Addendum from Portugal--how about an annotated IFFS surveillance for the millennium?
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Cryopreservation↗
Biomedical subjects
M Sousa
Publications and source records attributed to M Sousa.
Chromatin condensation during Scrobicularia plana spermiogenesis: a controlled and comparative enzymatic ultracytochemical study.
In Scrobicularia plana testis, a nuclear acid phosphatase (ACPase) activity was detected in mid and late spermatids with the improved Gomori-chloride procedure. Lead deposits were first observed in mid spermatids at focal points over condensed chromatin strands, increasing in density as chromatin further condensated. In late spermiogenesis, lead deposits became concentrated between chromatin aggregates, and after total DNA compaction were transfered to the nuclear periphery and then shed into the cytoplasm. The specificity of the nuclear ACPase was tested against different pH values (3.9, 7.2, 7.8, 9.0), substrates (TPP, IDP, TMP, p-NCS, ATP, GTP, AMP, ADP, AMP-PNP) and inhibitors (NaF, levamisole, Zn, vanadate, theophylline). To further specify the nature of this nuclear ACPase, other enzymes were comparatively studied at their optimal pH values and at pH 5.0: nucleoside-diphosphatase, thiamin-pyrophosphatase, inorganic trimetaphosphatase, lysosomal arylsulfatases A and B, ATPase, GTPase, 5'-nucleotidase, adenylate kinase, and adenylate cyclase. Several other controls were introduced to exclude artefactual deposits induced by lead ions and tissue molecules. The results showed that the enzyme has an optimal pH at 5.0, a high specific affinity for beta-GP, and is inhibited by NaF, which suggests that it behaves as a type B-ACPase, and all controls demonstrated the specificity of the enzymic activity. Because lead deposits were specifically and temporally associated with spermatid chromatin condensation, when DNA and RNA synthesis, histones, phosphoproteins and RNA molecules strongly decrease, it is possible to suggest that the nuclear ACPase could be associated with DNA processing during chromatin compaction or involved in the hydrolysis of 2' and 3' nucleotides resulting from nuclear RNase action during RNA degradation.
Acid Phosphatase↗
Total hip arthroplasty for tuberculous coxitis.
We report a case of tuberculous arthritis of the hip in a 22 year old male patient, treated with arthrotomy and antituberculous antibiotic therapy for 9 months; the joint deteriorated and 2 years later he underwent uncemented total hip arthroplasty. He received antibiotic therapy for 3 months preoperatively and for 6 months postoperatively. At 5 year follow-up there was no evidence of recurrent infection.
Adult↗
Clinical efficacy of spermatid conception: analysis using a new spermatid classification scheme.
Fertilization and pregnancy outcomes of 50 round spermatid injection (ROSI) and 20 elongated spermatid injection (ELSI) treatment cycles are related to various characteristics of the cycles, with particular reference to spermatid developmental stage as assessed by using a classification scheme adapted to this purpose. Although this classification includes eight stages, a complete block was mostly detected at the earliest stage (Sa1) or at the latest stages (Sd1 and Sd2). Thus, spermiogenesis was blocked at Sa1 stage in 50 cases (71%), at Sd1 stage in eight cases (11%) and at Sd2 stage in 10 cases (14%). Only in two cases (3%) was spermiogenesis blocked at an intermediate stage (Sb2). Globally, fertilization rates were higher for ELSI than for ROSI. No pregnancy was achieved in the ROSI cycles, whereas nine pregnancies resulted from the ELSI cycles. Two of them (both with Sd2 spermatids) ended in a first trimester spontaneous abortion. Of the seven ongoing pregnancies, five are singleton (two with Sd1 spermatids, two with Sd2 spermatids, and one after a mixed transfer after injection of Sa2 and Sd1 spermatids) and two are twin (one with Sd1 and the other with Sd2 spermatids). No pregnancy was achieved in the two cycles with Sb2 spermatids. One of the two twin pregnancies has already resulted in the birth of two healthy children.
Adult↗
In-vitro maturation of round spermatids using co-culture on Vero cells.
In an attempt to determine whether co-culture could promote sperm maturation, three patients with non-obstructive azoospermia, two with maturation arrest at the level of primary spermatocytes and one patient with <1% tubules showing complete spermatogenesis, and one patient with total globozoospermia, gave consent to experimentally co-culture round spermatids retrieved from the testicle on Vero cell monolayers. In all azoospermic patients elongating spermatids could be obtained from round spermatids. In one case of maturation arrest, of 37 round spermatids co-cultured for up to 5 days, 30% developed flagella, 46% matured to elongating and 19% to elongated spermatids, with one mature spermatozoon also obtained (3%). In the same patient, primary cultures of three round spermatids with flagella enabled development of one further mature spermatozoon. In the case with total globozoospermia, of six round spermatids co-cultured for up to 5 days, one mature spermatozoon was obtained, with a flagellum and normal head morphology. These preliminary findings suggest that it may be possible to overcome the round spermatid block, and even the triggering of morphological abnormalities arising at the spermiogenic level, by in-vitro maturation under special environmental conditions.
Animals↗
Short-term intensive consolidation therapy after all-trans retinoic acid in acute promyelocytic leukemia.
Complete remission induced by all-trans retinoic acid (ATRA) in acute promyelocytic leukemia is short lived, and several consolidation chemotherapy courses usually are given to reduce the relapse rate. To assess the value of short-term intensive consolidation, 38 patients with newly diagnosed acute promyelocytic leukemia entered a prospective study in which induction therapy with ATRA immediately was followed by a single course of mitoxantrone plus high-dose cytarabine (3 g/m2 every 12 hours, days 1-4), with no further treatment. Complete remission was achieved in 31 patients (81.6%) after a median time of 49 days of ATRA (to which chemotherapy was added at entry in 10 patients with leukocytosis). Thirty patients received the planned consolidation course. After a median follow-up of 36 months, four of these patients have relapsed and 24 are still in first complete remission, for an estimated disease-free survival of 75% at 60 months. The authors conclude that this single course consolidation of ATRA-induced remission provides excellent long-term control of acute promyelocytic leukemia.
Adult↗
Current problems with spermatid conception.
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Female↗
Successful pregnancy after spermatid injection.
We present nine cases of spermatid intracytoplasmic injection for the treatment of non-obstructive azoospermia. In eight cases, no elongated spermatids or spermatozoa were found in previous spermiograms or testicular biopsies. In these patients, treatment was performed using ejaculated (n = 6) and testicular (n = 2) retrieved round spermatids (Sa type). In cases where ejaculated round spermatids were used, they were isolated on the day before oocyte retrieval and left in culture for 24 h before intracytoplasmic sperm injection (ICSI). No pregnancy was obtained in either group, although culturing seemed to increase the fertilization rate. In one other case, elongated spermatids were observed in the previous spermiogram and thus a normal ICSI procedure was scheduled. However, on the day of oocyte retrieval, no spermatids could be recovered from fresh sequential ejaculates, and a testicular open biopsy was then performed. Both round and elongated spermatids were found in the testicular tissue, but only the more mature germinal cells (Sb2) were injected. From this case, a normal pregnancy was obtained which resulted in the birth by Caesarean section at 37 weeks of gestation of a normal healthy baby girl, weighing 2700 g.
Adult↗
Spermatids as gametes: indications and limitations.
The feasibility of achieving viable embryos, developing to term after transfer into the uterus, by fertilizing oocytes with spermatids has been demonstrated both in animal studies and in preliminary human clinical trials. Here we review the current clinical indications of spermatid conception and discuss the predictable success rates associated with each of these indications. Potential health hazards relating to the use of spermatids for conception are updated taking into account the risk of abnormal or incomplete epigenetic modifications of newly discovered human imprinted genes. We also add new experimental data showing the occurrence of spermatids in patients lacking spermatozoa and demonstrating that round spermatids recovered from patients with complete spermiogenesis failure (no elongated spermatids or spermatozoa ever detected in the patient's history) are often deficient in the factor(s) responsible for oocyte activation. The possible consequences of this deficiency for the occurrence of abnormal fertilization patterns and for the impairment of further preimplantation and post-implantation development are discussed. It is concluded that the development of diagnostic tests to assess the intrinsic quality of spermatids, with regard to their ability to act as gametes, is urgently needed as part of pre-treatment diagnosis before infertile couples are included in a spermatid conception programme. Centres wishing to use spermatids in human assisted reproduction should also be prepared to offer adequate diagnostic methods to control genomic imprinting abnormalities in the progeny.
Female↗
Proliferative activity in peripheral ossifying fibroma and ossifying fibroma.
A proliferative activity study analysing morphometric and quantitative aspects of nucleolar organizer regions (NORs) and proliferating cell nuclear antigen (PCNA) expression was conducted in 10 cases of peripheral ossifying fibroma (POF) and 10 cases of ossifying fibroma (OF). For NOR identification, the silver staining technique (AgNOR technique) was used. PCNA expression was determined by immunohistochemical staining using the PC10 antibody. The AgNOR analysis for the two lesions showed a profile characteristic of benign lesions. OF showed higher AgNOR number and PCNA expression than POF. Our results suggest increased proliferative activity in OF compared with POF.
Antibodies, Monoclonal↗
Granulocyte-macrophage colony-stimulating factor in post-remission therapy of acute myeloid leukemia.
The impact on occult leukemia of GM-CSF as a sensitizing agent has not been studied. We treated 41 adult patients with de novo acute myeloid leukemia, 25 of whom achieved complete remission and were given 1 to 3 post-remission courses, each course including GM-CSF begun 4 days prior to chemotherapy and given until day 3. After a median follow-up of 32 months, the probability of remaining in continuous complete remission was 17% at 46 months. GM-CSF in this setting was not associated with an improved outcome, arguing against a priming effect.
Acute Disease↗
Ultrastructure of oogenesis in Penaeus kerathurus (Crustacea, Decapoda). I. Previtellogenic oocytes.
Although Malacostracan species represent an important alimentary human resource, the ultrastructure of oogenesis in P. kerathurus remains unknown. Previtellogenic oocytes of Penaeus kerathurus possess a large nucleus with several peripheral nucleoli. The endoplasmic reticulum (ER) is originated from expansions of the nuclear envelope (NE) and contains small dense granules, which are first formed inside the intermembranous space of the NE but are later exported to the ER lumen. Direct vesiculation from the NE and ER then give rise to the Golgi complexes. Small yolk vesicles appear to be mainly formed by vesiculation of the ER, but also receive materials from the Golgi complexes. They contain a fine fibrillar content which seems to originate from decondensation of the small dense granules. Small vesicles and small multivesicular bodies originated from the NE, ER and Golgi complexes, as also myelin figures directly shedded from the NE, fuse together to give origin to large multivesticular bodies (MVB). These organelles, which have an incomplete membrane and appear meshed within nuage materials, give origin, at a later stage, to lipid droplets that are thereafter extruded into the cytoplasm. Neighbouring oocytes exhibit intercellular bridges, the remaining of their surface being surrounded by a single layer of flattened follicular cells. These results show for the first time in Malacostraca the existence of oocyte intercellular bridges, that the ER and Golgi complexes arise from NE activity, that early yolk formation is endogenous and derives from the activity of the NE, ER and Golgi complexes, and that lipid droplets are products of intracellular membrane recycling activity occurring within large multivesicular bodies.
Animals↗
Aging, hyaluronidase removal of the cumulus, and microinjection do not affect the sperm binding potential of human oocytes.
PURPOSE: We studied the influence of aging, hyaluronidase removal of the cumulus, and microinjection on the sperm binding potential of human oocytes under intact zona assay conditions to determine the safe use of unstored aged unfertilized ICSI oocytes on zona binding tests. Results were also compared with those for aged IVF oocytes under the same conditions. METHODS: To avoid the large variation in sperm binding scores, we compared the ratios of the number of sperm bound to nontreated versus to treated oocytes. Treated oocytes were those whose zonae were exposed to hyaluronidase, an agent that decreases sperm binding. Experimental groups were fresh oocytes, experimentally aged fresh oocytes, fresh oocytes whose cumulus was removed either mechanically or with hyaluronidase, aged ICSI oocytes, and aged IVF oocytes. RESULTS: Statistical analysis within and between groups showed that aging, previous insemination, cumulus removal by hyaluronidase, and microinjection did not affect the sperm binding potential of human oocytes. CONCLUSIONS: As the zona binding ability of fresh and unfertilized aged ICSI oocytes is similar, they can be used safely in zona assays.
Aging↗
Pregnancy and birth after intracytoplasmic sperm injection with totally immotile sperm recovered from the ejaculate.
OBJECTIVE: To report the birth of two healthy children after intracytoplasmic sperm injection (ICSI) with totally immotile spermatozoa recovered from the ejaculate. DESIGN: Retrospective case report. SETTING: University-based hospital. PATIENT(S): Four couples in whom spermatozoa recovered from the ejaculate were totally immotile but presented normal vitality scores. INTERVENTION(S): Therapeutical IVF-ET attempts coupled with ICSI. MAIN OUTCOME MEASURE(S): Fertilization and pregnancy results after ICSI. RESULTS: With random sperm injection, 19 of the 36 injected oocytes showed normal fertilization and cleavage. One of four patients had a twin pregnancy that resulted in birth of two healthy children. CONCLUSION(S): In cases in which totally immotile ejaculated sperm present normal vitality scores, normal clinical outcomes can be achieved by using the usual random sperm selection during conventional ICSI.
Abortion, Spontaneous↗
Efficient modification of intracytoplasmic sperm injection technique for cases with total lack of sperm movement.
A rapid, simple and efficient method for selecting living spermatozoa for intracytoplasmic sperm injection (ICSI) in cases with total lack of sperm movement is described. The selection is based on a characteristic deformation of living spermatozoa exposed to hypo-osmotic conditions during short sequential exposures to modified culture medium and polyvinylpyrrolidone solution; the osmolarity of both of these solutions is reduced by one half by diluting them with an equal amount of water. The application of the sperm viability selection step in six ICSI treatment cycles with total absence of sperm movement resulted in a fertilization rate of 41.9% and the establishment of two ongoing clinical pregnancies. The method described for the selection of living spermatozoa makes it possible to reach acceptable fertilization rates and to obtain ongoing pregnancies by ICSI in cases with total lack of sperm movement. Because of its simplicity, this method can easily be improvised when the total lack of sperm movement is an unexpected finding made on the day of the planned ICSI.
Cell Separation↗
Developmental changes in calcium content of ultrastructurally distinct subcellular compartments of preimplantation human embryos.
The ultrastructural localization of mobilizable Ca2+ in different subcellular compartments of human oocytes and preimplantation embryos was studied using the potassium-pyroantimonate technique and transmission electron microscopy; the specificity was confirmed by chelation experiments and X-ray microanalysis. In unfertilized oocytes, Ca2+ was detected in small vesicles beneath the plasma membrane as well as in other forms of smooth endoplasmic reticulum (SER) and in mitochondria but not in cortical granules. In pronuclear zygotes and blastomeres of cleaving embryos, Ca(2+)-rich vesicles were no longer present close to the plasma membrane, and the entire periphery was poor in Ca(2+)-containing organelles which, however, were abundant in the perinuclear region. The uneven Ca2+ loading of SER and mitochondria from the pronuclear stage onwards suggests that Ca2+ release from both these types of organelle contributes to the embryonic Ca2+ signals. During mitosis, less Ca2+ was detected with organelles, but the antimonate reaction product was more abundant in the cytosol. These data suggest that, in addition to different forms of SER, mitochondria also act as a source of mobilizable Ca2+ in preimplantation human embryos. The previously described developmental and cell cycle related changes in the characteristics of Ca2+ signals are associated with the redistribution and structural reorganization of these organelles.
Antimony↗
Calcium responses of human oocytes after intracytoplasmic injection of leukocytes, spermatocytes and round spermatids.
Oocyte activation in mammals involves the action of a soluble sperm factor (SSF) that enables oocytes to develop a characteristic series of Ca2+ spikes (Ca2+ oscillations). SSF is also likely to be responsible for the Ca2+ oscillations driving oocyte activation after intracytoplasmic sperm injection [ICSI]. With an appropriate injection technique, Ca2+ oscillations do not develop spontaneously after ICSI but can be triggered by subsequent treatment of sperm-injected oocytes with Ca2+ ionophore. Here we show that Ca2+ oscillations, quite similar to those developing after ICSI, can be triggered by the ionophore treatment in human oocytes previously injected with human round spermatids. In contrast, oocytes injected with earlier spermatogenic cell (primary and secondary spermatocytes) and with non-germ cells (polymorphonuclear leukocytes) did not develop Ca2+ oscillations after the ionophore challenge although the subsequent injection of SSF did induce typical Ca2+ oscillations in these oocytes. Disintegration of the plasma membrane of the injected cells was detected in all cases by transmission electron microscopy. Thus, the absence of the typical oscillatory Ca2+ response in spermatocyte-injected oocytes was due to the actual deficiency of SSF in the spermatocytes rather than to a defective responsiveness of the injected oocytes or to the failure of SSF release into the oocyte cytoplasm. The ability of human round spermatids to induce a response to calcium in oocytes that is similar to that induced by mature spermatozoa may be important for normal embryonic development after spermatid conception.
Calcium↗
Developmental changes in calcium dynamics, protein kinase C distribution and endoplasmic reticulum organization in human preimplantation embryos.
Developmental changes in the Ca2+ dynamics of human zygotes and preimplantation embryos were related to changes in the distribution of endoplasmic reticulum (ER) and protein kinase C (PKC). The fertilization-induced Ca2+ oscillations were typically observed over > 5 h, were ryanodine-sensitive and showed a periphery-to-centre propagation of Ca2+ waves. At the same time, ER and PKC were accumulated in the cell periphery. After the appearance of pronuclei, ryanodine-sensitive Ca2+ oscillations of lower amplitude and frequency were observed until the pronuclear breakdown. However, Ca2+ waves then began in the perinuclear region, in the area of ER and PKC accumulation and spread towards the cell periphery. During the second to fourth cell cycle, small sinusoidal Ca2+ fluctuations were observed; sparse higher-amplitude Ca2+ spikes, superimposed on these basal fluctuations, appeared shortly before cell division. The sinusoidal Ca2+ fluctuations were asynchronous in individual blastomeres and disappeared progressively in arrested embryos. The direction of Ca2+ wave propagation and the distribution of ER and PKC were similar to the situation observed in pronuclear zygotes. In contrast to the zygotes, ryanodine did not arrest the Ca2+ oscillations but augmented their amplitude and frequency. These data suggest that human pre-embryos use different mechanisms of Ca2+ signalling in the early post-fertilization period, during the pronuclear development and during cleavage.
Calcium↗