The educational and professional status of clinical embryology and clinical embryologists in Europe.

STUDY QUESTION: What is the recognition of clinical embryology and the current status of clinical embryologists in European countries, regarding educational levels, responsibilities and workload, and need for a formal education in assisted reproductive technology (ART)? SUMMARY ANSWER: It is striking that the profession of clinical embryology, almost 40 years after the introduction of IVF, is still not officially recognized in most European countries. WHAT IS KNOWN ALREADY: Reproductive medicine has developed into a sophisticated multidisciplinary medical branch since the birth of Louise Brown 37 years ago. The European Board & College of Obstetrics and Gynaecology (EBCOG) has recognized reproductive medicine as a subspeciality and has developed a subspeciality training for gynaecologists in collaboration with the European Society for Human Reproduction and Embryology (ESHRE). However, nothing similar exists for the field of clinical embryology or for clinical embryologists. STUDY DESIGN, SIZE, DURATION: A questionnaire about the situation in clinical embryology in the period of 2012-2013 in the respective European country was sent to ESHRE National representatives (basic scientists only) in December 2013. At this time, 28 European countries had at least one basic scientist in the ESHRE Committee of National Representatives. PARTICIPANTS/MATERIALS, SETTING, METHODS: The survey consisted of 46 numeric, dichotomous (yes/no) or descriptive questions. Answers were obtained from 27 out of 28 countries and the data were tabulated. Data about the numbers of 'ESHRE Certified Embryologists' were taken from the ESHRE Steering Committee for Embryologist Certification. MAIN RESULTS AND THE ROLE OF CHANCE: In 2012, more than 7000 laboratory staff from 1349 IVF clinics in 27 European countries performed over 700 000 fresh and frozen ART cycles. Despite this, clinical embryology is only recognized as an official profession in 3 out of 27 national health systems. In most countries clinical embryologists need to be registered under another profession, and have limited possibilities for organized education in clinical embryology. Mostly they are trained for practical work by senior colleagues. ESHRE embryologist certification so far constitutes the only internationally recognized qualification; however this cannot be considered a subspecialization. LIMITATIONS, REASONS FOR CAUTION: Data were obtained through different methods, by involving national embryologist societies and cycle registers, collecting information from centre to centre, and in some cases by individual assessment of the situation. For these reasons, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: This paper presents the current status of clinical embryology and clinical embryologists in Europe and is an important step towards implementation of clinical embryology as an officially recognized profession. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: No.

Design of a drug delivery system with bimodal pH dependent release of a poorly soluble drug.

A delivery system which provides bimodal pH dependent release of poorly water soluble carvedilol in gastric and intestinal environment was designed. Preparation of solid dispersion with porous silica ensured a significantly higher dissolution rate of carvedilol in acidic and alkaline media in comparison to pure drug, while granulation of that solid dispersion with enteric polymer dispersion resulted in diminished immediate release in acidic media and fast release of the remaining drug in alkaline media. The ratio in quantities of first vs. second release was controlled with amount of enteric polymer dispersion used for granulation process. Desired 25 mg release of carvedilol at pH values 1.2 and 6.8 was achieved when 1.80 g of polymer per 1.0 g of solid dispersion (drug to silica ratio= 0.25 g : 2.0 g) was used.

Influence of atmospheric versus reduced oxygen concentration on development of human blastocysts in vitro: a prospective study on sibling oocytes.

Numerous studies show the beneficial effect of reduced oxygen on the culture of animal embryos in vitro. However, few similar studies have been carried out in humans, and the conclusions from these were contradictory. Using sibling human oocytes, a prospective study was carried out to analyse the effect of 5 and 20% oxygen on prolonged development of embryos. The outcomes measured were fertilization rate and proportion of morphologically optimal embryos, blastocysts and optimal blastocysts developing on day 5. The results were analysed separately for the group of IVF (n = 988 oocytes) and ICSI (n = 928 oocytes) cycles. It was found that low oxygen did not influence fertilization, but in comparison with 20% oxygen, it resulted in a significantly higher proportion of embryos being optimal on day 3 after IVF (59 versus 43.2%; P < 0.001) as well as after ICSI cycles (51.2 versus 28.5%; P < 0.001). In both methods, the lower oxygen concentration improved the blastulation rate (73.2 versus 63.1%; P < 0.05 and 67.4 versus 54.7%; P < 0.001) and increased the proportion of embryos reaching the stage of expanded blastocyst with normal inner cell mass on day 5 (31.1 versus 14.6%; P < 0.001 and 18.9 versus 11.4%; P < 0.01). The ratio of successful embryo development to optimal blastocyst stage on day 5 of culture, calculated for two oxygen concentrations, was 2.1 for IVF and 1.7 for ICSI, in favour of lower oxygen tension.

Morphokinetic Characteristics and Developmental Potential of In Vitro Cultured Embryos from Natural Cycles in Patients with Poor Ovarian Response.

Background. Patients with poor ovarian response to ovarian hyperstimulation represent an interesting group for studying the impact of embryo cleavage irregularities on clinical outcome since all embryos, regardless of their quality, are usually transferred to the uterus. The aim of our study was to follow the morphokinetics of fertilized oocytes from natural cycles in poor responders. Methods. Zygotes from 53 cycles were cultured in vitro for 3 days. The morphokinetics of their development and transfer outcomes were retrospectively analyzed for the normally and irregularly cleaved embryos. Results. Of all embryos, 30.2% had single and 20.8% multiple cleavage irregularities with the following prevalence: developmental arrest 30.2%, direct cleavage to more than two cells 24.5%, chaotic cleavage 13.2%, and reverse cleavage 11.3%. These embryos had longer pronuclear phases, first cytokinesis, second embryo cell cycles, and less synchronized divisions. The transfer of normally developing embryos resulted in an implantation rate of 30.8% and a delivery rate of 23.1%, but irregularly cleaved embryos did not implant. Conclusions. The use of time-lapse microscopy in poor responder patients identified embryos with cleavage abnormalities that are related with no or extremely low implantation potential. Gained information about embryo quality is important for counselling patients about their expectations.

Lack of clinically significant improvement of patients with tardive dyskinesia following phosphatidylcholine therapy.

A double-blind controlled study was undertaken to examine the value of phosphatidylcholine as a treatment for tardive dyskinesia (TD) in 19 psychiatric patients. All patients were maintained on their usual psychotropic medication throughout the entire study. In addition, they were given either phosphatidylcholine (30 g/day) or placebo for 6 weeks. Thirteen of the patients received the crossover treatment for 6 weeks, after which 10 of the 13 were continued on the crossover medication for an additional 6 weeks. At the end of the study, 5 patients had received phosphatidylcholine for 12 weeks and another 12 patients had received the drug for only 6 weeks. Plasma and red blood cell choline levels were monitored every 3 weeks as a measure of compliance. Although some patients showed clinical improvement of their TD, the results did not differ significantly between active drug and placebo. This was in spite of a marked elevation of plasma and red blood cell choline (up to 300% for the Lafayette Clinic patients and up to 400% for the patients from the Ypsilanti Regional Psychiatric Hospital) during treatment with phosphatidylcholine. Side effects of the drug included occasional gastrointestinal upsets and diarrhea but, in general, the medication was tolerated very well. The results indicate that large doses of phosphatidylcholine of soya origin are of no clinical value in treating symptoms of TD in spite of very large increases in blood choline.

Fluphenazine-induced acute and tardive dyskinesias in monkeys.

Five Cebus apella monkeys were treated with biweekly injections of fluphenazine enanthate (0.1-3.2 mg/kg IM). Three of these completed 1 full year of treatment, one injured its leg after 6 months of treatment and was killed, and another died of unknown causes after 9 months of treatment. All monkeys displayed abnormal movements corresponding to the early appearing extrapyramidal symptoms of neuroleptic-treated patients. These consisted initially of slowing or absence of volitional movement, trembling of the hands, trembling of the entire body, and general drowsy behavior. As treatment progressed, a variety of abnormal postures and movements appeared after each injection that were not exacerbated by drug withdrawal and, as tested at the end of the year, could be abolished or prevented with benztropine mesylate (0.2-0.5 mg/kg IM). The three monkeys that completed 1 year of treatment with fluphenazine were then withdrawn from the drug. After withdrawal, all three developed movements similar in appearance to those of patients with tardive dyskinesia (TD). Reinstitution of fluphenazine treatment, as tested in one monkey, abolished all movements resembling TD.

Naloxone enhancement of DMT and LSD-25 induced suppression of food-rewarded bar pressing behavior in the rat.

The narcotic antagonist naloxone was tested to determine its possible interaction with N,N-dimethyltryptamine (DMT) and lysergic acid diethylamide-25 (LSD) in adult male Holtzman rats trained to press a bar on a fixed-ratio four schedule (FR4), i.e., every fourth press earned a reward of 0.01 ml sugar sweetened milk. LSD (0.1 mg/kg) or increasing doses of DMT (1.0, 3.2, and 10.0 mg/kg) were administered i.p. to disrupt food-rewarded fixed ratio bar pressing in a dose related fashion. Pretreatment (5--10 min) with behaviorally ineffective doses of naloxone (1.0--5.6 mg/kg) dramatically enhanced the effects of DMT and LSD. The content of DMT in the brain and liver of rats injected with DMT alone (10 mg/kg) and with a 5 min pretreatment of naloxone (3.2 mg/kg) was determined by radiochemical analysis at 30 and 90 min after 14C-DMT injection. There was no significant difference for either brain or liver 14C-DMT levels when control DMT rats were compared with the naloxone pretreated rats. These results seem to rule out interference by naloxone with the metabolism of DMT as a mechanism of the observed behavioral potentiation.

Interactions of partial LSD analogs with behavioral disrupting effects of LSD and DMT in the rat.

Adult male Holtzman rats were trained to barpress on a schedule whereby every fourth press earned a reward of 0.01 ml of sugar-sweetened milk (FR4). After an i.p. injection of LSD (0.1 mg/kg) or DMT (3.2 or 10 mg/kg) such barpressing is abolished completely and resumed, usually within an hour, at a rate near the preinjection control rate of pressing. It continues at a steady, uninterrupted pace until the animals are removed from the operant chamber one-half hour later. A series of N,N-diethylnipecotamide derivatives were synthesized and tested for their ability to modify the disruptive effect of these hallucinogens. N,N-diethylbutyramide (DBA) and 1-methyl-1,2,5,6-tetrahydropyridine-3-(N,N-diethylcarboxamide) (THPC) were also tested. Pretreatment with a single i.p. injection of any of these compounds (5--40 mg/kg) either had no effect on or else prolonged the duration of hallucinogen-induced cessation of barpressing.

Value of the serum estradiol level on the day of human chorionic gonadotropin injection and on the day after in predicting the outcome in natural in vitro fertilization/intracytoplasmic sperm injection cycles.

OBJECTIVE: To predict the risk of premature ovulation and possibility of pregnancy in natural IVF/ICSI cycles on the basis of the estradiol (E2) level on the day of hCG injection and on the day after. DESIGN: A prospective study. SETTING: Hospital research program. PATIENT(S): One hundred sixty-four women undergoing 305 IVF/ICSI natural cycles. INTERVENTION(S): Serum E2 levels were measured approximately 12 h before (day 0) and 12-17 h after hCG administration (day 1). MAIN OUTCOME MEASURE(S): E2 levels on day 0 and day 1, the ratio of the day 1 to day 0 levels, and cycle outcome. RESULT(S): In cycles with premature ovulation and in conception cycles, the average E2 level on day 0 was statistically significantly higher than in other cycles, whereas the E2 ratio was statistically significantly lower. Multiple logistic regression was used to determine the connection of the E2 level on day 0 (B0 = -0.742, B = 2.147, P =.01) and the E2 ratio (B0 = -0.742, B = -3.135, P<.001) with premature ovulation. Only the E2 ratio (B0 = 0.659, B = -2.209, P =.0068) was significantly connected with conception. CONCLUSION(S): In predicting the outcome of natural IVF/ICSI cycles, the importance lies not in the E2 level on the day of hCG administration or on the day after, but rather in the E2 ratio.

In vitro fertilization program based on programmed cycles monitored by ultrasound only.

OBJECTIVE: Programmed oocyte retrieval was performed in order to make the in vitro fertilization (IVF) program cheaper and work of the IVF team easier. METHOD: In a group of 77 patients included in the IVF program, the menstrual cycle was modified with estrogen-progesterone contraceptive pills. For this reason, it was possible to start the stimulation protocol in all patients on the same day. The stimulation protocol was a combination of clomiphene (100 mg) for 5 days and HMG (150 IU) every other day. Cycles were monitored by ultrasound only. RESULT: The implantation rate per embryo transfer was 22.4%. The number of embryos per embryo transfer was low (2.6 +/- 1.4) and eliminated the need for cryopreservation. Fertilization rate (82%) and embryo transfer rate (87%) were high. The take home baby rate was 14.3%. CONCLUSION: Seventy percent of all punctures were performed in 3 days in the middle of the week. In our conditions, programmed oocyte retrieval is associated with significant economic benefits.

Simplification of the clinical phase of IVF and ICSI treatment in programmed cycles.

OBJECTIVE: To evaluate the success of a protocol for controlled ovarian hyperstimulation allowing patient self-selection into groups for ovulation stimulation planned 8 weeks and more in advance following cycle synchronization, drug self-administration as well as a reduced number of folliculometries. METHODS: A total of 714 patients received the same stimulation protocol. In 260 cases GnRH-a was applied daily and in 454 as depot. In all patients FSH-HP was self-administered subcutaneously for ovarian stimulation. In 316 patients IVF and in 398 patients ICSI was performed. RESULTS: The delivery rate per started cycle was higher in patients receiving depot GnRH-a in the IVF and ICSI group (30.2 vs. 23.4) than in those receiving subcutaneous GnRH-a (20.2 vs. 22.1). CONCLUSION: Programming of the IVF/ICSI cycle greatly simplifies treatment. A comparison of pregnancy rate and delivery rate per cycle between depot and subcutaneous daily application of GnRh-a did not confirm any statistically significant difference.

Number of oocytes retrieved and resulting pregnancy. Risk factors for ovarian hyperstimulation syndrome.

OBJECTIVE: To test whether the risk of developing clinically significant ovarian hyperstimulation syndrome (OHSS) is related to the number of oocytes retrieved by puncture and the resulting pregnancy and to determine the most suitable cutoff limit of the number of oocytes in predicting OHSS. STUDY DESIGN: The study included 973 patients who underwent ovarian stimulation for in vitro fertilization or intracytoplasmic sperm injection. Using the classification of Schenker and Weinstein, we identified patients who developed moderate and severe OHSS. By multiple logistic regression we established the risk of moderate or severe OHSS development in relation to the number of oocytes retrieved and the resulting pregnancy. A receiver operator characteristic curve was constructed to describe the relation between sensitivity and the false positive rate for the number of oocytes retrieved in the prediction of OHSS. RESULTS: We identified 35 (3.6%) patients who developed OHSS, 8 (0.8%) severe and 27 (2.8%) moderate. The risk of developing OHSS increased with the number of oocytes retrieved (odds ratio = 1.14) and with pregnancy (odds ratio = 1.14). The most suitable limit for predicting OHSS was 10 oocytes, with 81.9% specificity and 48.6% sensitivity. The risk of OHSS development in cycles with < or = 10 oocytes and no conception was 1.31% and, with conception, 5.12%. The risk of OHSS development in cycles with > 10 oocytes and no conception was 4.43% and with conception, 15.93%. CONCLUSION: The risk of OHSS development increases with the number of oocytes retrieved and with pregnancy. The most suitable limit for predicting OHSS is 10 oocytes; however, due to low sensitivity, it is also necessary to consider other factors when establishing the increased risk of OHSS.

Three protocols for monitoring follicle development in 587 unstimulated cycles of in vitro fertilization and intracytoplasmic sperm injection. A comparison.

OBJECTIVE: To test the adequacy of unstimulated cycles for intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) and to evaluate implantation and pregnancy rates in three monitoring protocols. STUDY DESIGN: A retrospective chart review of 587 patients undergoing IVF and ICSI in unstimulated cycles was performed. In the first group (protocol A), all cycles were monitored by ultrasound only. Human chorionic gonadotropin (hCG) was given when the mean follicle diameter reached 18 mm. In protocol B, hCG was given when serum estradiol (E2) or follicle diameter reached the critical value (0.91 nmol/L and 18 mm). With a smaller follicle diameter, the E2 level had to be higher, and vice versa. In protocol C, hCG was administered when the serum E2 was > 0.49 nmol/L and follicle diameter at least 15 mm. Cycles with positive luteinizing hormone in urine before hCG was given were cancelled. RESULTS: The cancellation rate was lower in protocol C (33/335, 9.8%) than protocol B (42/151, 27.8%) and A (41/101, 40.5%). In protocol C the pregnancy rate per cycle was higher with IVF (n = 219) and ICSI (n = 116) cycles (10.5% and 12.1%) than when protocol B was used (3.8% and 4.3%). The pregnancy rate per transfer was highest when protocol C was used in the IVF (23/105, 21.9%) and ICSI group (14/53, 26.4%). CONCLUSION: Unstimulated cycles monitored by serum E2, urinary luteinizing hormone and ultrasound can produce an acceptable pregnancy rate after IVF and ICSI.

Intra-oral PTH administration promotes tooth extraction socket healing.

Intermittent parathyroid hormone (PTH) administration increases systemic and craniofacial bone mass. However, the effect of PTH therapy on healing of tooth extraction sites is unknown. The aims of this study were to determine the effect of PTH therapy on tooth extraction socket healing and to examine whether PTH intra-oral injection promotes healing. The mandibular first molars were extracted in rats, and subcutaneous PTH was administered intermittently for 7, 14, and 28 days. In a second study, maxillary second molars were extracted, and PTH was administered by either subcutaneous or intra-oral injection to determine the efficacy of intra-oral PTH administration. Healing was assessed by micro-computed tomography and histomorphometric analyses. PTH therapy accelerated the entire healing process and promoted both hard- and soft-tissue healing by increasing bone fill and connective tissue maturation. PTH therapy by intra-oral injection was as effective as subcutaneous injection in promoting tooth extraction socket healing. The findings suggest that PTH therapy promotes tooth extraction socket healing and that intra-oral injections can be used to administer PTH.

Configuration of maternal and paternal chromatin and pertaining microtubules in human oocytes failing to fertilize after intracytoplasmic sperm injection.

The microtubules and chromosomes of 180 human oocytes failing to fertilize after intracytoplasmic sperm injection were observed in order to establish how sperm chromatin and sperm astral microtubule configuration is related to the phases of oocyte cell cycle, and to find the defects in those structures causing fertilization arrest. As many as 125 (69%) oocytes were arrested at metaphase II. In one-fourth of them, damages of the second meiotic spindle were noted. In their cytoplasm intact sperm were found in 38 (30%) cases, a swollen sperm head in 36 (29%) and prematurely condensed sperm chromosomes (G1-PCC)-a result of active mitosis promoting factor (MPF)-in 51 (41%) cases. G1-PCC were mostly (73%) surrounded by the bipolar paternal spindle instead of astral microtubules. A male pronucleus was never presented in metaphase II oocytes. In 19 (11%) oocytes, arrested at anaphase II, no intact sperm were found. As many as 9 (47%) oocytes contained sperm in G1-PCC form, which proves that anaphase II oocytes mostly retain active MPF, despite oocyte activation. As many as 78% of 36 monopronucleate oocytes contained sperm, with delay in the process of sperm nucleus decondensation. Sperm in G1-PCC form and a bipolar paternal spindle were never found in monopronucleate oocytes. From this we conclude that sperm that does not activate the oocyte may continue decondensing the chromatin, but the oocyte prevents male pronucleus formation before the female one, mostly by causing PCC in the sperm and by duplicating the sperm centrosome. Mol. Reprod. Dev. 55:197-204, 2000.

[Evaluation of embryotoxicity of materials used in the IVF laboratory]. / Procjena embriotoksicnosti materijala upotrebljavanih u radu IVF laboratorija.

It has been established that the quality of water for the preparation of culture media has an essential influence on the percentage of two cell-stage mouse embryos developing successfully until the blastocyst stage. The quality of media prepared with bidistilled water (p less than 0.001) is inferior to that prepared with bidestilled demineralized water (p less than 0.02). Best results were attained by using the medium prepared with Nanopure water (Barnstead), in which 73.2% of embryos developed into blastocysts. There was no statistically significant difference between this medium and the commercial liquid medium (Sigma), in which 83.1% of embryos developed into blastocysts. The age of the medium and the percentage of added serum have no statistically significant influence on the outcome of the test. Contact of the medium with the syringe containing a black rubber piston proved explicitly embryotoxic (p less than 0.001).

Effect of neuroleptics and of potential new antipsychotic agents (MJ 13859-1 and MJ 13980-1) on a monkey model of tardive dyskinesia.

Two adult female cebus apella monkeys with persistent tardive dyskinesia (TD) were given acute i.m. injections of reference neuroleptics (chlorpromazine, haloperidol, thioridazine, and clozapine) or of potential new antipsychotic agents (MJ 13859-1 and MJ 13980-1). The drugs were assessed for their ability to modify TD symptoms or to produce acute neurologic reactions. Effects of three doses of MJ 13859-1 administered orally were also examined. At the doses used, thioridazine and clozapine had little or no effect. Chlorpromazine, haloperidol, MJ 13859-1 and MJ 13980-1 reduced or abolished TD and concomitantly produced hypokinesia, akinesia, mask expression, trembling, and reduced response to stimuli. Haloperidol also produced a mildly abnormal posture. In addition to the above effects, MJ 13859-1 also produced "slow motion" movement, sustained bizarre postures, sudden falls, and episodes of severe rigidity with trembling.

Tolerance and limited cross-tolerance to the effects of N, N-dimethyltryptamine (DMT) and lysergic acid diethylamide-25 (LSD) on food-rewarded bar pressing in the rat.

Lysergic acid diethylamide=25 (LSD) and N,N-dimethyltryptamine (DMT) abolish food-rewarded, fixed-ratio bar pressing by rats in a dose-related fashion. Adult male Holtzman rats trained to press a bar (respond) for milk reward on a 4-response fixed-ratio schedule were given i.p. injections of 3.2 or 10 mg/kg of DMT every 2 hours for 21 days. Every 24 hours the animals were placed in operant chambers for 30 minutes before a scheduled injection and were left in the chambers for 30 to 80 minutes after. During the first week of chronic treatment, daily bar pressing worsened progressively until the 6th day of the series, at which time rats in the 10 mg/kg group did not bar press at all. As the chronic injections continued, rates of bar pressing gradually increased until responding was not disrupted at all by an injection of DMT. Rats in the 3.2 mg/kg group showed cross-tolerance to an injection of LSD (0.1 mg/kg). Another group of rats was made partially tolerant to the disruptive effects of LSD (0.1 mg/kg i.p.) on bar pressing with a series of injections given once per day for 21 days and then three times per day for the next 4 days. Cross tolerance was not demonstrated to a challenge injection of 10 mg/kg of DMT. The LSD injections were continued for another 3 to 5 days until the animals were completely tolerant to the LSD. They then displayed cross-tolerance to 3.2 mg/kg of DMT.

A monkey model of tardive dyskinesia (TD): evidence that reversible TD may turn into irreversible TD.

Three Cebus apella monkeys were treated with biweekly injections of fluphenazine enanthate for 1 year. Two distinct motor syndromes were produced. The first consisted of acute dystonic, dyskinetic, parkinsonian, and akathisia-like reactions, which worsened after each injection, were not exacerbated by drug withdrawal, and could be abolished or prevented with benztropine. The second appeared after cessation of neuroleptic treatment and consisted of abnormal movements very similar to those of patients with tardive dyskinesia (TD). This syndrome was abolished completely by reinstitution of fluphenazine treatment. One monkey was given a second and third course of fluphenazine, each course lasting 4 weeks. The second syndrome was seen after but not during each course and remained robust for a longer period after the third than after the first or second courses of treatment. We conclude that the first syndrome is analogous to the early appearing extrapyramidal symptoms of neuroleptic-treated patients and that, according to preliminary evidence, reversible TD may turn into irreversible TD with continued periods of on and off neuroleptic treatment.