4-Hydroxyestradiol improves mouse embryo quality, epidermal growth factor-binding capability in vitro and implantation rates.

Embryo implantation in the uterus is a critical step to achieve success following ART. Despite favorable uterine conditions, a great number of good quality embryos fail to implant, often for reasons that are unknown. Hence, improving the implantation potential of embryos is a subject of great interest. 4-Hydroxyestradiol (4-OH-E2), a metabolic product of estradiol produced by endometrial cells, plays a key role in endometrial-embryonic interactions that are necessary for implantation. Nonetheless, the effects of 4-OH-E2 on embryos obtained in vitro have not been yet described. This study was designed to determine whether culture media enriched in 4-OH-E2 could improve the quality and implantation rate of embryos obtained in vitro, using both in vitro and in vivo models. We also analyzed its effects on the epidermal growth factor (EGF)-binding capability of the embryos. Our results showed that the presence of 4-OH-E2 in the culture media of embryos during the morula to blastocyst transition increases embryo quality and attachment to endometrial cells in vitro. 4-OH-E2 can also improve viable pregnancy rates of mouse embryos produced in vitro, reaching success rates that are similar to those from embryos obtained directly from the uterus. 4-OH-E2 improved the embryos' ability to bind EGF, which could be responsible for the increased embryo implantation potential observed. Therefore, our results strongly suggest that 4-OH-E2 is a strong candidate molecule to supplement human IVF culture media in order to improve embryo implantation. However, further research is required before these findings can be translated with efficacy and safety to fertility clinics.

Laparoscopic uterine graft procurement and surgical autotransplantation in ovine model.

Currently, uterus transplantation (UTx) is a clinical option for infertile women. Over the past three decades, treating benign or malignant gynecological diseases with minimally invasive gynecological surgery has improved, providing significant advantages over conventional open surgery. This study addresses the method used for laparoscopic live-donor ovariohysterectomy and graft harvest from a sheep model. Using a microsurgical practice, ten grafts were autotransplanted after uterine perfusion. End-to-end anastomosis techniques were used to approximate veins and arteries. Follow-ups were carried out 2-months after surgery and postoperative studies included ultrasound scan, diagnostic hysteroscopy, vascular angiography, and exploratory laparoscopy. All transplants were completed without complications. After vascular anastomosis, total reperfusion of the tissue was accomplished in all animals without confirmation of arterial or venous thrombosis. Angiographic explorations did not show any statistically significant dissimilarity in the arterial diameters between the different examination times. 3-months after uterine transplantation all animals underwent assisted reproduction techniques. Patent uterine arteries were observed 4, 8 and 12 months after the transplant. 6-months after transplantation, six sheep (60%) became pregnant with assisted reproduction practices. We noticed an increase in the degree of fibrosis of the cervix samples in non-pregnant animals of the transplant group. Laparoscopic surgery can be an advantageous approach for the uterus retrieval procedure during uterine transplantation. However, larger sample sized reports are needed in order to accomplish validation, standardization and wider use of this route.

Composition and biting activity of Anopheles (Diptera: Culicidae) in the Amazon region of Colombia.

To provide information for public health policy on mosquito nets in the Amazon region of Colombia, we conducted landing catches to estimate Anopheles species composition and biting activity. Two hundred twenty person-nights of catches were done in seven locations over a period of 14 mo. A total of 1,780 Anopheles mosquitoes were caught (8.1 per person-night). Among the nine species found, An. oswaldoi Peryassú was the most common (776 mosquitoes, 44%), followed by An. darlingi Root s.l. (498, 28%). An. oswaldoi was the most common species collected outdoors, where its biting rate dropped steadily from a peak of >15 bites/person-night at the start of the night (1800-1900 hours) to approximately equal to 2 bites/person-night before dawn. An. darlingi was the most common species collected indoors, with a biting rate of approximately equal to 3-4 bites/person-night until about midnight, when the rate dropped below 1 bite/person-night, before showing a secondary peak before dawn. Sixty-four mosquito nets were analyzed by the technique of high-performance liquid chromatography (HPLC) for levels of deltamethrin (DM). All but two (62) of these were reported by their owners to have been impregnated with insecticide, and 53 were found by HPLC to have deltamethrin. However, one half (32) of the nets had concentrations <4 mg/m2 and therefore were likely to have been inadequately protective. An inverse association was found between the reported time between washes and deltamethrin concentration. These findings show a need for additional protection from mosquitoes when not inside nets, as well as for more effective impregnation, possibly through wash-resistant insecticide formulation.

Contribution of culture media to oxidative stress and its effect on human oocytes.

The adverse effects of reactive oxygen species (ROS) on many aspects of reproduction are well documented. However, much less is known regarding the contribution of culture media to the oxidative stress of gametes during assisted reproductive techniques. This study measured the generation of ROS by culture media during IVF procedures and its effects on human oocytes. Commercially supplied culture media generated ROS at various rates, depending on the composition, whereas follicular fluid generated ROS at a much lower level. The incubation of cumulus-oocyte complexes (COC) in culture media induced marked lipid peroxidation compared with levels found in freshly retrieved COC. This plasma membrane damage, measured with the quenching of cis-parinaric acid fluorescence assay, was attenuated by supplementation of the medium with alpha-tocopherol or catalase. Moreover, there was an association between ROS production by culture medium and thiolic content consumption within the oocytes, suggesting that the intracellular reduced glutathione pool was partially depleted during in-vitro manipulation. The results show that culture medium could damage oocytes (and consequently embryo development) depending on their composition, and it is proposed that current IVF protocols could be revised in order to decrease ROS generation.

Store-operated calcium entry in human oocytes and sensitivity to oxidative stress.

Calcium signaling is a cellular event that plays a key role at many steps of fertilization and early development. However, little is known regarding the contribution of extracellular Ca(2+) influx into the cell to this signaling in gametes and early embryos. To better know the significance of calcium entry on oocyte physiology, we have evaluated the mechanism of store-operated calcium entry (SOCE) in human metaphase II (MII) oocytes and its sensitivity to oxidative stress, one of the major factors implicated in the outcome of in vitro fertilization (IVF) techniques. We show that depletion of intracellular Ca(2+) stores through inhibition of sarco(endo)plasmic Ca(2+)-ATPase with thapsigargin triggers Ca(2+) entry in resting human oocytes. Ba(2+) and Mn(2+) influx was also stimulated following inhibition, and Ca(2+) entry was sensitive to pharmacological inhibition because the SOCE blocker 2-aminoethoxydiphenylborate (2-APB) reduced calcium and barium entry. These results support the conclusion that there is a plasma membrane mechanism responsible for the capacitative divalent cation entry in human oocytes. Moreover, the Ca(2+) entry mechanism described in MII oocytes was found to be highly sensitive to oxidative stress. Hydrogen peroxide, at micromolar concentrations that could mimic culture conditions in IVF, elicited an increase of [Ca(2+)](i) that was dependent on the presence of extracellular Ca(2+). This rise was preventable by 2-APB, indicating that it was mainly due to the enhanced influx through store-operated calcium channels. In sum, our results demonstrate the occurrence of SOCE in human MII oocytes and the modification of this pathway due to oxidative stress, with possible consequences in IVF.

Case-control study of mosquito nets against malaria in the Amazon region of Colombia.

The degree of effectiveness of mosquito nets against malaria in the Americas has remained uncertain. We carried out a case-control study of net use and mild malaria in the Amazonas state of Colombia. Two hundred ninety cases were enrolled via the Health Department services, and 977 community-based controls matched for age, sex, and place of residence. We found that a large proportion of the population (96% of controls) slept under nets. Nevertheless, we found a benefit of impregnated nets compared with no net use: adjusted odds ratio (OR) for mild malaria 0.44, 95% confidence interval (CI) 0.20-0.98. Nonimpregnated nets had a benefit that was only slightly smaller but not statistically significant (OR for mild malaria 0.54, 95% CI 0.25-1.18). Travel in the previous month had an odds ratio of 6.2 (95% CI 3.1-8.8) and a population attributable fraction of 13% compared with 11% for failure to use an impregnated net. We conclude that, in the Amazon region, promotion of mosquito net use and impregnation is justified, and that there is a need for measures to protect travelers from malaria.