Transabdominal punction of follicular fluid in IVF cyclus in a patient after ovarian transposition.

OBJECTIVE: Description of punction of follicular fluid in a patient after ovarian transposition. DESIGN: Case report. SETTING: Department of Obstetrics and Gynaecology, 1st Faculty of Medicine, Charles University and the General Faculty Hospital, Prague. CASE REPORT: We present a case of IVF treatment in a patient with ovarian transposition undergoing punction of follicular fluid and difficulties during this procedure acording to transabdominal route. CONCLUSION: Transabdominal punction od follicular fluid is possible, but with technical difficulities and smaller amount of obtained oocytes. We recomend to aplicate IVF procedures prior to surgical solution.

[HYPERPARATHYROIDISM AND PARATHYROID LESIONS IN THE ChNPP ACCIDENT SURVIVORS]. / GIPERPARATYREOZ TA URAZhENNIa PRYShchYTOPODIBNYKh ZALOZ U OSIB, OPROMINENYKh VNASLIDOK AVARIÏ NA ChAES.

OBJECTIVE: Study of the status of parathyroid glands in individuals exposed to ionizing radiation as a result of the ChNPP accident and comparison with the general population of Ukraine. MATERIALS AND METHODS: Subjects exposed as a result of the ChNPP accident (n = 1,348) and people from the general population of Ukraine (n=655) were examined. Diagnostic ultrasound scan of thyroid and parathyroid glands (PTG) was conducted in all study subjects. The technique of parathyroid ultrasound screening was developed, which led to an increase in the efficiency of their imaging. Additionally, the 25-hydroxyvitamin D (25(OH)D), parathyroid hor- mone, ionized calcium and some other parameters were selectively assayed in serum. RESULTS: High incidence of parathyroid hyperplasia was detected 27-32 years after the irradiation in persons ex- posed as a result of the ChNPP accident, especially in evacuees from the 30-km exclusion zone (71.4%; χ2Yates' = 24.1; р = 0) and residents of radilogically contaminated territories (41.7%; χ2Yates' = 6.45; p < 0.01) having no primary hyperparathyroidism. High prevalence of vitamin D insufficiency and deficiency was revealed in all study subgroups, namely in 83.1 % of the general population of Ukraine along with a bit better vitamin D status in the ChNPP acci- dent survivors i.e. the vitamin insufficiency and deficiency was found in 78.7 % of them. Incidence of hyperparathy- roidism, predominantly of the secondary (normocalcemic) one, was 33.8 % among persons exposed as a result of the ChNPP accident (p > 0.3) being somewhat higher than in the general population of Ukraine (26.1%), despite above- mentioned better supply of vitamin D. CONCLUSIONS: There is a widespread insufficiency or deficiency of vitamin D (over 78.7%) in the population of Ukraine in general providing an unfavorable background for the higher prevalence of health disorders associated with calcium and phosphorus metabolism. The latter features parathyroid hyperplasia and musculosceletal, immune, cardiovascular, and endocrine system comorbidities. Such disorders should exacerbate with a secondary increase in parathyroid hormone secretion (26.1 %). Higher incidence of normocalcemic hyperparathyroidism (33.8% versus 26.1%) against a background of better vitamin D status among irradiated individuals indicates the existence of other factors, where the past combined effects of Chornobyl radioactive fallout and external parathyroid exposure are most likely to be involved. This could explain the greater number of cases of parathyroid hyperplasia and hyper- parathyroidism among the exposed subjects. However the additional precise studies are required here with clarifi- cation of the personal data in population groups of the ChNPP accident survivors. Participants of the ChNPP acci- dent clean-up work in the «iodine period¼ of 1986 are of especial concern here. Besides that, the study population should be expanded with inclusion of subjects exposed in prenatal period.

Initial healthcare to pediatric population under the radiation events. / MEDYChNA DOPOMOGA DYTIaChOMU NASELENNIu PRY RADIATsIY̆NYKh PODIIaKh NA PERShYKh ETAPAKh.

The objective of this paper is to analyze the data from scientific literature and available recommendations for health professionals on healthcare providing to pediatric population in the events associated with risk of radiation exposure. Over the past sixty years there were several large scale radiological events with a large number of chil dren affected, namely the atomic bombing of Hiroshima and Nagasaki, accident at the Chornobyl nuclear power plant, contact to 137Cs radiation source unutilized at the hospital shutdown in Brazil etc. Further research has crys tallized injuries and health disorders in the survived children in all cases being much more significant vs. other pop ulations. Analysis of circumstances and features of a number of emergency situations or incidents in the nuclear industry for several decades has shown a high probability of radioactive materials release into the environment. The danger of terrorism with the use of ionizing radiation sources resulting in a considered inevitable hazard to the chil dren is estimated as serious in recent years and deserves an especial mention here.

[Successful treatment of azoospermia diagnosed as "Sertoli cells only syndrome with maturation arrest"--developing a quality embryo]. / Uspesné resení azoospermie s diagnózou "Sertolli cells only syndrome" (SCO sy.) a Maturation Arrest (MA)--získání kvalitního embrya.

OBJECTIVE: The transfer of good quality embryo in the program of assisted reproduction in the case of azoospermia, dg. Sertolli cells only syndrome (SCO sy) + maturation arrest (MA). Testes were assessed and found to have a high occurrence of Sertolli cells and very low occurrence of germinal cells, which were arrested at the round spermatid level. The histological evaluation was hypospermatogenesis gr. 3 (minimum 1 spermatid/sample). DESIGN: Case report. SETTING: Laboratory IVF, Iscare, a. s., Department of Biology and Biochemistry of Fertilization, Institute of Molecular Genetics, Czech Academy of Sciences, Prague. SUBJECT AND METHOD: The successful integration of three methods provides a solution for this case of azoospermia. Immunology and histology can more exactly diagnose the degree of azoospermia. Detection and visualisation of spermatids using monoclonal antibodies against sperm proteins predicts the eventual occurrence of spermatogenesis, and histological evaluation confirms these immunological findings. Using the information of both methods it is possible to use special in vitro cultivation of testicular cells and so obtain injectable spermatozoa, or precursors of sperm, for the ICSI method. CONCLUSION: The probability of acquisition of good-quality embryo in the program of assisted reproduction is higher when these three methods are applied in combination.

Somatic and embryonic cell nucleus transfer into intact and enucleated immature mouse oocytes.

BACKGROUND: The aim of our study was to evaluate the possibility of embryonic or somatic cell haploidization after fusion with intact or enucleated immature oocytes which were subsequently cultured in vitro. Embryonic or somatic cell nuclei do not undergo premature chromosome condensation when fused to intact or enucleated immature oocytes whose maturation is prevented by dibutyryl cyclic AMP (dbcAMP). The presence of dbcAMP permits, however, the completion of DNA replication in somatic cell nuclei. METHODS AND RESULTS: The chromosomes condensed when the reconstructed cells were released from the dbcAMP block. When somatic or embryonic nuclei were introduced into intact immature meiotically competent oocytes and subsequently cultured their chromosomes assembled on a common spindle with meiotic chromosomes and proceeded through the meiotic-like division, judged according to the presence of the first polar body extruded. When embryonic cell nuclei were introduced into cytoplasts obtained from immature meiotically competent oocytes, polar bodies were extruded in about 75% of reconstructed cells but the metaphase plates were abnormal in almost all cases. When somatic cell nuclei were inserted into the above cytoplasts, polar bodies were extruded only very exceptionally and in these cells chromosomes were arranged in abortive metaphase plates. CONCLUSIONS: Our results suggest that somatic cell nuclei are unable to proceed through the reduction division (haploidization) when introduced into an immature oocyte meiotic cytoplasm.

[Surgical treatment of male infertility]. / Taktika chirurgické lécby muzské infertility.

OBJECTIVE: In the Czech Republic in the treatment of male infertility the most up-to-date technologies of assisted reproduction have also become the methods of choice: MESA--microsurgical epididymal sperm aspiration, TESE--testicular sperm extraction and ICSI--intracytoplasmic sperm injection. The objective of the present study was an attempt to find a reliable predictor of results of planned TESE as prediction of possible successful sperm extraction can prevent unnecessary ovarian stimulation. DESIGN: Retrospective analysis of a group of 202 men operated in the authors centre on account of azoospermia--65 patients with obstructive azoospermia and 137 patients with testicular azoospermia. SETTING: Andromeda, Biolab, Iscare IVF, Prague. METHOD: The following parameters were evaluated: testicular volume, FSH serum level and histological finding and their values were compared with results of TESE. RESULTS: Elevated FSH levels and low testicular volume do not rule out a positive result of TESE. The most accurate predictor is histological examination. Elongated spermatids were found in the histological material in 59.3% of the examined cases which correlates significantly with the 60.2% TESE yield in the evaluated group. CONCLUSION: The material assembled on operation was divided into three parts. One serves preparation of the perIerative native preparation, the second one is dispatched for histological examination and the largest part is frozen. Simultaneous collection of oocytes and sperm cells is done only in selected cases. The histological finding of elongated spermatids indicates a positive finding of sperm during TESE with an accuracy of 91%. Assessment of the causes of azoospermia, evaluation of the state of spermatogenesis and treatment are thus implemented by a single operation.

[Use of human sperm protein monoclonal antibodies in the diagnosis of sperm pathology and selection of a suitable assisted reproduction method for fertilization]. / Vyuzití monoklonálních protilátek proti proteinum lidských spermií k diagnostice patologie spermií a výberu vhodné metody fertilizace v asistované reprodukci.

OBJECTIVE: Use of monoclonal antibodies against sperm proteins in human medicine. DESIGN: Experimental and clinical studies. SETTING: Dep. Biology and Biochemistry of Fertilization, Institute of Molecular Genetics, Prague, Laboratory IVF, Iscare IVF, Prague, Dep. of Immunobiology, Institute for the Care of Mother and Child, Prague. METHODS: Monoclonal antibodies against human sperm intra-acrosomal and cell surface proteins were used for quantitative analysis of these proteins by the immunofluorescence test in samples of human sperm of good and poor qualities. RESULTS: The detection of intra-acrosomal proteins was decreased and, on the other hand, detection of surface proteins was the same or higher in pathological spermatozoa. CONCLUSIONS: Monoclonal antibodies can be used for diagnostics of sperm pathology (quantitative detection of proteins) and for evaluation of the physiological state of sperm cells (state of acrosome before or after acrosome reaction). Finally, monoclonal antibodies could be useful for selection of a suitable method of fertilization (IUI, standard IVF, ICSI) in the laboratories of assisted reproduction.

[MESA, TESA, TESE + ICSI: results of the first 50 cases]. / MESA, TESA, TESE + ICSI: výsledky u prvních 50 prípadu.

During the period between February 1996 and August 1997 51 infertile men were operated. The authors evaluate the results from 47 couples--25 cases of testicular azoospermia and 22 cases of obstructive azoospermia. The mean age of the men in the group was 33 years (22-48 years). The follow-up period is 1-18 months. In 17 cases, microsurgical epididymal sperm aspiration (MESA) was made, in five cases testicular sperm aspiration (TESA) and in 25 men, testicular sperm extraction (TESE) was performed. In the group with testicular azoospermia it proved possible to obtain sperm in 12 of 25 cases, i.e. in 48%. In men with obstructive azoospermia all aspirations were successful, i.e. the yield was 100%. In this group five children were born, seven pregnancies are under way. Thus regardless of the etiology of male infertility 12 of 47 cases, i.e. 25.5% were resolved successfully. When the results of the two groups are differentiated, the outcome is as follows: in the group with testicular azoospermia one child was born and two pregnancies are under way, i.e. 12%, in the group with obstructive azoospermia four children were born and five pregnancies are under way, i.e. 40.9%.

[Microsurgery and micromanipulation in the treatment of male infertility: results of a one-year investigation]. / Mikrochirurgie a mikromanipulace v lécbe muzské infertility: výsledky rocního sledování.

A one-year study of 51 infertile couples, 47 couples evaluated--25 cases of testicular azoospermia and 22 cases of obstructive azoospermia. The mean age of the men in the group is 33 years (22-48 years). The follow-up period is 1-18 months. In 17 instances microsurgical epididymal sperm aspiration--MESA was made, five times testicular sperm aspiration--TESA and in 25 men testicular sperm extraction--TESE was used. In the group with testicular azoospermia it proved possible to obtain sperm in 12/25 cases, i.e. in 48%. In men with obstructive azoospermia all aspirations were successful, i.e. the yield was 100%. In this group five infants were born, another seven pregnancies are under way. Thus regardless of the etiology of male infertility 12/47 cases, i.e. 25.5%, were successfully resolved. When using differentiated evaluation of the two groups the results are as follows: in the group with testicular azoospermia one infant were born and five pregnancies are under way, i.e. 40.9%.

Beneficial influence of Vero cells on in vitro maturation and fertilization of bovine oocytes.

The aim of this study was to examine the effects of Vero cells and other somatic cells on in vitro maturation of bovine oocytes. Both denuded oocytes and oocytes with intact cumuli (COCs) were cultured on monolayer of Vero cells, cumulus cells and granulosa cells. The effect of gonadotropins was investigated after the addition of gonadotropins to the culture medium. The evaluation using analysis of variance revealed that removal of cumulus cells generally reduced the percentage of oocytes completing their maturation in vitro and that this effect could not be overcome by the addition of gonadotropins to the culture medium. However, in individual experiments, when oocytes were co-cultured with different monolayers of somatic cells, Vero cells were able significantly support the maturation of denuded oocytes, and their beneficial effect was further enhanced by the addition of gonadotropins (76 vs 80.9%). We did not observe a similar effect after the co-culture of oocytes with a monolayer of cumulus cells (65.3 and 53%, respectively). Granulosa cell monolayer delayed maturation in the both COCs and denuded oocytes (10.5 and 16.5%, respectively). In vitro fertilization was successful in most of the experimental groups. However, when denuded oocytes were cultured without any somatic cell support, they did not decondense the penetrated sperm head after in vitro fertilization. This study demonstrates that 1) Vero cells beneficially affect the in vitro maturation of bovine oocytes; 2) cumulus cells in the form of monolayer lose their beneficial influence on in vitro maturation of bovine oocytes; and 3) granulosa cells and FSH and LH alone (without somatic cells) do not show positive effects on in vitro maturation of bovine oocytes.

Inhibition of meiotic maturation in growing pig oocytes by factor(s) from cumulus cells.

Total inhibition of germinal vesicle breakdown (GVBD) was observed in growing pig oocytes (internal diameter 80, 90 and 100 microns) when they were cultured in a medium conditioned by cumulus oocyte complexes (COCs) of fully grown oocytes. In denuded growing oocytes, only partial inhibition was observed. The inhibitory effect was fully reversible. The addition of heparin (300 IU/ml) could overcome the effect of the conditioned medium. Transient exposure (6 h) of oocytes to dibutyryl cyclic adenosine monophosphate (dbcAMP) (1 mg/ml) could also partly reverse the effect of factor (s) produced by cumulus cells of fully grown oocytes. Follicle-stimulating hormone (5 micrograms/ml) was able to increase the percentage of maturing oocytes. The addition of luteinizing hormone (5 micrograms/ml) had no effect on GVBD inhibition by cumulus-conditioned medium.

Vitrification of mouse 8-cell embryos with glycerol as a cryoporotectant.

Glycerol at a concentration of 6.85 M has been used for cryopreservation of 8-cell mouse embryos with the aim to induce formation of ice-free glass after plunging into liquid nitrogen. Before treatment with this concentration at approximately 0 degrees C, embryos were pre-equilibrated in 1.37 M glycerol at ambient temperature. It was found that the main source of damage to embryos is due to treatment with a high concentration of glycerol and osmotic events during its dilution. Cooling and warming of embryos per se induce little or no harm to their capacity to form expanded blastocysts after 48 h in vitro. Best results (together 169/198, 85.4%) were obtained, when both time (not more than 15 min) and temperature (approximately 0 degrees C) of exposure of embryos to vitrification media were controlled properly.

Cryopreservation of mouse 8-cell embryos in microdrops.

Eight-cell embryos collected from superovulated inbred strains and F1 hybrid mice were frozen by the microdrop technique developed in our laboratory. The technique based on pre-equilibration in medium with 10% glycerol, before transfer into vitrification solution, expel of embryos in 5 microliters to 20 microliters of vitrification solution directly into liquid nitrogen and thawing of microdrops in medium with 0.5 M sucrose was used. The behavior and morphological appearance of embryos during pre-freezing and post-thawing periods was documented. The efficiency of cryopreservation in microdrops was high, as documented by 90% to 100% of intact embryos after the freezing and thawing cycle. Furthermore, no zona pellucida damage was observed. The developmental potential of embryos frozen in microdrops was comparable with development of unfrozen embryos of the same genetic origin. After freezing and storage 83% to 93% of embryos developed to blastocysts and 73% to 92% embryos underwent "implantation" after 48 h and 96 h of in vitro culture, respectively.

Construction of aggregation chimaeras from 8-cell mouse embryos stored for several years in liquid nitrogen.

Eight-cell embryos of 7 different inbred strains frozen and stored for 2 to 4 years in liquid nitrogen were used as the source of embryos and individual 1/8-blastomeres for construction of different types of aggregation chimaeras. Overall survival of stored embryos, evaluated by in vitro culture, ranged from 63% in A/By to 85% in C57BL/10By embryos. Chimaeric embryos prepared by aggregation of frozen/thawed zona-free 8-cell embryos continued in normal development through expanding blastocysts (77% to 93%) to in vitro implantations. Construction of directed chimaeras by total rebuilding of 8-cell embryos previously stored in liquid nitrogen was successful and resulted in normal development. Of the aggregates made by regular mixing of 1/8-blastomeres obtained from BALB/c and C57BL/6J embryos thawed from liquid nitrogen, 90% reached the blastocyst stage and 77% implanted after 48 h and 96 h of in vitro culture, respectively. Addition of 1/8 blastomeres from frozen/thawed BALB/c embryos to four 1/8-like blastomeres from nuclear transplantation experiments documented, as all of the 30 constructed aggregates formed expanding blastocysts within 48 h of in vitro culture, the possibility of using embryos from embryo banks for the rescue of products of genetic manipulations at the embryo level.