Is there a future for spermatid injections?

Microinjection of spermatids into oocytes has proven to be a successful assisted reproduction procedure in the animal model. In the human, low fertilization and cleavage to the 4-cell stage were reported after intracytoplasmic sperm injection (ICSI) with round spermatids. In comparison with a conventional ICSI-testicular sperm extraction (TESE) programme, the implantation rate after round spermatid injection is dramatically low. Different problems have been encountered during the development of the spermatid injection technique and they could be partially responsible for the lower outcome when using round spermatids. Compared with the round spermatid cells, spermatids in the elongation phase are easy to isolate and identify from other round cells present in a wet preparation. The morphological identification does not reveal anything about the viability or the genetic normality of the round spermatids. Severe testicular dysfunction may have consequences on the quality of the few spermatogenic cells present. Others factors, such as the pathology of the patient, play an important role in the successful treatment. Even if the results are extremely low, spermatid injection seems more favourable for men who have already proven their capacity to produce some spermatozoa. A spermatogenic block at the round spermatid level has led to early abortions, increasing the suspicion of the role of a genetic factor. In order for this technique to be safe for use in clinics, more intensive work is needed to improve the selection and handling of cells and to ascertain the genomic imprinting and gene expression necessary for embryonic development. Hence, when using immature cells for conception, the screening of the patient and the follow-up of the pregnancies and babies should be mandatory.

Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation.

Spermatid microinjection into oocytes has proven to be a successful assisted reproduction procedure in the animal model and in the human species, since in the latter a few full-term pregnancies were actually obtained. Patients entering our spermatid injection study included those with a total absence of spermatozoa in the testicular tissue notwithstanding previous positive biopsies (n = 29): an obstructive problem (n = 3), secretory azoospermia (n = 26), and those with total arrest at the spermatogenesis level in previous explorative biopsies (n = 15). In the latter group, absence of spermatids was recorded in four cases. Mature, elongated, elongating and round spermatids (ROS) were injected in respectively 3, 2, 3, and 32 attempts. A total of 260 metaphase II oocytes were injected with ROS, 36 oocytes with spermatids at other stages of maturity. The rates of oocytes showing two pronuclei (2PN) and two polar bodies reached 22% and 64% respectively after injection of round or elongated-mature spermatids. The fertilization rate after ROS injection was influenced by the percentage of spermatozoa observed in a previous biopsy. Patients with a positive preliminary biopsy had significantly more 2PN (33%) when compared to those with a severe spermatogenic dysfunction and in whom no spermatozoa were found (only 11%) (P < 0.05). Incubation of oocytes in calcium ionophore after ROS injection had a positive effect on the rate of 2PN formation (36 versus 16%). Ninety per cent of all the normally fertilized oocytes cleaved. The percentage of grade A and B embryos depended on the type of injected cells: 12% after ROS and 30% with the other types of haploid cells. A total of 39 transfers resulted in five pregnancies: three full term with healthy babies delivered (one after ROS injection, and two after injection of an elongating and a mature spermatid), one 4 months ongoing (after elongating spermatid injection) and one miscarriage at 4 weeks (after elongated cell injection). Compared to our conventional intracytoplasmic sperm injection-testicular sperm extraction (ICSI-TESE) programme, the implantation rate after ROS injection was very low (5.5 versus 10.5%).

[Medically assisted reproduction with immature spermatozoa. Clinical examination and surgical technics]. / La procréation médicalement assistée avec spermatozoïdes immatures. Examens cliniques et techniques chirurgicales.

The authors report their experience with the use of spermatids in TESE programs where mature spermatozoa could not be isolated from testicular biopsies. The details of the indications for spermatid insemination, the technicity of the procedure and the results are exposed.

Fertilizing ability of immotile spermatozoa after intracytoplasmic sperm injection.

Sometimes spermatozoa from ejaculate, epididymis or testis show a total absence of motility. For some patients, however, very few spermatozoa with very poor motility can be found after several hours of incubation (initially immotile spermatozoa). Other samples show no motility at all even after extended culture (totally immotile spermatozoa). Intracytoplasmic sperm injection (ICSI) is the only method available to select and retrieve a single immotile or initially immotile spermatozoon and inject it into the oocyte. A total of 103 patients with asthenozoospermia underwent ICSI in this study. It was shown that initially immotile and totally immotile spermatozoa, whatever their origin, have the capacity to fertilize an oocyte after ICSI. No significant difference could be observed between the fertilizing capacity of testicular or epididymal spermatozoa. Totally immotile ejaculated spermatozoa, however, fertilized significantly fewer oocytes after ICSI when compared with initially immotile ejaculated spermatozoa. Embryos of lower quality tended to be produced when totally immotile spermatozoa of any origin were used, compared with embryos resulting from initially immotile spermatozoa. Ongoing pregnancies were conceived after ICSI with initially immotile spermatozoa from any origin and totally immotile spermatozoa retrieved from testis only. One biochemical pregnancy was the result of embryo transfer after ICSI with totally immotile ejaculated spermatozoa. No supernumerary embryos could be cryopreserved for patients with totally immotile spermatozoa from ejaculate or epididymis. For a Kartagener patient, subzonal insemination (SUZI) seemed to be a better approach for obtaining fertilization and pregnancy than ICSI because no fertilization occurred after ICSI on sibling oocytes. Hence a healthy pregnancy was obtained after SUZI.

Differentiation of pancreatic carcinoma induced by retinoic acid or sodium butyrate: a morphological and molecular analysis of four cell lines.

The antiproliferative and differentiation-inducing effects of all-trans retinoic acid (RA) and sodium butyrate (SB) were investigated in four pancreatic ductal adenocarcinoma cell lines, two poorly differentiated ones (PT45 and PaTu-II), one moderately to poorly differentiated one (Panc-1) and one highly differentiated one (A818-1). Treatment with 20 microM RA resulted in moderate inhibition of cell growth in all cell lines, but clear evidence of cytodifferentiation (including elongated cell processes, increased rough endoplasmic reticulum, intensified immunostaining for the mucin marker (M1) was found only in PT45 and Panc-1. These phenotypic changes were paralleled by upregulation of RAR (retinoic acid receptor)-alpha and -gamma mRNA. SB (1 and 2 mM) treatment inhibited the cell growth of all cell lines much more prominently than RA. Cytodifferentiation was also largely restricted to PT45 and Panc-1. A noticeable phenomenon was enhancement of the expression of the neuroendocrine markers synaptophysin and Lcu7 in Panc-1 cells. In conclusion, it is evident that the original differentiation status of cells and their responsiveness to the agents are not clearly associated, and that RA responsiveness correlates with upregulation of RAR-alpha and -gamma mRNA.

Expression of the jun family of genes in human ovarian cancer and normal ovarian surface epithelium.

The jun genes (c-jun, jun-B and jun-D) play a role in critical cell functions such as proliferation, differentiation and apoptosis. We documented jun expression at the mRNA and protein level in human ovarian cancer tissues (n=28), surface epithelial cells of normal ovaries (n=14) and ovarian cancer cell lines (n=6). Almost all of ovarian tumors as well as normal ovaries concomitantly express c-jun, jun-B, and jun-D mRNA. Immunohistochemistry was less sensitive and revealed nuclear c-Jun and Jun-B proteins in the malignant epithelial cells of respectively 38% and 11% of ovarian tumors and in the surface epithelium of a normal premenopausal ovary. In cultured ovarian cancer cells, c-jun and jun-B expression is inducible by serum and TPA and is therefore not constitutive. The c-jun and jun-B proteins therefore play a role both in differentiation of the normal ovarian surface epithelium, as well as in the proliferation of epithelial ovarian cancer cells. High jun-B expression relates to a more malignant phenotype both in vitro and in vivo. The jun-D gene is suppressed in ovarian cancer cells as compared to normal ovarian surface epithelial cells in situ and in vitro. Downregulation of jun-D might therefore be part of the malignant ovarian epithelial cell phenotype.

Two essential steps for a successful intracytoplasmic sperm injection: injection of immobilized spermatozoa after rupture of the oolema.

A total of 740 cycles of intracytoplasmic sperm injection (ICSI) were performed: 625 cycles when < 6 x 10(5) total motile spermatozoa were harvestable from the ejaculate and 115 cycles in cases with a history of previous fertilization failure after classic in-vitro fertilization or subzonal sperm injection. An average of two pronuclei were observed in 63% of the injected oocytes, allowing 725 transfers of a maximum of three embryos (98%). Of 214 pregnancies initiated, 179 were established (25% of ICSI attempts). Because the fertilization rates from our initial 80 ICSI cycles were 2-fold less than those achieved previously, we changed the injection procedure and analysed, in 740 ICSI attempts, the importance of interfering technical factors and how to establish a successful ICSI programme. A remarkable change in the fertilization rate up to 68% (595 cycles) occurred when two steps in the injection procedure were performed well, i.e. immobilization of the spermatozoon and placement of the spermatozoon into the ooplasm after cytoplasmic aspiration into the pipette until oolema rupture. This immobilization, by touching the tail with the pipette, is mandatory for increasing the percentage of fertilization, even with totally non-motile spermatozoa (41%). Because aspiration of the cytoplasm is an invasive part of the ICSI procedure and influences the quality of the embryos, it is essential to reduce the amount of cytoplasm drawn into the pipette. This could be attained by using a spikeless injection pipette with the smallest possible internal diameter.

Frequent deletion of chromosome 19 and a rare rearrangement of 19p13.3 involving the insulin receptor gene in human ovarian cancer.

Human ovarian cancer cells usually have multiple specific chromosomal deletions which can be detected by cytogenetic analysis or molecular techniques. Tumour suppressor genes might be located in these deleted chromosomal segments. The importance of these different loci is usually estimated from the frequency with which they are deleted. Here we report a 59% loss of heterozygosity for chromosome 19 in the DNA of human invasive epithelial ovarian cancer from a series of 37 patients. In all cases informative on both chromosomal arms a subchromosomal loss is observed. Analysis of the same tumours for chromosome 17p and 11p loss suggests that loss of chromosome 19p/q is less important than 17p loss, but more important than 11p loss. The deletion of chromosome 19q seems to be associated with distant, hematogeneous metastasis (stage IV). In two patients with high grade tumours, the deletion involves a rearrangement of the insulin receptor locus (19p13.3). This suggests that some of the previously described frequent cytogenetic 19p+ markers and 19p13.3 breaks observed in high grade ovarian cancers, might actually occur in the insulin receptor gene.

New therapeutic possibilities handling residual azoospermia.

Obviously, medical therapy of secretory azoospermia or microsurgical therapy of excretory azoospermia are not always successful. The unsolvable cases therefore can be grouped as residual azoospermias. Both the medical and microsurgical approaches are reviewed and their success rates analyzed. Some problems are unsolvable after work-up of the diagnosis. Till the early nineties, such patients were discouraged to undergo further medical therapeutic approaches and were advised to consider either adoption or donor insemination. At the present time, new possibilities have risen since the use of epididymal spermatozoa for performing assisted fertilization has considerably altered the picture. Furthermore, the last breakthrough of using testicular spermatozoa combined with the ICSI procedures have offered solutions that were unthinkable only a few years ago. The impact of these new approaches is discussed and the future development of microsurgery versus assisted reproduction techniques is also considered.

Benign angiomyolipoma involving the renal vein and vena cava as a tumor thrombus: case report.

A 53-year-old woman presented with a renal angiomyolipoma extending as a thrombus into the renal vein and vena cava. This case is most unusual and we are unaware of any report of a benign angiomyolipoma that has presented as a tumor thrombus. The intravascular growth of a benign angiomyolipoma has previously been described.

Immunomodulatory activity of two new aza alkyl phospholipid antineoplastic drugs.

The present work reports the modulation of immunocompetent cell functions by two aza alkyl phospholipids (AAP), BN 52205 and BN 52211. Each compound was compared with 1-O-octadecyl-2-O-rac-glycero-3-phosphocholine (ET-18-OCH3) and/or three drugs used for cancer treatment, i.e. cisplatyl (CIS), 5-fluorouracil (5-FU) and cytosine arabinoside (ARA-C). Interleukin (IL)-1 release from P388D1 cells was increased 2-fold in the presence of 5 micrograms/ml BN 52205 or BN 52211. However, these stimulations were lower than those obtained with ARA-C, 5-FU and CIS. Compared with ET-18-OCH3, CIS and 5-FU, BN 52205 and BN 52211 were more efficient in increasing tumor necrosis factor production induced by lipopolysaccharide (LPS) from human monocytes. In vitro, all compounds exhibited similar activity in enhancing IL-6 production from human monocytes stimulated with LPS, with the exception of 5-FU and CIS that were inactive. At 20 mg/kg (i.v.), a peak of IL-6 production was reached 2 h after injection of ET-18-OCH3 [> 1280 U/ml (n = 4, p < 0.001) versus 3.5 +/- 0.2 U/ml (n = 7)], whereas BN 52211 induced a maximum of IL-6 production after 4 h (77 +/- 27 U/ml, n = 5, p < 0.001). BN 52205 induced peaks of IL-6 production after 3 and 6 h (90 +/- 62 and 68 +/- 35 U/ml, respectively, p < 0.001, n = 4). The proliferation of rat splenocytes was abolished in the presence of BN 52205 and BN 52211 at 10 micrograms/ml, corresponding to only a partial reduction of IL-2 production at the same concentration. The production of interferon-gamma was stimulated 6- to 10-fold in the presence of 1-5 micrograms/ml BN 52205, BN 52211 and ARA-C. BN 52211 and BN 52205 were also potent enhancers of IL-3 production, whereas 5-FU and ARA-C were inhibitory. These results indicate that in addition to a direct antitumoral effect, AAP may also exhibit immunomodulatory activity both in vitro and in vivo.

Deletion of chromosome 11p13-11p15.5 sequences in invasive human ovarian cancer is a subclonal progression factor.

In human ovarian cancer, multiple specific chromosomal deletions can be found by cytogenetic analysis or molecular techniques such as restriction fragment length polymorphism probing. This work confirms the loss of HRAS alleles in 1 out of 2 cases of invasive ovarian cancer as determined in 32 samples or cell lines derived from 19 patients. Results with other polymorphic probes indicate that a consensus deletion probably includes 11p15.5-11p13. Tumor suppressor genes might be located in the deleted area, and deletion of the gene might then play a role in disease progression. Examination of DNA from distinct tumor sites of individual patients indicates clonal heterogeneity in the malignant cell population, indicating that loss of 11p sequences is a late event in the disease progression. Loss of alternate 11p alleles at different disease sites in one patient is inconsistent with the current model of tumor suppressor gene inactivation. The 11p deletion seems to be limited to ovarian cancers in younger patients. Eight novel permanent ovarian cancer cell lines, previously not described in the literature and derived from tumor sites from five patients, were included in the current analysis.

Cytostatic activity of new synthetic anti-tumor aza-alkyllysophospholipids.

Alkyllysophospholipids are analogues of the naturally occurring 2-lysophosphatidylcholine which have been reported to have selective in vitro/in vivo anti-tumor activity. Their antiproliferative effect has been found against a variety of animal and human tumor cell lines. We have characterized the cytostatic activity of 2 newly synthetized aza-alkyllysophospholipids (AALPs), the BN52205 and the BN52211, on a human tumor cell line derived from a colon adenocarcinoma, the HT29. We used 3 different flow cytometric approaches to study which phase of the cell cycle was the most sensitive to the antiproliferative activity of the 2 AALPs. By applying the biparametric analysis of 5'-bromo-2-deoxyuridine incorporation vs. DNA content we have been able to demonstrate that the 2 AALPs do not interfere with the S phase of the cell cycle. The simultaneous measurement of total nuclear protein vs. DNA content in isolated HT29 nuclei enabled us to exclude a block in the M phase of the cell cycle. Finally, stathmokinetic analysis enabled us to show that cytostatic activity of the 2 new AALPs is characterized by multiple "terminal points" as the drugs' action results in a G1 block, in a slow-down of the transition from late S to G2 followed by an accumulation of HT29 cells in the G2 phase of the cell cycle.

Perichondrial microvascular free transfer: creation of a compound flap for laryngeal reconstruction in rabbits.

A free revascularized compound perichondrial flap was used over an intralaryngeally placed stent for reconstruction of a frontal laryngeal defect. The microvascular flap replaced the cartilaginous and mucosal defect. Short-term results showed successful reconstruction with a patent airway and viable mucosa. The vascularized sheet of perichondrium was not chosen for its neochondrogenetic effect, but it served as a vascular bed, nourishing the mucous and cartilaginous components in the compound flap. It is suggested that for clinical purposes the reliable fascia forearm flap, which is available in large amounts, can be used as a transferable vascularized bed with a thickness comparable to that of the perichondrial flap.

The role of fluorinated pyrimidine analogues in the induction of the in vitro expression of the fragile X chromosome.

The modes of action of 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluoro-2'-deoxycytidine (FdCyd) were studied in PHA-stimulated lymphocytes from normal volunteer donors and a fragile X patient. In both cell types, FdUrd and FdCyd inhibited cell proliferation at concentrations of 3 x 10(-8) M. Thymidylate synthetase was identified as the decisive target for the action of both FdUrd and FdCyd, as judged from the following observations: First, addition of thymidine to the culture medium was able to counteract both FdUrd and FdCyd toxicities, whereas addition of dCyd had no observable effect. Second, inhibition of the in situ thymidylate synthetase activity measured as an increase in the level of [3H]-dThd incorporation coincided with the inhibition of cell proliferation. Third, the inhibition of the thymidylate synthetase-dependent incorporation of [3H]-dUrd into newly synthesized DNA coincided with the inhibition of cell proliferation. The effects of FdUrd and FdCyd on the in vitro expression of fragile site Xq27 of fragile X chromosomes was shown to be based on the depletion of the intracellular pool of thymidine-5'-monophosphate (dTMP), as judged from the following observations: First, both the FdUrd- and FdCyd-dependent induction of site Xq27 coincided with the antiproliferative effects of the respective fluoropyrimidines. Second, addition of thymidine (dThd) to the culture medium both prevented the expression of site Xq27 and neutralized the cytotoxicity of FdUrd and of FdCyd. On the basis of these findings, we provide further evidence for the concept that the fragile X site is located in an AT-rich region.

Dorsal defect of the patella: concept of its origin and relationship with bipartite and multipartite patella.

In a series of 2286 single radiographic examinations of the knee in 1985, 6 dorsal defects of the patella (DDP) were detected. The diagnosis was made if a round lucent lesion of the dorsal superolateral surface of the patella was found abutting against articular cartilage. In four of our patients, an association with a multipartite patella (MP) was found. Biopsy of one lesion showed dense connective tissue and areas of bone necrosis. In one patient, the pattern of reossification of the lesion could be demonstrated. Our observations provide further evidence that the DDP is a stress-induced anomaly of ossification rather than a post-traumatic subarticular cyst of the patella, a diagnosis sometimes suggested by the clinical context. The initial lesion is probably a traction lesion at the insertion of the vastus lateralis muscle rather than ulceration of articular cartilage. We suggest a possible relationship between dysfunction of the quadriceps mechanism, patellar subluxation, and the genesis of the DDP.