Loredana Di Matteo

Intracytoplasmic injection of morphologically selected spermatozoa (IMSI) improves outcome after assisted reproduction by deselecting physiologically poor quality spermatozoa

PurposeWe used computer assisted sperm selection (MSOME) during cycles of intracytoplasmic sperm injection to test whether this technique improves results over traditional ICSI protocols. We also used the TUNEL assay to test whether MSOME could deselect physiologically abnormal spermatozoa.MethodsIndividual spermatozoa were examined with MSOME. Normal and abnormal spermatozoa were tested for the level of DNA fragmentation using TUNEL assay. In a prospective, randomized trial, patients were selected for standard ICSI, or IMSI techniques. We tested the two groups for biological and clinical parameters.Results64.8% of spermatozoa, otherwise selectable for ICSI, were characterized by abnormalities after computer-assisted sperm analysis. These sperm were also characterized by an increase in the level of DNA fragmentation. We noted an increase in embryo quality, pregnancy and implantation rates after computerized sperm selection during ICSI procedures.ConclusionsComputerised selection of spermatozoa during ICSI procedures deselects physiological abnormal spermatozoa and improves clinical results.

Cell Biology of the Harderian Gland

The harderian gland is an orbital gland of the majority of land vertebrates. It is the only orbital gland in anuran amphibians since the lacrimal gland develops later during phylogenesis in some reptilian species. Perhaps because it is not found in man, little interest was paid to this gland until about four decades ago. In recent years, however, the scientific community has shown new interest in analyzing the ontogenetic and morphofunctional aspects of the harderian gland, particularly in rodents, which are the preferred experimental model for physiologists and pathologists. One of the main characteristics of the gland is the extreme variety not only in its morphology, but also in its biochemical properties. This most likely reflects the versatility of functions related to different adaptations of the species considered. The complexity of the harderian gland is further shown in its control by many exogenous and endogenous factors, which vary from species to species. The information gained so far points to the following functions for the gland: (1) lubrication of the eye and nictitating membrane, (2) a site of immune response, particularly in birds, (3) a source of pheromones, (4) a source of saliva in some chelonians, (5) osmoregulation in some reptiles, (6) photoreception in rodents, (7) thermoregulation in some rodents, and (8) a source of growth factors.

Intratesticular feedback mechanisms in the regulation of steroid profiles in the frog, Rana esculenta.

Testosterone (T), 5 alpha-dihydrotestosterone (DHT), estradiol-17 beta (E), and progesterone (P) were measured in the plasma of the frog, Rana esculenta, during the annual cycle. Moreover, in vitro experiments were carried out in order to investigate the local regulation of steroidogenesis. Testosterone and DHT showed high values during autumn and early spring and had a T/DHT ratio which increased during summer, while E peaked in midspring, remaining at detectable values thereafter. Progesterone increased in autumn, winter, and spring. In vitro incubations of minced testes showed that E, stimulated by pituitary factors, inhibited androgen synthesis while T did not. Our results indicate that paracrine and/or autocrine mechanisms operate in the frog testis to regulate annual steroid profiles.

Molecular forms of immunoreactive gonadotropin-releasing hormone in hypothalamus and testis of the frog, Rana esculenta.

The hypothalamus and the testis of the frog, Rana esculenta, contain gonadotropin-releasing hormone (Gn-RH)-like peptides which are recognized by an antiserum raised against mammalian Gn-RH. Two molecular forms which coelute with synthetic chicken II and salmon Gn-RH from reverse-phase HPLC were distinguished in the hypothalamus. A single peak coeluting with synthetic chicken II Gn-RH was present in the testis.

Mitochondria and human preimplantation embryo development

Human reproduction, like all biological systems, is characterised by a large level of variability. In this field, the variability is observed as a large difference in implantation potential of human embryos developing in vitro, despite similarities in observable parameters such as rate of development and morphology of these embryos. One of the underlying factors that determines developmental potential in these embryos is the availability of energy in the form of ATP for development. Here, we suggest that, despite the evidence suggesting that mitochondrial metabolism is relatively inactive during preimplantation embryo development, aerobic (mitochondrial) metabolism contributes a major role in the supply of ATP. A second pathway, anaerobic respiration, is also active and the two pathways work in synchrony to supply all the ATP necessary. We discuss the differences in the two forms of energy production and suggest that, although anaerobic respiration can supplement deficiencies in the energy supply in the short term, this is not sufficient to substitute for aerobic respiration over long periods. Therefore, we suggest that deficiencies in the levels of aerobic respiration can explain variability in the implantation potential of apparently equivalent embryos.

An oocyte score for use in assisted reproduction.

PurposeIn this work, we describe a system for the morphological scoring of human oocytes prior to fertilisation and use this system to test whether oocyte morphology is an indicator of fertilisation, embryo development and implantation potential.MethodsThe study is a prospective trial of the use of oocyte morphological scores in 822 patients undergoing their first cycle of ICSI. Analyses of oocytes were performed prior to ICSI procedures and the scores compared with fertilisation rates, embryo quality and clinical results.Results‘Top quality’ oocytes had a significantly higher level of fertilisation (96%) as compared to low scoring oocytes (25.6%). Where top quality oocytes formed top quality embryos, we noted a clinical success rate of 63.4%.ConclusionsClinical success rates were increased in cases where top quality oocytes formed top quality embryos after ICSI. The analysis of oocyte morphology may represent a positive selection feature during ICSI.

Localization of GnRH molecular forms in the brain, pituitary, and testis of the frog, Rana esculenta

In the amphibian brain four molecular forms of GnRH have been identified so far: mammalian GnRH (m- and hydroxyproline9m-), chicken II GnRH (cII), and a salmon (s) GnRH-like peptide. In Rana esculenta, cII- and s-GnRH-like molecules have been partially characterized in the brain extracts using HPLC combined with radioimmunoassay. Moreover, since cII-GnRH-like material has been detected in Rana esculenta testis, the present study describes the localization of the above peptides in the brain and testis of the frog. Immunoreactive cII-GnRH and m-GnRH neurons and fibers were identified in the anterior preoptic area (APOA) and in the median septal area (MSA). A population of cells located on the dorsal side of the caudal preoptic region was also stained. Immunopositive fibers were seen to overlap the median eminence before ending within the pars nervosa. Moreover, densely packed fibers made close contact with the vascular complex in the median eminence. Conversely, immunoreactive s-GnRH-like material was absent in APOA and MSA, but weakly scattered elements were detected by the anti-s-GnRH serum in the dorsal side of the caudal preoptic region. Using m-GnRH antiserum, a strong immunopositivity was observed in the median eminence but not within the pars nervosa, indicating that, besides cII-GnRH and s-GnRH-like material, also m-GnRH-like material is present in Rana esculenta brain. In the testis, cells of the interstitial and germinal compartment were detected by anti-cII-GnRH during different periods of the annual cycle. In particular, in October and February interstitial tissue was intensely stained, coinciding with periods of increased androgen production and the onset of the new spermatogenic wave, respectively.

Human cleavage-stage embryo vitrification is comparable to slow-rate cryopreservation in cycles of assisted reproduction

ObjectivesTo compare embryo survival, pregnancy and implantation rates after cryopreservation of human cleavage-stage embryos with slow-rate cryopreservation or vitrification.Study design262 patients, attending for assisted reproduction, were prepared for oocyte retrieval using standard controlled ovarian hyperstimulation protocols. Excess embryos were cryopreserved on day 3 either by vitrification, or slow-rate cryopreservation in a programmable freezer. Cycles of thawing were monitored for thaw efficiency, pregnancy and implantation rates.ResultsClinical pregnancy and implantation rates were highly comparable between cycles in which day 3 embryos were thawed either after slow-rate cryopreservation or vitrification.ConclusionsThese data suggest that vitrification of human embryos during assisted reproduction cycles achieves comparable success rates to fresh cycles and therefore can be applied in the laboratory of assisted reproduction.

Morphological and hormonal changes in the frog, Rana esculenta, testis after administration of ethane dimethane sulfonate.

Apart from mice, in rodents ethane dimethane sulfonate (EDS) selectively destroys Leydig cells. This has been indicated as a new method for the study of seminiferous interstitial compartment interaction. No information on the possible destruction and repopulation of Leydig cells exists in lower vertebrates. This study deals with EDS effects in the frog, Rana esculenta. Animals received a single intraperitonial dose (100 mg/kg body wt) and were sacrificed at 0, 12, and 24 hr and 3, 4, 7, 14, and 28 days postinjection. Androgens (testosterone + DHT) were measured in plasma and right testes. Moreover, left testes were fixed and examined for histological observation. Plasma androgen levels were extremely low on Day 4 after EDS treatment and remained unchanged thereafter. In testes, androgen levels decreased on Day 4 but increased to control levels on Day 14. Leydig cells were damaged within 3 days post-treatment and were completely destroyed on Days 4 and 5. Germinal compartment damage appeared only where the adjacent interstitial tissue presented complete destruction. Pale primary spermatogonia (stem cells) were always present. Testes restored to normal on Day 14 and spermatogenesis resumed to the regenerating interstitial tissue. These results show that regenerating testes in R. esculenta retain androgens and that interstitial-germinal compartment communications may have a role in maintaining spermatogenesis.

Morphology of the Harderian gland of the Gecko, Tarentola mauritanica.

ABSTRACT The Harderian gland of the gecko, Tarentola mauritanica, was studied at the histological, histochemical, and ultrastructural levels. It is a nonlobate compound acinar gland surrounded by a thin capsule of connective tissue. Numerous connective tissue‐type mast cells, ultrastructurally similar to those described in other higher vertebrates, were identified in the interstitial tissue between the acini. Pyramidal or columnar‐shaped secretory glandular cells were observed in the acini. In the glandular cells, two types of structures could be distinguished on the basis of their high or low electron density. Lipid droplets were found in the cytoplasm of the Harderian gland of both sexes. Histochemical tests showed that the Harderian gland of the gecko is a seromucous gland. The secretion is essentially merocrine, although an apocrine type of secretion is sometimes observed. J. Morphol. 244:137–142, 2000.