvious in BR001 followed by GK30 and Coker 312. Changes in morphology of chloroplast, increase in number and size of starch grains as well as increase in number of plastoglobuli were the noticed qualitative effects of Cd on photosynthetic organ. Cd in the form of electron dense granules could be seen inside the vacuoles and attached to the cell walls in all these cultivars. From the present experiment, it can be well established that both apoplastic and symplastic bindings are involved in Cd detoxification in these cultivars. Absence of tonoplast invagination reveals that Cd toxic levels did not cause water stress in any cultivars. Additionally, these cultivars possess differential capabilities towards Cd accumulation and its sequestration.

Human p53 is phosphorylated on serines 6 and 9 in response to DNA damage-inducing agents.

To characterize the sites in human p53 that become phosphorylated in response to DNA damage, we have developed polyclonal antibodies that recognize p53 only when it is phosphorylated at specific sites. Several attempts to generate an antibody to p53 phosphorylated at Ser(6) using a phosphoserine-containing peptide as an immunogen were unsuccessful; however, phosphorylation-specific antibodies were produced by using the phosphoserine mimetic, l-2-amino-4-phosphono-4, 4-difluorobutanoic acid (F(2)Pab), in place of phosphoserine. Fmoc-F(2)Pab was prepared by an improved synthesis and chemically incorporated using solid phase peptide synthesis. Affinity-purified antibodies elicited by immunizing rabbits with an F(2)Pab peptide coupled to keyhole limpet hemocyanin recognized a p53(1-39) peptide phosphorylated only at Ser(6) but not the unphosphorylated peptide or the same peptide phosphorylated at Ser(9), Ser(15), Ser(20), Ser(33), or Ser(37). Untreated A549 cells exhibited a background of constitutive phosphorylation at Ser(6) that increased approximately 10-fold upon exposure to either ionizing radiation or UV light. Similar results were obtained for Ser(9) using antibodies raised against a conventional phosphopeptide. Ser(9) was phosphorylated by casein kinase 1 in vitro in a phosphoserine 6-dependent manner. Our data identify two additional DNA damage-induced phosphorylations in human p53 and show that F(2)Pab-derivatized peptides can be used to develop phosphorylation site-specific polyclonal antibodies.

[A study on the genetic epidemiology of congenital heart disease].

In order to explore the genetic model of congenital heart disease (CHD), 375 individuals of CHD were investigated. The results showed that the incidence of primary relatives was 6.9%, the heritability was 66.13 +/- 1.13%, the incidence of secondary relatives was 1.7% and the heritability was 38.25 +/- 8.3% respectively. The average heritability among primary, secondary relatives was 65.65 +/- 1.09% which suggested that the genetic factor played an important role in pathogenesis. The sequence of the incidence in the relatives showed: primary relatives (6.9%) > secondary relatives (1.7%) > distant relatives (0.86%) and was in accordance with polygenic incidence, suqqesting that gene play an important role in the liability variation of CHD.

[Synthesis of receptor reagent--estradiol-polyethyleneglycol-fluorescein isothiocyanate].

Estradiol conjugate has been used as a tracer to detect estrogen receptor of human mammary cancer cells. In order to look for more stable carrier of fluorescent estradiol conjugate, we substituted polyethyleneglycol (PEG) for bovine serum albumin (BSA) and synthsized 17 beta-estradiol-6-carboxymethyl-oxime-PEG-fluorescein isothiocyanate. The structure of the title compound was confirmed by IR and UV. In this paper, we also describe another procedure to convert the terminal hydroxyl groups of PEG to the more reactive primary amino PEG by tosylation, azide group substitution and catalytic hydrogenation. The ratio of conversion is about 87%.