Endometrial Receptivity Analysis - a tool to increase an implantation rate in assisted reproduction.

INTRODUCTION: A successful embryo implantation is crucial for a positive outcome of in vitro fertilization. But there is only a short period during which the endometrium is receptive for embryo, this so called implantation window can be detected by a molecular diagnostic method endometrial receptivity analysis (ERA). OBJECTIVE: To find out the percentage of patients with a non-receptive endometrium in the time of ERA and to learn what part of them got pregnant after the identification of their personalized implantation window. DESIGN: A retrospective study. SETTING: REPROMEDA Biology Park, Centre of Reproductive Medicine and Preimplantation Genetic Diagnosis, Brno. METHODS: A cohort of 85 patients undergoing ERA from August 2015 to October 2018 was studied. 74 patients experienced a previous implantation failure, the average number of preceding unsuccessful frozen embryo transfers was 2,5 in this group, 11 women went through ERA due to the preventive reason before the first FET. In all women one euploid embryo was transferred. 48 patients were prepared either for ERA or FET in a natural menstrual cycle, 37 women in HRT cycle. We were interested in a percentage of non-receptive patients in the time of ERA and wanted to discover what part of non-receptive women got pregnant after the identification of their personal implantation window. The average number of frozen embryo transfers needed to achieve the pregnancy was also calculated. RESULTS: 31 of 85 patients (36.5%) were found to have a non-receptive endometrium. In the natural cycle 13 of 48 (27.1%) were non-receptive: five were pre-receptive, three early receptive, two late receptive and three post-receptive. In the HRT cycle 18 of 37 patients (48.6%) were non-receptive: 12 were pre-receptive, four early receptive, one late receptive, one post-receptive. Personalized FET was done in 26 of total 31 initially non-receptive patients, 18 of them got pregnant (69.2%). In the natural cycle 6 of 11 (54.5%) achieved the pregnancy, in the HRT cycle 12 of 15 women (80.0%) got pregnant. To achieve the clinical pregnancy 1.5 frozen embryo transfer in average was needed. CONCLUSION: A displaced implantation window was found in more than 1/3 of patients undergoing an assisted reproductive treatment. After the personalized FET the clinical pregnancy was noticed in 69.2% of them. This result supports an individual approach to patients in IVF programme besides other at the timing of embryo transfer after the identification of pWOI.

[Complete molar pregancy with development of non-metastatic persistent trophoblastic diseaseA case report, reflection of rules of care and decision-making processes Forensic responsibility and the position of doctor in the general gynecologic and obstetric outpatient practice]. / Mola hydatidosa completa s prechodem do nemetastázující perzistující trofoblastické nemociKazuistika, poucení a úvaha nad pravidly dispenzarizace a rozhodovacích mechanismu Forenzní zodpovednost a postavení spádového gynekologa.

OBJECTIVE: To define the forensic responsibility and the position of doctor in the general gynecologic and obstetric outpatient practice in care of the complete molar pregancy. DESIGN: Case report and review article. SETTING: General gynecologic and obstetric outpatient practice Velké Mezirící; Sanatorium REPROMEDA, Centre of reproductive medicine and preimplantation genetics, Brno; Histopatology department of Hospital Jihlava. CASE REPORT: The changing clinical presentation of complete molar pregnancy with development of non-metastatic gestational trophoblastic disease: management. Subsequent early pregnancies outcome following complete hydatiform molar pregnancy. DISCUSSION: Discussed are the forensic responsibility and the position of doctor in the general gynecologic and obstetric outpatient practice with the collaboration of Trophoblastic Disease Center based on the detail expert knowledges: rules of care and decision-making processes and potential controversies, the pitfalls of the histopathologic diagnosis, the genetics of complete hydatiform mole: new lights on a disease, outpatient follow-up and possibility and the risks of the subsequent pregnancy. CONCLUSION: The conclusion is trying to guide quickly a doctor in the general gynecologic and obstetric outpatient practice in the decision-making processes through the crossings of any situation of the complete molar pregnancy and outpatient follow-up, alternatively with the collaboration of Trophoblastic Disease Center.

[Assisted Reproduction and Preimplantation Genetic Diagnosis in Patients Susceptible to Breast Cancer]. / Asistovaná reprodukce a preimplantacní genetická diagnostika u pacientek ohrozených karcinomem prsu.

BACKGROUND: Assisted reproduction, as well as pregnancy itself, in patients with breast cancer or other hereditary type of cancer, is a widely discussed topic. In the past, patients treated for breast cancer were rarely involved in the discussion about reproductive possibilities or infertility treatment. However, current knowledge suggests, that breast cancer is neither a contraindication to pregnancy, nor to assisted reproduction techniques. On the contrary, assisted reproduction and preimplantation genetic diagnosis methods might prevent the transmission of genetic risks to the fetus. AIM: In this review we summarize data concerning pregnancy risks in patients with increased risk of breast cancer. In addition, we introduce current possibilities and approaches to fertility preservation prior to assisted reproduction treatment as well as novel methods improving the safety of fertility treatment. In the second part of this review, we focus on karyomapping--an advanced molecular genetic tool for elimination of germinal mutations in patients with predisposition to cancer. Moreover, the rapid development of preimplantation genetic diagnosis methods contributes to detection of both chromosomal aneuploidy and causal mutations in a relatively short time-span.

[Guidlines for the good laboratory practice in assisted reproduction - clean environment]. / Smernice ke správné laboratorní praxi pri asistované reprodukci - cistota prostredí

The guidlines are the recommendation for good laboratory practice in embryological laboratories. In this first part the requirements of the clean environment from the point of view of the oocytes, sperm, and embryos protection against infection and from point of view of the embryological laboratory staff health protection were described.

[Activities and responsibilities of workers in embryologic and andrologic laboratories in assisted reproduction centers]. / Cinnosti a odpovednosti pracovníku embryologické a andrologické laboratore v centrech asistované reprodukce.

This paper presents the current status and rules for the laboratory staff activities and their competences in the centers of assisted reproduction. The rules were processed by the members of the Association of Reproductive Embryology (ARE) committee under the current legislation. Committee members of the Czech Sterility and Assisted Reproduction Society and Czech Gynecology and Obstetric Society approved these rules as obligatory for assisted reproduction centres in Czech Republic.

[Current issues of assisted reproduction in the Czech Republic]. / Aktuální otázky asistované reprodukce v Ceské republice.

INTRODUCTION: Participants of the lunch table discussion held during the 22nd symposium of assisted reproduction in Brno discussed some current topics of assisted reproduction. DISCUSSED TOPICS: More than 150 participants at round tables discussed 10 topics: 1. IVF in native AR cycle,2. observation of the embryo development dynamics, 3. evaluation and support of endometrial receptivity,4. increased number of elective single embryo transfers (eSET), 5. transport of gametes and embryos in the Czech Republic and between the Czech Republic and abroad, 6. National registry of assisted reproduction,7. new view on sperm pathology, 8. problems with the SAR membership records, 9. surogacy motherhood and 10. preimplantation genetic diagnosis and preimplantation genetic screening. CONCLUSIONS: All findings were presented in the afternoon session. Some of the topics brought concrete results. Some topics could not be clearly concluded and will be the subject of further discussions. A brief summary of those discussion conclusions presents this paper.

[Theoretic and practical content of the clinical embryology]. / Teoretická a praktická nápln oboru klinická embryologie.

Clinical embryology is in whole Europe respected health care specialization. The specialists in this field are certified by the European Society of Human Reproduction and Embryology (ESHRE). In the Czech Republic the specialization has been formally established by the Government Order No 31/ 2010 after 30 years unformal existence. Concurrently with the wide development of new techniques in assisted reproduction, there is necessary to define the content of clinical embryology as a health care, scientific and educational discipline. The definition authorized by the Committee of Association of Reproductive Embryology (ARE) is given in this article..

[Preimplantation genetic diagnosis (PGD) of chromosomal aberrations using the fluorescence in situ hybridization method (FISH)--introduction to problems, sampling methods and examination techniques]. / Preimplantacní genetická diagnostika (PGD) chromosomálních aberací metodou fluorescencní in situ hybridizace (FISH)--úvod do problematiky, prehled metod pri odberu materiálu a technika vysetrení.

OBJECTIVE: Presentation of preimplantation genetic diagnosis and the set of laboratory processes like aspiration, preparation and evaluation of polar bodies, sperm cells and blastomeres using FISH method (fluorescent in situ hybridization) in ART. DESIGN: Review. SETTING: Sanatorium REPROMEDA, Brno, Veterinary Research Institute, Brno. METHODS: Overview of published data and own clinical experience with the cell aspiration methods, evaluated sample preparation and the proper chromosomes visualisation using FISH method. CONCLUSION: The review brings an overview of conditions and methods including sample obtaining, FISH analysis preparation and implementation, processed during PGD.

[The effect of spermatozoal ultrastructure on the outcome of fertilization in vitro]. / Vliv ultrastruktury spermií na výsledek fertilizace in vitro.

UNLABELLED: The ultrastructure of sperm cells in cumulus oophorus of the human oocyte 20 hrs after in vitro fertilization (IVF) was studied. MATERIALS AND METHODS: Mechanically removed cumular cells were processed for electron microscopy and the spermatozoa deposited in the intracellular spaces as well as those penetrating into follicular cells were evaluated. The samples were divided into three groups. Group I contained cumuli from unfertilized oocytes, group II involved those from fertilized oocytes, but not pregnancy after embryo transfer. Group III was formed by cumuli from fertilized oocytes of patients, where pregnancy had been achieved. RESULTS: In group I numerous anomalies of spermatozoal ultrastructure were found, such as head malformations, incompleted chromatin condensation, large cytoplasmatic droplets, etc. In groups II and III, about 70% normal sperm cells were found. CONCLUSIONS: These findings provide evidence of the important role of the morphological quality of spermatozoa in the failure of in vitro fertilization, also in cases where anomalies could not be detected by the usual methods of sperm examination.

[Prolonged culture of human embryos: comparison of coculture on human tubal epithelium and culture in synthetic media]. / Prodlouzená kultivace lidských embryí: srovnání kokultivace na lidských tubárních epiteliích a kultivace v syntetickém médiu.

UNLABELLED: The aim of this work was to evaluate prolongation of cultivation time of human embryos. The prolongation above the standard limit was implemented (1) by coculture of embryos on a monolayer of human epithelial cells and (2) by using a synthetic medium. MATERIAL AND METHODS: Ovarian stimulation, oocyte recovery, insemination and cultivation up to the pronuclear stage were done in our centre in the usual way. Group I: 104 women, prolonged culture by the coculture method. The zygotes were placed on the monolayer and cultivated for an other 24 or 48 hours. Group II: 249 women, prolonged culture in a synthetic medium. The zygotes were cultivated up to the 2- to 4-cell stage in standard IVF medium, and then put into M3 medium for the next 24 or 48 hours. A transfer of a maximum of 4 embryos was done. RESULTS: In group I in 104 women from 341 zygotes 181 embryos (53.1%) reached the eight- and more than eight-cell stage, and 76 transfers were done. 15 pregnancies were achieved (19.4% pregnancy rate). In group II in 249 patients from 672 embryos 49.7% of them reached 8- and more than 8-cell stage. 51 pregnancies were achieved (22.6 pregnancy rate). In the control group of 250 IVF after 48 hours cultivation 165 transfers (66.0%) were done, and 16.4% pregnancies were achieved, i.e. 6.2% less compared to synthetic medium and 3.3% less than in cocultivation. CONCLUSION: There is evidence of better IVF/ET results in case of prolonged culture time. The experience in our centre has shown the need of reevaluation of the coculture method. The exacting character of its preparation and manipulation will have to be replaced by synthetic media in spite of their high price.

[Assisted hatching--a useful micromanipulation technic in women after repeated failure of embryo transfer]. / Asistovaný hatching--prínosná mikromanipulacní technika pro zeny s opakovane neúspesným embryotransferem.

One reason for the low implantation rate after IVF/ET is the limiting ability of blastocysts to escape from the zona pellucida, and of nidation. This procedure can be assisted by the creation of an artificial opening in the zona pellucida before the transfer (assisted hatching). An artificial slot in the zona of 58 embryos (20 patients) was created mechanically by using Leitz Fluovert microscope and Narishige micromanipulators. From 20 embryo transfers 6 clinical pregnancies resulted (30.0% clin. preg. rate). In 4 cases women with three or more unsuccessful transfers were involved. This method should be used in classical IVF, specially in cases of repeated high quality embryo transfers which failed.

Determination of intracytoplasmic filaments in the chondrocytes of articular cartilage.

Intracytoplasmic filaments were demonstrated in the chondrocytes of articular cartilage of cattle at 82 days post partum and at 3 and 4 years of age using transmission electron microscopy and immunocytochemistry. Samples of cartilage for electron microscopy were processed in a standard way. Immunocytochemical procedures were performed with semithin sections made from the material prepared for electron microscopy after embedding medium and osmium compounds had been eliminated. Vimentin filaments were demonstrated by means of anti-vimentin monoclonal antibody visualized by peroxidase-labelled immunoglobulin. Both techniques showed that intracytoplasmic filaments were situated around the nucleus and only occasionally at the cell periphery, e.g. in cell projections.

[Oocyte removal in conjunction with pelvic microsurgery]. / Oozytenentnahme in Verbindung mit Beckenmikrochirurgie.

The paper evaluates results of gamete transfer into the oviduct or embryos into the uterus in connection with pelvic microsurgery. Between August 1981-December 1989 225 microsurgical operations have been performed in a stimulated cycle. Ovaries have been stimulated by clomiphene-citrate or in combination with human menopausal gonadotropin. The operation was timed 34 hours after application of human chorionic gonadotropin. In a group of 156 women we recovered at least one oocyte. From 77 gamete transfers into the oviduct we achieved 11 pregnancies, i.e. 14.3 per cent. 46 embryo transfers into the uterus resulted in 5 pregnancies, i.e. 10.9 per cent. From the total number of 16 pregnancies we achieved 5 deliveries, 8 clinic abortions and 3 ectopic pregnancies. The connection of sterility microsurgery with oocyte retrieval increases the success rate of operations without another stress of a patient and has also its economic significance.

[Gamete and embryo transfer in conjunction with microsurgical treatment of sterility]. / Gameto- a embryotransfer ve spojení s mikrochirurgickou lécbou sterility.

The authors evaluate the results of the programme of transfer of gametes into the oviduct or of embryos into the uterus in conjunction with microsurgery of sterile women. During the period between August 1981 and December 1988 a total of 211 microsurgical operations were made in stimulated cycles. The ovaries were stimulated with clomiphene citrate or in combination with human menopausal gonadotropin. The operation was made 36 hours after administration of human choriogonadotropic hormone. In 146 women at least one oocyte was obtained. A total of 72 gametotransfers into the oviduct were made and in eight, i. e. 11.1%; pregnancy was achieved. Of 43 embryotransfers five pregnancies were achieved, i. e. 11.6%. A total of 13 pregnancies were achieved with four spontaneous deliveries, seven miscarriages and two extrauterine pregnancies. The combination of selected cases of microsurgery of sterility with the IVF programme increases the rate of successful operations without a further burden for the patient, and it extends the possibilities of sterility treatment.

[Initial experience with cryopreservation of human embryos]. / První zkusenosti s kryokonzervací lidských embryí.

We present our first experience with human embryo cryopreservation. In our hands the best results were obtained with cryopreservation of pronuclear-staged embryos using propanediol as cryoprotectant. In this group 3 embryos out of 4 continued their development after thawing. The advantages of embryo cryopreservation in IVF programs are discussed.

[Developmental capacity of human embryos after cryopreservation in the unicellular zygote stage]. / Vývojová zpusobilost lidských embryí po kryokonzervaci ve stadiu jednobunecné zygoty.

This study deals with the developmental competence of human embryos after cryopreservation at the one-cell stage using propanediol as cryoprotectant. From 11 frozen zygotes, nine underwent cleavage after thawing. The occurrence of multinuclear blastomeres was the most frequent abnormality which, however, can often be observed in fresh embryos as well. The results of an ultrastructural analysis of the developing frozen-thawed embryos, with special attention to markers of embryonic genome expression, did not show any noticeable impairment of the developmental competence of these embryos as compared with parameters previously described for unfrozen embryos. The findings are discussed with particular reference to current freezing policies in human in-vitro fertilization programmes.

[A simple microphotographic method for the evaluation of sperm motility]. / Jednoduchá mikrofotografická metoda pro objektivní hodnocení pohybu spermií.

A simple objective method for examination of sperm quality, both for practical and research purposes, is described. The results obtained show that the evaluation of samples must be performed by 15 minutes after the removal of the spermatozoa from the culture system and by 5 minutes after the placement on the microscope slide, in order to avoid the influence of external conditions on sperm motility. From the comparison of parameters examined (percentage of sperm motility, mean velocity, lateral head displacement, number of waves per second, distribution in individual speed intervals) it follows that media used for sperm incubation (B2 and F 10) do not affect negatively sperm motility and may be exchanged with each other if necessary. The results contribute to the improvement of diagnostic and treatment of andrological disturbances. The study will be used in our programme of treatment of infertile couples.

Specific antibody against the Aujeszky's disease virus in the complex cumulus oophorus-oocyte of sows.

Oocytes collected from sows vaccinated and revaccinated with an inactivated vaccine against Aujeszky's disease (AD) were examined for the presence of specific antibodies of the IgG class against AD virus in the complex cumulus oophorus-oocyte by means of immunocytochemical methods. No specific antibody of the IgG class was detected in the cumulus-oocyte complexes collected from sows before vaccination. On the other hand, the specific IgG antibody was found in all immunocytochemically examined complexes cumulus-oocyte collected from revaccinated sows. The specific antibody of the IgG class against AD virus and the porcine IgG were immunocytochemically visualized under both light and electron microscopes as a fine granular product disseminated in oocytes and follicular cells.

Zona pellucida resistance to sperm penetration before the completion of human oocyte maturation.

Human oocytes exposed to capacitated spermatozoa in vitro when at metaphase of the 1st meiotic division (metaphase I) were not penetrated, even though some subsequently progressed to metaphase of the 2nd meiotic division (metaphase II). When the non-penetrated oocytes that had reached metaphase II during the incubation with spermatozoa were freed from the zona pellucida and reinseminated, two or more pronuclei developed in most of them. By contrast, no penetration was observed when the oocytes were reinseminated in the zona-intact state. When compared with metaphase II oocytes, metaphase I oocytes had a similar zona-binding capacity for spermatozoa, but fewer spermatozoa were found within the zona. These results indicate that the zona pellucida of human oocytes undergoes important maturational changes during the transition from metaphase I to metaphase II. Ultrastructural and previous histochemical findings suggest that these changes involve secretions from both the oocyte and cumulus cells and that the increased zona resistance at metaphase I may be due to relative insufficiency of cumulus cell-secreted 'softening' factors. If the integrity of the cumulus oophorus is disturbed at this stage, this condition becomes irreversible.