Bortezomib Treatment can Overcome Glucocorticoid Resistance in Childhood B-cell Precursor Acute Lymphoblastic Leukemia Cell Lines.

BACKGROUND: The response to initial glucocorticoid (gc) treatment is a reliable stratification factor in childhood acute lymphoblastic leukemia (ALL) and may predict the response to multi-agent chemotherapy. In a former study we detected that the valosin-containing protein (VCP, cdc48), a member of the ubiquitin proteasome degradation system (UPS), is altered in gc-resistant leukemic cells suggesting that the associated pathways might be involved in chemotherapy resistance in childhood ALL. METHODS: Human B-cell precursor leukemia cell lines, gc-resistant MHH-cALL-2 and gc-sensitive MHH-cALL-3, were treated with prednisolone and various concentrations of bortezomib. Viability and apoptosis rates were determined. RESULTS: Both cell lines showed a dose-dependent increase in caspase activity after bortezomib single treatment. The gc-sensitive cells showed an additive effect after combined treatment with prednisolone and bortezomib. In contrast, both cell lines showed a reduced viability and enhanced propidium iodide positivity after combined treatment as determined by flow cytometry. Western blot analyses of poly-(ADP-ribose) polymerase 1 (PARP-1) suggested that combined treatment promote necrotic cleavage of PARP-1 in gc-resistant cells. Furthermore, after prednisolone treatment the UPS associated proteins VCP and NFκB-inhibitor IκBα were differentially modulated in gc-resistant cells. CONCLUSIONS: The proteasome inhibitor bortezomib seems to sensitize gc-resistant childhood ALL cells for prednisolone-induced cell death.

IVF versus ICSI for the fertilization of in-vitro matured human oocytes.

Traditional dogma suggests that intracytoplasmic sperm injection (ICSI) should be performed to ensure successful oocyte fertilization in an in-vitro maturation (IVM) cycle. This study postulated that there would be no difference in the fertilization rate when ICSI was compared with IVF. This hypothesis was tested in a randomized trial of IVF versus ICSI in IVM. A total of 150 immature oocytes were collected in eight cycles of IVM for patients diagnosed with polycystic ovarian syndrome (PCOS). Patients were primed with minimal FSH before transvaginal oocyte aspiration. Sibling oocytes were inseminated by 50% IVF and 50% ICSI. There was no significant difference in fertilization, useable or total blastocyst development between the two insemination technique groups. Clinical pregnancy results for combined fresh and cryopreserved transfers were identical between the two insemination techniques with a total of two fresh and five cryopreserved IVF-inseminated embryos resulting in three clinical pregnancies (42.9%) and five fresh and two cryopreserved ICSI-derived embryos resulting in three clinical pregnancies (42.9%). This research has shown IVF to be a legitimate fertilization technique for IVM oocytes in PCOS patients and provides a greater awareness of the use of a fertilization method previously not utilized with IVM. In-vitro maturation (IVM) is an alternative treatment method to traditional IVF. Due to the minimal use of stimulating hormones in this treatment, IVM has a lower risk of ovarian hyperstimulation syndrome, it can be used for fertility preservation in cancer patients and it is more cost conservative. Early research into the effects of IVM showed a hardening effect on the membrane surrounding the egg (the zona pellucida). It was initially believed that, to overcome this hardening in order to allow the egg to be fertilized, spermatozoa would need to be injected into the egg using intracytoplasmic sperm injection. Due to recent advances in hormonal stimulation protocols (FSH priming) and culture conditions, we postulated that, for patients suffering from polycystic ovarian syndrome (PCOS), fertilization, embryo development and clinical pregnancy would not be superior in the injected oocytes compared with those inseminated by IVF. We found that by using the two insemination techniques on sibling oocytes from eight PCOS patients, there was no significant difference in fertilization, useable or total blastocyst development (day 5 or 6 embryos) and that clinical pregnancy results were identical. This research provides a greater awareness of a fertilization technique which is not normally utilized for IVM treatment, providing a less invasive, more cost-effective approach for the patient.

Leg length inequality measured by ultrasound and clinical methods.

Leg length inequality (LLI) was determined by ultrasound and two clinical methods in 100 subjects without previous disorders of the lower extremities. The mean LLI by ultrasound was 4.1 mm (range: 0-16 mm). An LLI of 10 mm or more was found in 4% of the subjects. The mean inter-observer variation by ultrasound was 0.9 mm (range: divided by 5-8 mm), which was significantly less than that obtained by clinical methods. The 95% confidence interval of inter-observer variation (+/- 2 SD) by ultrasound was +/- 5 mm and this interval is assumed to represent the accuracy of the method. Because LLI measurement by ultrasound is more reliable than clinical methods, ultrasound is recommended for routine use in clinical practice.

[Evaluation of late results for surgical treatment of chronic lateral epicondylitis]. / Ocena odleglych wyników leczenia operacyjnego przewleklego zapalenia nadklykcia bocznego kosci ramiennej.

The results of surgical treatment of 25 patients with lateral epicondytilis (tennis elbow) were analysed. The mean time before surgery was 2 years and 4 months. The patients' negative attitude towards surgery and the prolonged conservative treatment by general practitioners were the reasons for such a long period of ineffective conservative procedures. Following Hohmann's surgery the flexion in elbow joint and dorsal flexion in radio-carpal joint limitation were the most common residual impairments. The loss of muscle circumference of the operated extremity was observed in 10 (40%) patients. Pain subsided in 20 (80%) and numbness in 11 (44%) patients. Two third of the patients were satisfied with the surgery outcome This moderately good general outcome was probably due to the prolonged time passed since the onset of symptoms and repeated local steroid injections. The majority of manual workers returned to their work.

Practitioner reporting of birth defects in children born following assisted reproductive technology: Does it still have a role in surveillance of birth defects?

BACKGROUND: National assisted reproductive technology (ART) data collections that rely on practitioners' reports of birth defects have consistently reported lower proportions of children with birth defects than record linkage studies that link ART infants to birth and malformation registers. METHODS: We compared the birth defect data reported to the national Australian Assisted Conception Data Collection (ACDC) by practitioners at three Western Australian ART clinics with the birth defect data identified on the Western Australian Birth Defects Registry (WABDR) through record linkage of all the pregnancies conceived at these clinics to the WABDR. Cases are reported to the WABDR by multiple statutory and voluntary sources. RESULTS: We found that the national ACDC significantly underestimated the prevalence of birth defects in WA-born ART infants. Less than one-third of ART children identified with a major birth defect on the WABDR were reported to the ACDC. CONCLUSIONS: Although national ART data collections provide valuable information on pregnancy rates and short-term pregnancy outcomes such as multiple birth and birth weight, we strongly recommend that birth defect information used for patient counselling is preferentially drawn from large studies that have used record linkage to high-quality birth defect registers.

Human embryo: a biological definition.

This paper defines a human embryo from a biological standpoint that takes into account emerging technologies in reproductive science. The paper does not consider legal, moral, religious or social views. As the definition of a human embryo must reflect the multifactorial processes of development, an approach has been adopted which combines recognition of observed events with potential for further development. This acknowledges that fertilization and development are not static processes, and as such embryo status can only be defined by observation of specific markers. The following biological definition of 'human embryo' is proposed. A human embryo is a discrete entity that has arisen from either: the first mitotic division when fertilization of a human oocyte by a human sperm is complete or any other process that initiates organized development of a biological entity with a human nuclear genome or altered human nuclear genome that has the potential to develop up to, or beyond, the stage at which the primitive streak appears, and has not yet reached 8 weeks of development since the first mitotic division.

[Epiphysiolysis of the femur head in hypogonadism of pituitary origin]. / Epiphyseolysis capitis femoris w przebiegu hipogonadyzmu pochodzenia przysadkowego.

A case of a twenty years old male patient with unilateral slipped upper femoral epiphysis has been presented. An accompanying pituitary hypogonadism was revealed on physical examination and laboratory tests. Reports from literature of hormone-related epiphyseolysis have been discussed. The authors believe that the presented case of slipping has its origin in the found hormone deficiency.

The effect on low back pain of shortening osteotomy for leg length inequality.

This study evaluates the effect on low back pain of shortening osteotomy in patients with leg length inequality (LLI). Twenty-two patients were followed up after an average of 5 years. The mean preoperative LLI was 32 mm and was 4.3 mm at follow up. The degree of low back pain was significantly reduced after the operation.

[Algodystrophy during pregnancy complicated by fracture of the femoral neck. Case report]. / Algodystrofia okolociazowa powiklana zlamaniem szyjki kosci udowej--opis przypadku.

A case of 33 years old female with pathologic fracture of the femoral neck that occurred in the third trimester of pregnancy is presented. Generalized osteoporosis has been found in post-delivery radiographic examination. The patient has been operated with the Zespol hip-plate fixator.

Effect of antispermatozoal antibodies in seminal plasma upon spermatozoal function.

The indirect immunobead test for antispermatozoal antibodies of the class IgA, IgG and IgM was applied to the seminal plasma of male partners of infertile couples. The presence of both IgA and IgG was associated with a decreased incidence of good post-coital test results and a reduced rate of fertilization of human oocytes. No significant differences were found for men with IgA or IgG alone when compared to men with no detectable antispermatozoal antibodies.

Birth from cryopreserved embryos following in-vitro maturation of oocytes and intracytoplasmic sperm injection.

This case report describes the birth of a baby following the transfer of cryopreserved embryos generated from intracytoplasmic sperm injection (ICSI) carried out on the second day after oocyte pick-up of in-vitro-matured metaphase I and germinal vesicle stage oocytes. The couple had a history of three failed intrauterine insemination attempts and reduced fertilization rates in two previous in-vitro fertilization (IVF) cycles. In the IVF-ICSI treatment cycle, 6/11 mature oocytes became fertilized following ICSI on the first day. However, the patient failed to conceive following the transfer of three embryos. Five oocytes were immature (two at metaphase I stage and three with a germinal vesicle) and these were cultured overnight. All had extruded a polar body by the following day and ICSI was therefore performed; four oocytes became fertilized, and were cryopreserved at the pronulear stage in propanediol. In the next treatment cycle, transfer of frozen embryos was planned. The pronuclear zygotes were thawed and cultured for 24 h prior to the transfer of two embryos in a cycle stimulated with low doses of follicle stimulating hormone. This resulted in a pregnancy and the delivery of a healthy baby boy. In-vitro maturation of metaphase I and germinal vesicle oocytes which are routinely collected in IVF-ICSI cycles, followed by second day ICSI fertilization, may provide a valuable source of embryos for infertile couples.

Cytogenetic abnormalities of unfertilized oocytes generated from in-vitro fertilization and intracytoplasmic sperm injection: a double-blind study.

In the present study we have assessed the cytogenetic abnormalities of unfertilized oocytes from in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) programmes during a one year period (July 1995 to July 1996) with the cytogenetic analysis being carried out in a double-blind manner. A total of 88 unfertilized ICSI and 85 unfertilized IVF oocytes were used for the study and of these 51 and 62 oocytes, in each respective group, were suitable for analysis. The haploidy, diploidy and aneuploidy rates between ICSI (62.7, 7.8 and 5.9%) and IVF (61.3, 9.7 and 14.5%) groups were similar. A significant inter-patient variation in the incidence of hypohaploidy was observed within the IVF group. Chromosomal fragmentation or breakage was observed at a similar rate in both groups of unfertilized oocytes (23.5 and 14.5% for ICSI and IVF respectively). A significantly higher proportion of ICSI oocytes contained sperm nuclei (27/51, 52.9%) than did IVF oocytes (20/62, 32.3%, P < 0.01). The distribution and state of sperm head chromatin in relation to oocyte chromosomal complement was studied in both groups. ICSI oocytes contained decondensed or swollen sperm nuclei in association with haploid oocyte chromosomes (12/27, 44.4%) or condensed sperm heads in oocytes showing no chromosomal complements (7/27, 25.9%). In IVF oocytes sperm heads were either arrested in the condensed state (5/20, 25%), metaphase stage (3/20, 15%) or had undergone premature chromosome condensation (PCC; 6/20, 30%) in association with haploid oocyte chromosomes. The incidence of PCC was similar in the two groups. A marked variation in the incidence of total chromosomal abnormality was observed between patients within both ICSI (0-75%) and IVF (0-71%) groups indicating a possible similarity in oocyte quality between the majority of male factor and tubal infertility patients. The type of sperm used in the two fertilization procedures showed an increased incidence of chromosomal breakage with ICSI-MESA (microepididymal sperm aspiration) spermatozoa (4/6, 67%) compared to the ICSI-ejaculated (6/35, 17.1%; P < 0.05), ICSI-testicular biopsy (2/10, 20%) and IVF-normospermic (9/62, 14.5%; P < 0.01) spermatozoa. Chromosomal fragmentation may be associated with the degree of difficulty experienced at sperm injection, especially with sperm retrieved from the reproductive tract. Thus chromosomal fragmentation in ICSI may need further investigation using a larger sample size in order to assess the possible causative factors.

Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme.

Mouse oocytes and embryos were obtained following ovulation induction of (C57B16 x CBA) F1 animals. Zonae pellucidae were exposed to alpha-chymotrypsin in phosphate-buffered medium (PB1) supplemented with 3 mg/ml bovine serum albumin upon a heated stage (37 degrees C) and were observed constantly through an inverted microscope. The endpoint of the bioassay was the limits of the zona no longer being seen clearly at x 200 magnification, and the time taken for each zona to dissolve was recorded. A dose-dependent response in dissolution time was clearly seen, with 1% alpha-chymotrypsin being chosen as the routine working solution. Cryopreservation of 2-cell mouse embryos using propanediol did not cause zona hardening but induced a small and significant softening, as gauged by the time taken for zona dissolution (2181 +/- 167 versus 1864 +/- 82 s). Zona hardening was not suspected to occur after the freezing of human embryos as there was no difference in implantation rates per embryo for in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles between fresh [IVF: 63/644 (9.7%); ICSI: 51/330 (15.5%)] and frozen embryos [IVF: 36/458 (7.9%); ICSI: 18/112 (16.1%)]. Conversely, significant hardening of the zonae of mature oocytes was seen following cryopreservation (747 +/- 393 s) compared with freshly ovulated oocytes (151 +/- 68 s). It is concluded that (i) the freezing of murine oocytes with propanediol results in zona hardening, implying a possible benefit of ICSI after the cryopreservation of human oocytes, and (ii) the cryopreservation of embryos is not associated with zona hardening or reduced implantation, making microdissection of the zona in such cases generally unwarranted.

Changes in motility patterns during in-vitro culture of fresh and frozen/thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection.

The present report describes the motility changes in vitro (percentage motile and progressively motile) of freshly collected testicular and epididymal spermatozoa and following freeze/thaw of the same spermatozoa from a man with obstructive azoospermia. Washed spermatozoa were cultured in micro droplets under paraffin oil or in test tubes using HEPES-buffered or bicarbonate-buffered medium containing 10% human serum. In fresh testicular sperm cultures 60-65% of the sperm cells became motile within 2 days of culture; the motility was maintained for a further 4-5 days before a decline was observed. The progressive motility improved markedly on the third day of culture and it peaked around day 5. Only a small number of frozen/thawed testicular spermatozoa became motile during in-vitro culture (15-20%) and the motility was maintained for only 2-3 days before it declined. Furthermore, only 10-12% of the spermatozoa showed progressive motility. Spermatozoa recovered from micro-epididymal sperm aspiration (MESA) showed a gradual decrease in progressive motility and in 5 days all sperm cells were found to be immotile in both freshly collected and frozen/thawed spermatozoa. All culture systems supported sperm motility. It is clear that testicular spermatozoa, particularly from men with obstructive azoospermia, can be collected and maintained in vitro for up to 1 week before the oocyte retrieval but when frozen testicular or epididymal spermatozoa are used it is more reliable to thaw these spermatozoa on the day of intracytoplasmic sperm injection.

Cytogenetic analysis of unfertilized oocytes following intracytoplasmic sperm injection using spermatozoa from a globozoospermic man.

A man with globozoospermia was treated in our in-vitro fertilization-intracytoplasmic sperm injection (ICSI) programme. In the treatment cycle, 24 oocytes were collected from his wife. All the oocytes were at metaphase II stage. The semen sample produced on the day had a normal sperm count, good motility, but with 100% globozoospermia. All oocytes were injected with randomly selected spermatozoa and of these, two oocytes showed two pronuclei and another contained a single pronucleus. The remainder were unfertilized. The normally fertilized oocytes (two pronuclear) cleaved to the four-cell stage and were transferred to the patient. At 48 h after ICSI, the 21 unfertilized oocytes were processed for cytogenetic analysis. All oocytes contained a haploid chromosome set. The only abnormality seen was a chromosome fragment in one metaphase. Eighteen oocytes contained decondensed sperm nuclei and of these, 14 nuclei were beginning to show signs of premature chromatin condensation (PCC) and the other four showed strong signs of PCC. Thus it appears that in some forms of globozoospermia, arrest of nuclear decondensation and/or PCC are another cause of fertilization failure. The most likely cause for this is the absence or down-regulation of spermatozoa associated activating factor in round-headed spermatozoa.

Fertilization of supernumerary oocytes following gamete intrafallopian transfer (GIFT): correlation with outcome of GIFT treatment.

In the course of 280 consecutive gamete intrafallopian transfer (GIFT) attempts, supernumerary oocytes in excess of those transferred were inseminated in vitro. Pregnancy resulted in 31.1% of patients following the GIFT treatment. The overall in vitro fertilization (IVF) rate of supernumerary oocytes was significantly higher in those who became pregnant (41.2%) than in patients who did not (34.9%). However, the failure to fertilize any supernumerary oocytes was not significantly different between those becoming pregnant (27.6%) and those in whom the treatment failed (37.3%). Failure to fertilize any supernumerary oocytes in vitro was of a higher rate if suboptimal sperm preparation was used or if only one oocyte was left over for in vitro insemination following the GIFT treatment. We concluded that the in vitro fertilization rate was higher in the pregnant group, but the total failure to fertilize or the fertilization of any supernumerary oocytes does not predict the outcome of GIFT. Embryos resulting following fertilization of excess oocytes may be cryopreserved for subsequent use.

The successful recovery and fertilization of oocytes from the pouch of Douglas.

Two groups of women were studied in whom a proportion of follicles had either ovulated spontaneously (7 women) or ruptured during manipulation at laparoscopy (30 women), and oocytes were recovered from the pouch of Douglas. There were no significant differences in the fertilization rates of oocytes collected in the pouch of Douglas from ovulated follicles, compared with those from the remaining intact follicles [15/20 (75%) vs 14/20 (70%)]. Also, there was no significant difference between the fertilization rate of oocytes from follicles ruptured at the time of oocyte collection and that of oocytes from intact follicles [25/38 (66%) vs 101/140 (72%)]. One woman became pregnant, following the transfer of four four-cell embryos, all derived from spontaneously ovulated oocytes found in the pouch of Douglas. She gave birth to a baby girl. The present study has shown that oocytes may still be retrieved from the pouch of Douglas, despite follicle dispersal; these oocytes can be fertilized; and the embryos derived from ovulated oocytes recovered from the pouch of Douglas may generate an ongoing pregnancy following in vitro fertilization and embryo transfer.

The fertilization of human oocytes by spermatozoa from men with antispermatozoal antibodies in semen.

Seventy-two couples, including 15 with antispermatozoal antibodies in the male partner's semen, were studied in a program of in vitro fertilization and embryo transfer. Cases were further subclassified as normospermic or oligospermic and antispermatozoal antibodies were assessed with categorization into the respective human immunoglobulin classes as determined using the indirect immunobead test. The study reveals that fertilization is significantly reduced (P less than 0.001) only if both IgA and IgG antibodies are present in semen but there is no reduction if either class is present alone. The fertilization rate of oocytes is significantly reduced (P less than 0.001) by sperm from oligospermic samples, and there is a further reduction in those cases with combined IgA/IgG antispermatozoal antibodies.