Artificial oocyte activation with calcium ionophore A23187 in intracytoplasmic sperm injection cycles using surgically retrieved spermatozoa

Objective: To evaluate the effect of artificial oocyte activation (AOA) on intracytoplasmic sperm injection (ICSI) cycles using surgically retrieved sperm.Design: Laboratory study.Setting: Fertility/assisted fertilization center.Patient(s): Couples undergoing surgical sperm retrieval for ICSI (n = 204).Intervention(s): Application of calcium ionophore A23187 for AOA.Main Outcome Measure(s): Cycles were divided into experimental groups according to the origin of the sperm used for injection and the type of azoospermia: [1] testicular sperm aspiration in nonobstructive-azoospermic patients (TESA-NOA group, n = 58), [2] TESA in obstructive-azoospermic patients (TESA-OA group, n = 48), [3] and percutaneous epididymal sperm aspiration in obstructive-azoospermic patients (PESA-OA, n = 98). For each experimental group, cycles where AOA was applied (subgroup: activation) were compared with cycles in which AOA was not applied (Subgroup: control). The fertilization, high-quality embryo, implantation, and pregnancy rates were compared among the subgroups.Result(s): For patients undergoing TESA, AOA did not improve ICSI outcomes for either type of azoospermia. However, for cases in which the injected sperm were retrieved from the epididymis, a statistically significantly increased rate of high-quality embryos was observed with AOA.Conclusion(s): Artificial oocyte activation may improve ICSI outcomes in azoospermic patients when epididymal, but not testicular spermatozoa, are injected. (Fertil Steril (R) 2009;92:131-6. (C)2009 by American Society for Reproductive Medicine.)

Relationship between DNA damage and sperm head birefringence

Birefringence or double refraction is the decomposition of a ray of light into two rays when it passes through an anisotropic material such as quartz. Sperm cells have been demonstrated to be optically anisotropic. The objective of this study was to evaluate the relationship between the pattern of human sperm head birefringence (SHBF) and DNA damage. A total of 26 patients with normal semen were included. DNA damage (fragmentation and denaturation) was evaluated in the sperm head in the context of birefringence, both total (SHBF-T) and partial (SHBF-P), by terminal deoxyribonucleotidyl transferase (TdT)-mediated dUDP nick-end labelling assay and acridine orange fluorescence, respectively. Positive DNA fragmentation in spermatozoa with SHBF-T (205/1053; 19.5%) was significantly higher (P < 0.0001) than in spermatozoa that presented SHBF-P (60/820; 7.3%). However, the percentage of denatured DNA in spermatozoa with SHBF-T (824/1256; 65.6%) was not significantly different from the ones with SHBF-P (666/1009; 66.0%). In conclusion, the data support a positive relationship between spermatozoa with total SHBF in their head and increased DNA fragmentation. (C) 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Evaluation of N-acetilglucosaminidase and myeloperoxidase activity in patients with endometriosis-related infertility undergoing intracytoplasmic sperm injection

Aim: Inflammation is as an important factor in ovulation with the active participation of leucocytes and their inflammatory mediators. The present study was performed to compare the activity of the inflammatory enzymes myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) in patients with endometriosis-related infertility and in normally ovulating women undergoing intracytoplasmic sperm injection (ICSI).Material and Methods: This prospective study included infertile women undergoing ICSI treatment. These women were divided into two groups: endometriosis anovulation (n = 18) and normally ovulating (n = 20). NAG and MPO activity was evaluated colorimetrically in serum and in follicular fluids obtained at the time of oocyte retrieval.Results: There was a significant correlation between the serum and follicular fluid activities of NAG and MPO (tau = 0.256, P = 0.025; and tau = -0.234, P = 0.041; respectively). Both serum and follicular fluid NAG activities were higher in patients with endometriosis compared to the control group (P < 0.001). MPO follicular fluid activity was lower in patients with endometriosis compared to normally ovulating women (P = 0.016).Conclusion: Infertile patients with endometriosis show a distinct pattern of serum and follicular fluid macrophage/neutrophil activation compared to normally ovulating women undergoing ICSI, which may reflect the role of immune and inflammatory alterations in endometriosis-related infertility.

Relationship between visualization of meiotic spindle in human oocytes and ICSI outcomes: a meta-analysis

The objective of this meta-analysis was to investigate the influence of meiotic spindle visualization in human oocytes on intracytoplasmic sperm injection (ICSI) outcomes. Search strategies included on-line Surveys of databases (MEDLINE, em BASE, Science Citation Index, Cochrane Controlled Trials Register and Ovid). The fixed effect was used for odds ratio. Ten trials fulfilled the inclusion criteria comparing in-vitro and clinical ICSI outcomes with or without visualization of meiotic spindle in fresh and in-vivo matured oocytes. According to the meta-analysis, the results showed statistically significant higher fertilization rate (P < 0.0001) when the meiotic spindle was viewed than when it was not. Moreover, the percentage of pro-nuclear-stage embyros with good morphology (P = 0.003), cleavage rate (P < 0.0001), percentage of day-3 top-quality embryos (P = 0.003) and percentage of embryos that reached the blastocyst stage (P < 0.0001) were statistically significantly better among, embryos derived from oocytes in which meiotic spindle was viewed compared with those in which meiotic spindle was not observed. However, these differences were not observed in the clinical pregnancy or implantation rates. This observation has clinical relevance mainly in countries where there is a legal limit on the number of oocytes to be fertilized. However, additional controlled trials are needed to further confirm these results.

Single-embryo transfer reduces clinical pregnancy rates and live births in fresh IVF and Intracytoplasmic Sperm Injection (ICSI) cycles: a meta-analysis

Background: It has become an accepted procedure to transfer more than one embryo to the patient to achieve acceptable ongoing pregnancy rates. However, transfers of more than a single embryo increase the probability of establishing a multiple gestation. Single-embryo transfer can minimize twin pregnancies but may also lower live birth rates. This meta-analysis aimed to compare current data on single-embryo versus double-embryo transfer in fresh IVF/ICSI cycles with respect to implantation, ongoing pregnancy and live birth rates.Methods: Search strategies included on-line surveys of databases from 1995 to 2008. Data management and analysis were conducted using the Stats Direct statistical software. The fixed-effect model was used for odds ratio (OR). Fixed-effect effectiveness was evaluated by the Mantel Haenszel method. Seven trials fulfilled the inclusion criteria.Results: When pooling results under the fixed-effect model, the implantation rate was not significantly different between double-embryo transfer (34.5%) and single-embryo transfer group (34.7%) (P = 0.96; OR = 0.99, 95% CI 0.78, 1.25). on the other hand, double-embryo transfer produced a statistically significantly higher ongoing clinical pregnancy rate (44.5%) than single-embryo transfer (28.3%) (P < 0.0001; OR: 2.06, 95% CI = 1.64,2.60). At the same time, pooling results presented a significantly higher live birth rate when double-embryo transfer (42.5%) (P < 0.001; OR: 1.87, 95% CI = 1.44,2.42) was compared with single-embryo transfer (28.4%).Conclusion: Meta-analysis with 95% confidence showed that, despite similar implantation rates, fresh double-embryo transfer had a 1.64 to 2.60 times greater ongoing pregnancy rate and 1.44 to 2.42 times greater live birth rate than single-embryo transfer in a population suitable for ART treatment.

Glutamate-induced obesity leads to decreased sperm reserves and acceleration of transit time in the epididymis of adult male rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Lung morphology and growth of rats exposed to tobacco smoke and alcohol

OBJETIVO: Investigar os efeitos morfológicos da exposição crônica à inalação de fumaça do tabaco e o do consumo de álcool nos pulmões e no crescimento de ratos. MÉTODOS: Sessenta ratos Wistar machos foram distribuídos em quatro grupos: controle, tabaco, álcool e tabaco + álcool, e acompanhados por um período de 260 dias. No final do periodo foi realizada análise morfológica dos pulmões por microscopia óptica e eletrônica. O crescimento dos ratos foi investigado através da medição do comprimento focinho-ânus, peso corporal e índice de massa corporal. RESULTADOS: Os três grupos expostos às drogas apresentaram peso e comprimento significativamente menores que os do grupo controle. As percentagens de bronquiolite e alveolite, e o diâmetro alveolar médio foram maiores nos grupos expostos à fumaça do tabaco, mas sem significancia estatística quando comparadas ao grupo controle. A microscopia eletrônica revelou apoptose mais intensa e lesões degenerativas no grupo de fumantes, enquanto lesões degenerativas nos corpos lamelares foram mais intensas com a associação de ambas as drogas. CONCLUSÕES: Este modelo experimental mostrou alterações morfológicas observadas por microscopia eletrônica, principalmente devido à exposição ao tabaco. Tanto o alcool como o tabaco prejudicaram o crescimento dos animais, o tabaco mostrando um efeito maior sobre o comprimento e o álcool sobre o peso corporal.

Selection of X chromosome of buffaloes sperm with Percoll gradients

The aim of the present study was to evaluate the selection of X chromosome of buffaloes sperm with Percoll gradients. The stock solution of Percoll was prepared in the proportion of 1:11 (1 part of Percoll:11 parts of a solution containing KCl 1M, NaH(2)PO(4) 0.1M, NaCl 1.5M and sodium HEPES 23.8 g/ml). In order to prepare 9 different gradients were added to the stocked Percoll the A solution (glicine-yolk extender) in the following proportions: 90, 80, 72, 65, 57, 49, 34 and 25%. A sample of 0.7 ml of the fresh semen was deposited at 2 ml of Percoll 80% for the sperm wash. The precipitate was put in tube with 0.7 ml of each gradient. Then, the precipitated was washed in TES solution by centrifugation (500xg for 10 minutes), and collected again and diluted in TES solution to be freeze. The presence of the F body in the spermatozoa was observed in 58.7 +/- 5.4% of the control group and in 41.2 +/- 5.4% of the treated group (p<0.01). This result showed an increment of 17.55 of male sperm in the Percoll's group. The reduction of the centrifugation force did not improve the percentage of X sperm.

Effects of incubation temperature on muscle morphology and growth in the pacu (Piaractus mesopotamicus)

This study describes the influence of incubation temperature during initial development phase on the morphology and muscle growth characteristics in the pacu (Piaractus mesopotamicus). Pacu eggs were incubated at 25, 27, and 29 degreesC until hatching. After day 5, fish from each temperature were transferred to 5001 tanks. At hatching and after 5, 25, and 60 days, muscle samples were collected, some were frozen in liquid nitrogen and others fixed in 4% paraformaldehyde or 2.5% glutaraldehyde. These samples were used for morphological, histochemical, immunohistochemical, and morphometric analysis. At hatching, we observed a superficial monolayer of small diameter fibers, lying just beneath the skin surrounding several round cells. From day 5, we observed two distinct populations of muscle fibers distributed in two layers: (1) red-in a superficial region with aerobic activity, and following acid preincubation, high mATPase activity, and 2) white-with anaerobic activity, and following alkaline preincubation, high mATPase activity. Twenty-five days after hatching, an intermediate layer and cell proliferating zones could be seen in the dorsal fin muscle region, with intermediate characteristics. Throughout the experimental period, there was an increase in muscle mass due to new fiber recruitment in the cell proliferating zones and between the more differentiated fibers in red, intermediate, and white muscles. This was more obvious from day 25, and at 29 degreesC than at 25 and 27 degreesC. Fiber hypertrophy occurred from hatching to 60 days and was more evident from 5 to 25 days. The number of proliferating nuclei (PCNA-labelling) increased from hatching to 60 days, and was more obvious in the 29 degreesC group at 60 days. Our results show that at incubation temperatures of 25, 27 and 29 degreesC, hypertrophy was predominantly from hatching to 25 days, after that muscle growth by hyperplastic mechanism increased. The interaction of muscle hypertrophic and hyperplastic growth processes in the 29 degreesC group produced the largest fish at the end of the experiment. (C) 2004 Elsevier B.V. All rights reserved.

Importance of sperm genotype (indicus versus taurus) for fertility and embryonic development at elevated temperatures

Heat stress has negative effects on bovine reproduction, particularly for European breeds (Bos taurus taurus) that are less thermotolerant than zebu cattle (Bos taurus indicus). Here, the evidence that spermatozoa and oocyte both contribute to early embryonic resistance to heat shock is demonstrated. In addition, the use of reproductive biotechnologies to improve bovine thermotolerance, are outlined by comparing data from taurus, indicus and crossbred genotypes. (c) 2005 Published by Elsevier B.V.

Fertility-associated antigen on Nelore bull sperm and reproductive outcomes following first-service fixed-time AT of Nelore cows and heifers

The objective was to determine whether the presence of fertility-associated antigen (FAA) on sperm collected from Nelore (Bos indicus) bulls can be used to assess potential fertility of sperm for use at first-service fixed-time AI (TAI). Six Nelore bulls were selected based on FAA status (FAA-negative: N = 3; FAA-positive: N = 3) and the ability to produce neat semen with >= 70% morphologically normal sperm and 60% estimated progressive motility before cryopmservation. In Experiment 1, suckled multiparous Nelore cows (N = 835) were evaluated for body condition score (BCS) and received an intravaginal progesterone device (CIDR) and 2.0 mg of estradiol benzoate (Day 0). on Day 9 the CIDR was removed, 12.5 mg of PGF(2 alpha) and 0.5 mg of estradiol cypionate were administered, and calves were removed for 48 h. All cows received TAI on Day II (48 h after CIDR removal). Pregnancy per TAI (P/TAI) was not different between FAA-positive and FAA-negative bulls (41.5% vs. 39.3%, respectively). There was an effect of AI technician on P/TAI (36.0% vs. 43.9%; P < 0.05) and BCS tended to affect P/TAI (P = 0.09), as cows with BCS >= 2.75 were 1.4 times more likely to become pregnant compared with cows with BCS < 2.75. In Experiment 2, nulliparous Nelore heifers (N = 617) were evaluated for BCS and received a CIDR and estradiol benzoate (2.0 mg) on Day 0. on Day 7, all heifers received PGF(2 alpha) (12.5 mg). on Day 9, CIDR inserts were removed and all heifers received estradiol cypionate (0.6 mg) and 200 IU eCG. All heifers received TAI on Day 11 (48 h after CIDR removal). Pregnancy/TAI was different (P = 0.04) between FAA-positive and FAA-negative bulls (33.7% vs. 40.7%, respectively). Presence of FAA on sperm was unsuccessful in assessing the potential fertility of sperm for use in TAI. (C) 2012 Elsevier B.V. All rights reserved.

Cryopreservation effects on domestic cat epididymal versus electroejaculated spermatozoa

Frozen-thawed epididymal spermatozoa have already been successfully used in artificial insemination in the domestic cat, proving to be a valuable resource for the reproduction of felid species, which are threatened with extinction. The aim of this study was to compare the effects of freezing and thawing on domestic cat semen collected by electroejaculation (EL) and from the epididymides (EP) and vasa deferentia. Ten adult cats were anesthetized, electroejaculated and immediately thereafter, orchiectomized. Epididymal spermatozoa were collected through the compression of caudae epididymidis and vasa deferentia. Spermatozoa were frozen-thawed following a single protocol. Sperm motility, sperm progressive status (0-5), plasma membrane integrity and morphology (light and transmission electron microscope) were assessed on two occasions, immediately after collection and after freezing and thawing. There were no significant differences between the electroejaculated and epididymal fresh or frozen-thawed spermatozoa for any of the variables. However, the incidence of acrosome defects after freezing and thawing increased by 19% based on light microscopy, whereas ultrastructural images revealed acrosome damages in most sperm cells. Since these acrosomal changes are known to affect sperm fertilising capacity, further studies are needed to optimize cryopreservation techniques for epididymal as well as electroejaculated domestic cat spermatozoa. (c) 2006 Elsevier B.V. All rights reserved.