Assisted reproduction - principles, procedures, and their effectiveness.

Assisted reproduction is an integral part of infertility treatment. The basic method is in vitro fertilisation (IVF), and intracytoplasmic sperm injection into the oocyte, cryopreservation of sperm, oocytes and embryos is also commonly used. The proportion of embryos tested with preimplantation genetic testing (PGT) for chromosomal aneuploidy or defects in specific genes is increasing. The use of donated oocytes is also significant, particularly because of the depletion of ovarian reserve in women over 40 years of age. The effectiveness of treatment is always very fundamentally dependent on the age of the woman. Assisted reproductive treatment is very well available in the Czech Republic, as is fertility preservation by sperm/oocyte freezing.

ART in Europe, 2019: results generated from European registries by ESHRE†.

STUDY QUESTION: What are the data and trends on ART and IUI cycle numbers and their outcomes, and on fertility preservation (FP) interventions, reported in 2019 as compared to previous years? SUMMARY ANSWER: The 23rd ESHRE report highlights the rising ART treatment cycles and children born, alongside a decline in twin deliveries owing to decreasing multiple embryo transfers; fresh IVF or ICSI cycles exhibited higher delivery rates, whereas frozen embryo transfers (FET) showed higher pregnancy rates (PRs), and reported IUI cycles decreased while maintaining stable outcomes. WHAT IS KNOWN ALREADY: ART aggregated data generated by national registries, clinics, or professional societies have been gathered and analyzed by the European IVF-Monitoring (EIM) Consortium since 1997 and reported in a total of 22 manuscripts published in Human Reproduction and Human Reproduction Open. STUDY DESIGN, SIZE, DURATION: Data on medically assisted reproduction (MAR) from European countries are collected by EIM for ESHRE each year. The data on treatment cycles performed between 1 January and 31 December 2019 were provided by either national registries or registries based on initiatives of medical associations and scientific organizations or committed persons in one of the 44 countries that are members of the EIM Consortium. PARTICIPANTS/MATERIALS, SETTING, METHODS: Overall, 1487 clinics offering ART services in 40 countries reported, for the second time, a total of more than 1 million (1 077 813) treatment cycles, including 160 782 with IVF, 427 980 with ICSI, 335 744 with FET, 64 089 with preimplantation genetic testing (PGT), 82 373 with egg donation (ED), 546 with IVM of oocytes, and 6299 cycles with frozen oocyte replacement (FOR). A total of 1169 institutions reported data on IUI cycles using either husband/partner's semen (IUI-H; n = 147 711) or donor semen (IUI-D; n = 51 651) in 33 and 24 countries, respectively. Eighteen countries reported 24 139 interventions in pre- and post-pubertal patients for FP, including oocyte, ovarian tissue, semen, and testicular tissue banking. MAIN RESULTS AND THE ROLE OF CHANCE: In 21 countries (21 in 2018) in which all ART clinics reported to the registry 476 760 treatment cycles were registered for a total population of approximately 300 million inhabitants, allowing the best estimate of a mean of 1581 cycles performed per million inhabitants (range: 437-3621). Among the reporting countries, for IVF the clinical PRs per aspiration slightly decreased while they remained similar per transfer compared to 2018 (21.8% and 34.6% versus 25.5% and 34.1%, respectively). In ICSI, the corresponding PRs showed similar trends compared to 2018 (20.2% and 33.5%, versus 22.5% and 32.1%) When freeze-all cycles were not considered for the calculations, the clinical PRs per aspiration were 28.5% (28.8% in 2018) and 26.2% (27.3% in 2018) for IVF and ICSI, respectively. After FET with embryos originating from own eggs, the PR per thawing was at 35.1% (versus 33.4% in 2018), and with embryos originating from donated eggs at 43.0% (41.8% in 2018). After ED, the PR per fresh embryo transfer was 50.5% (49.6% in 2018) and per FOR 44.8% (44.9% in 2018). In IVF and ICSI together, the trend toward the transfer of fewer embryos continues with the transfer of 1, 2, 3, and ≥4 embryos in 55.4%, 39.9%, 2.6%, and 0.2% of all treatments, respectively (corresponding to 50.7%, 45.1%, 3.9%, and 0.3% in 2018). This resulted in a reduced proportion of twin delivery rates (DRs) of 11.9% (12.4% in 2018) and a similar triplet DR of 0.3%. Treatments with FET in 2019 resulted in twin and triplet DR of 8.9% and 0.1%, respectively (versus 9.4% and 0.1% in 2018). After IUI, the DRs remained similar at 8.7% after IUI-H (8.8% in 2018) and at 12.1% after IUI-D (12.6% in 2018). Twin and triplet DRs after IUI-H were 8.7% and 0.4% (in 2018: 8.4% and 0.3%) and 6.2% and 0.2% after IUI-D (in 2018: 6.4% and 0.2%), respectively. Eighteen countries (16 in 2018) provided data on FP in a total number of 24 139 interventions (20 994 in 2018). Cryopreservation of ejaculated sperm (n = 11 592 versus n = 10 503 in 2018) and cryopreservation of oocytes (n = 10 784 versus n = 9123 in 2018) were most frequently reported. LIMITATIONS, REASONS FOR CAUTION: Caution with the interpretation of results should remain as data collection systems and completeness of reporting vary among European countries. Some countries were unable to deliver data about the number of initiated cycles and/or deliveries. WIDER IMPLICATIONS OF THE FINDINGS: The 23rd ESHRE data collection on ART, IUI, and FP interventions shows a continuous increase of reported treatment numbers and MAR-derived livebirths in Europe. Although it is the largest data collection on MAR in Europe, further efforts toward optimization of both the collection and the reporting, from the perspective of improving surveillance and vigilance in the field of reproductive medicine, are awaited. STUDY FUNDING/COMPETING INTEREST(S): The study has received no external funding and all costs are covered by ESHRE. There are no competing interests.

Solved problems of reproductive medicine in the Czech Republic 2020.

INTRODUCTION: During the 30th symposium of assisted reproduction held on November 11, 2020 in Brno, the solved problems in reproductive medicine in the Czech Republic in 2020 were presented. The selected topics have concerned not only current issues in the field of clinical embryology and genetics as well as gynecology, but also legislation and ethics. Discussed topics: 1. How much time does the doctor have in the CAR (centrum of assisted reproduction) outpatient clinic per patient and how does the embryologist communicate with clients? 2. Reproduction and PGT-M in oncology patients and patients at risk with hereditary oncogenic mutations. 3. Non-invasive genetic testing of embryos from culture medium. 4. Genome editing. 5. What is the need to monitor hormonal levels in stimulation protocols? 6. Monitoring and embryo selection for transfer/kryo. 7. Is it time to change the law on donor remuneration? METHODS: The topics were prepared in advance by authorized members of our company with the task of elaborating theses, which they presented in a separate conference block. The presentation and the discussion were broadcast directly from the broadcast studio at Hotel International via an online connection. After the conference, all discussion topics and comments were incorporated. CONCLUSION: The work presents the state of the solved problems of reproductive medicine in the Czech Republic.

Association between latent toxoplasmosis and fertility parameters of men.

BACKGROUND: About a third of people in the world are infected with Toxoplasma gondii. This parasite has been found in the reproductive organs and semen of males of many animal species as well as humans. The effects of toxoplasmosis on sperm count, motility and morphology were confirmed in rats. A higher prevalence of toxoplasmosis has been observed in infertile men. On the other hand, no significant effect of infection on semen parameters in men was found in one already published study. OBJECTIVES: To compare the prevalence of toxoplasmosis in men with and without semen pathology and to examine in detail the possible impact of infection on semen volume, sperm count, motility and morphology. MATERIALS AND METHODS: The pre-registered cross-sectional study included 669 men who visited the Centre for Assisted Reproduction in Prague from June 2016 until June 2018. RESULTS: The incidence of fertility problems was significantly higher in the 163 Toxoplasma-infected men (48.47%) than in the 506 Toxoplasma-free men (42.29%), τ = 0.049, P = 0.029. After correction for multiple tests, we found significantly lower sperm concentration, concentration of progressively motile sperm, and concentration of non-progressively motile sperm in Toxoplasma-positive men than in Toxoplasma-negative men using partial Kendall correlation with age controlled. In addition, toxoplasmosis correlated with sperm quality in smokers but not in non-smokers. DISCUSSION AND CONCLUSION: Our results suggest that latent toxoplasmosis affects certain semen parameters (sperm count and motility), but does not seem to affect sperm morphology and semen volume. Impairment of semen parameters may be either a side effect of the presence of Toxoplasma gondii in male reproductive organs or a product of manipulation activity of the parasite aimed to increase the efficiency of the sexual route of its transmission. Tobacco smoking also appears to exacerbate the negative impact of toxoplasmosis on semen parameters.

Male-to-Female Presumed Transmission of Toxoplasmosis Between Sexual Partners.

Toxoplasmosis is one of the most widespread human parasitoses in developed countries. Sexual transmission has been confirmed in several animal species, and indirect evidence suggests it may occur in humans. We compared the seropositivity to Toxoplasma gondii in couples who visited the Center for Assisted Reproduction in Prague from June 2016 to June 2018 and analyzed various risk factors including the serological status of sexual partner. By comparing the risk factors in men and women, we tested the hypothesis of male-to-female sexual transmission of toxoplasmosis. The prevalence of toxoplasmosis in women with infected male partners (25.6%; n = 156) was higher than in women with uninfected male partners (18.2%; n = 477; P = 0.045). Therefore, a partner's seropositivity seems to be a risk factor for infection in women (n = 593; prevalence ratio = 1.418; P = 0.045) but not in men (n = 573; prevalence ratio = 1.058; P = 0.816). Our results support the hypothesis of the sexual transmission of T. gondii from men to women. The risk may seem relatively low, but transmission can occur during unprotected sexual intercourse, which may be at the time of conception. Because of the risk of congenital toxoplasmosis, a lower risk of infection than that observed in our study can represent a serious health problem.

Recurrent Microdeletions at Xq27.3-Xq28 and Male Infertility: A Study in the Czech Population.

BACKGROUND: Genetic causes of male infertility are hypothesized to involve multiple types of mutations, from single gene defects to complex chromosome rearrangements. Recently, several recurrent X-chromosome microdeletions (located in subtelomeric region of the long arm) were reported to be associated with male infertility in Spanish and Italian males. The aim of our study was to test their prevalence and infertility association in population of men from the Czech Republic. METHODS: 107 males with pathological sperm evaluation resulting in nonobstructive infertility were compared to 131 males with normal fecundity. X-chromosome microdeletions were assessed by +/- PCR with three primer pairs for each region Xcnv64 (Xq27.3), Xcnv67 (Xq28) and Xcnv69 (Xq28). The latter microdeletion was further characterized by amplification across the deleted region, dividing the deletion into three types; A, B and C. RESULTS: We detected presence of isolated Xcnv64 deletion in 3 patients and 14 controls, and Xcnv69 in 3 patients and 6 controls (1 and 1 patient vs.4 and 1 control for types A and B respectively). There was one control with combined Xcnv64 and Xcnv69 type B deletions, and one patient with combination of Xcnv64 and Xcnv69 type C deletions. The frequency of the deletions was thus not higher in patient compared to control group, Xcnv64 was marginally associated with controls (adjusted Fisher´s exact test P = 0.043), Xcnv69 was not associated (P = 0.452). We excluded presence of more extensive rearrangements in two subjects with combined Xcnv64 and Xcnv69 deletions. There was no Xcnv67 deletion in our cohort. CONCLUSION: In conclusion, the two previously reported X-linked microdeletions (Xcnv64 and Xcnv69) do not seem to confer a significant risk to impaired spermatogenesis in the Czech population. The potential clinical role of the previously reported patient-specific Xcnv67 remains to be determined in a larger study population.

Proliferative potential and phenotypic analysis of long-term cultivated human granulosa cells initiated by addition of follicular fluid.

PURPOSE: The aim of this study was to develop and optimize a strategy for long-term cultivation of luteinizing human granulosa cells (GCs). METHODS: GCs were cultivated in DMEM/F12 medium supplemented with 2% fetal calf serum. In vitro proliferation of GCs was supported by follicular fluid as well as FSH and growth factors. RESULTS: The cultured GCs were maintained for 45 days with a doubling time of 159 ± 24 h. GCs initiated by the addition of follicular fluid and cultivated under low serum conditions reached 10 ± 0.7 population doublings. GCs maintain the typical phenotypic expression and the telomere length according to specific culture conditions. CONCLUSION: Our present study has demonstrated that GCs can be maintained in vitro for at least 45 days and this cell model can be beneficial when studying hormonal regulation associated with follicular maturation and preparation of oocytes for fertilization.

Development of ovarian hyperstimulation syndrome: interrogation of key proteins and biological processes in human follicular fluid of women undergoing in vitro fertilization.

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication and potentially life-threatening condition resulting from excessive ovarian stimulation during assisted reproductive technologies. Our aim was to identify candidate proteins in follicular fluid (FF) using various proteomic approaches which may help to identify patients at risk of OHSS. We analysed the proteome alterations in FF from patients suffering from severe forms of OHSS (OHSS+) compared with a control group of women without or with only mild signs of OHSS (OHSS-). The 12 abundant proteins of FF were removed using an immunoaffinity system. Pools of remaining depleted proteins were applied to the two-dimensional (2D) electrophoresis and 2D liquid chromatography and proteins in differentially expressed protein spots/fractions were identified by mass spectrometry. Among a total of 19 candidate proteins differentially expressed (P< 0.05) between OHSS+ and OHSS- FF samples, three proteins, namely ceruloplasmin, complement C3 and kininogen-1, were found using both 2D techniques. Computer modelling highlighted the important role of kininogen-1 as an anchor for mediated interactions with other identified proteins including ferritin light chain and ceruloplasmin, hepatocyte growth factor-like protein, as well as complement C3 and gelsolin, thus linking various biological processes including inflammation and angiogenesis, iron transport and storage, blood coagulation, innate immunity, cell adhesion and actin filament polymerization. The delineation of such processes may allow the development of informed corrective therapeutic intervention in patients at risk of OHSS and a set of key proteins of the FF may be helpful as potential biomarkers for monitoring IVF therapy.

Concentrations of sRAGE in serum and follicular fluid in assisted reproductive cycles--a preliminary study.

BACKGROUND: To investigate inflammatory processes during ovarian hyperstimulation, we have studied the soluble receptor for advanced glycation end products (sRAGE) levels in sera and follicular fluids of women undergoing in vitro fertilisation (IVF) cycle. METHODS: A total of thirty-three women undergoing IVF treatment were recruited, the number of follicles in investigated IVF cycles was 18 +/- 10 (mean +/- SD), oocytes 12 +/- 8, and the clinical pregnancy rate was 42% (14/33). The control group of serum samples was comprised of 35 healthy female blood donors. Sera and follicular fluids were examined for sRAGE levels by enzyme-linked immunosorbent assay (sRAGE ELISA, Quantikine, R&D Systems). RESULTS: Serum levels of sRAGE in women after ovarian hyperstimulation and induction of ovulation (1039 +/- 493 pg/mL) were significantly lower than in healthy blood donors (1535 +/- 438 pg/mL), p = 0.045. Follicular sRAGE levels (4355 +/- 1100 pg/mL) were significantly higher than serum levels (1039 +/- 493 pg/ml), p < 0.001. Serum sRAGE levels showed significant negative correlation with the number of stimulated follicles (r = -0.71, p = 0.01) and retrieved oocytes (r = -0.54, p = 0.048). Women who successfully conceived after the IVF showed significantly higher sRAGE levels in follicular fluid (4595 +/- 925 pg/mL) compared to women who did not conceive (3986 +/- 806 pg/mL), p = 0.031. CONCLUSIONS: Concentration of sRAGE in follicular fluid is several-fold higher compared to serum and most other biological fluids investigated until this time. It supports the hypothesis that mammalian ovulation can be compared to an inflammatory event. A significant negative correlation of serum sRAGE with the yield of follicles and oocytes, together with the high follicular sRAGE levels, in particular in women who conceive after the IVF, could be explained by the essential outflow of sRAGE to the follicular compartment.

Proteome mining of human follicular fluid reveals a crucial role of complement cascade and key biological pathways in women undergoing in vitro fertilization.

In vitro fertilization (IVF) is fraught with problems and currently proteomics approaches are being tried out to examine the microenvironment of the follicle in order to assess biological and immunological parameters that may affect its development. Additionally, better understanding of reproductive process may help increase IVF birth rate per embryo transfer and at the same time avoid spontaneous miscarriages or life threatening conditions such as ovarian hyperstimulation syndrome. The primary aim of this study was to search for specific differences in protein composition of human follicular fluid (HFF) and plasma in order to identify proteins that accumulate or are absent in HFF. Depletion of abundant proteins combined with multidimensional protein fractionation allowed the study of middle- and lower-abundance proteins. Paired comparison study examining HFF with plasma/serum from women undergoing successful IVF revealed important differences in the protein composition which may improve our knowledge of the follicular microenvironment and its biological role. This study showed involvement of innate immune function of complement cascade in HFF. Complement inhibition and the presence of C-terminal fragment of perlecan suggested possible links to angiogenesis which is a vital process in folliculogenesis and placental development. Differences in proteins associated with blood coagulation were also found in the follicular milieu. Several specific proteins were observed, many of which have not yet been associated with follicle/oocyte maturation. These proteins together with their regulatory pathways may play a vital role in the reproductive process.

Successful pregnancy after thermal balloon endometrial ablation followed by in vitro fertilization and embryo transfer.

OBJECTIVE: To describe a case of successful pregnancy after IVF-ET in a patient with previous thermal ablation of the endometrium by uterine balloon therapy (UBT). DESIGN: Case report. SETTING: University hospital. PATIENT(S): Polymorbid patient who received UBT and became pregnant after IVF-ET. INTERVENTION(S): UBT, IVF-ET, and caesarean section. RESULT(S): After UBT, the patient underwent IVF-ET and gave birth to a healthy newborn at 36 weeks' gestation with a birth weight 2900 g and placenta accreta. CONCLUSION(S): Patients who wish to become pregnant after endometrial ablation should undergo preconception consultation and hysteroscopic examination.

Comparison of follicular fluid and serum levels of Inhibin A and Inhibin B with calculated indices used as predictive markers of Ovarian Hyperstimulation Syndrome in IVF patients.

BACKGROUND: Ovarian Hyperstimulation Syndrome (OHSS) is a severe health complication observed in some patients undergoing hormonal stimulation during IVF. Presence of OHSS is often associated with a high count of growing follicles responding to FSH hyperstimulation. However, the number of responding follicles may not be sufficient enough to predict the onset and severity of OHSS. The aim of this study was to find whether follicular fluid (FF) and serum concentrations of Inhibin A and Inhibin B in patients undergoing IVF treatment may serve as a predictor of OHSS status independent of the growing follicles count. METHODS: Serum and follicular fluid of fifty-three women undertaking the IVF program were separated into four groups according to their OHSS status and growing follicles count and analyzed for serum and FF concentrations of Inhibin A and Inhibin B. The resulting data were combined with clinical and demographic data to calculate indices independent of the growing follicles count. RESULTS: Serum Inhibin A and Inhibin B concentrations showed no significant difference between the severe OHSS group and the control group without OHSS. Moreover, the serum concentrations of Inhibin A and Inhibin B were strongly correlated with the growing follicles count. Their concentrations in the high responders group (>18 follicles) were significantly higher (p < 0.00001, p < 0.0001) when compared with normal and low responders (<18 follicles). To suppress the dependence on the growing follicle count, three indices were constructed and calculated. The best association with OHSS status and independence of the growing follicle count was achieved by using the Inhibin B TFF/SBM index calculated as follows: [concentration in FF] x [growing follicle count]/[concentration in serum] x [body mass]. The Inhibin B TFF/SBM index showed a clear difference (p = 0,00433) between the group with severe OHSS and the control group, while showing no apparent correlation with the growing follicle count. CONCLUSION: These observations demonstrated that while neither serum nor FF concentrations of Inhibin A nor Inhibin B can be used as an OHSS predictor independent of the growing follicle count, calculated indices may meet the criteria.

Follicular fluid and serum levels of inhibin A and pregnancy-associated plasma protein A in patients undergoing IVF.

OBJECTIVE: To elucidate transport of intrafollicular proteins Inhibin A and pregnancy-associated plasma protein A (PAPP-A) across the follicular fluid (FF)/blood barrier. DESIGN: A retrospective study. SETTING: IVF lab at a university hospital, academic, and industrial research labs. PATIENT(S): Fifty-five women undertook the IVF program. INTERVENTION(S): Follicular fluid aspirations and analysis, blood sample drawing, and serum analysis. MAIN OUTCOME MEASURE(S): Concentrations of Inhibin A, PAPP-A, and major serum proteins in FF and serum, total amount of PAPP-A, and Inhibin A in FF. RESULT(S): The FF/blood barrier permeability was calibrated using major serum proteins. The FF/serum ratio decreased with the molecular mass of proteins, and their FF and serum concentrations were well correlated. In contrast, concentrations of Inhibin A in paired serum and FF samples showed a weak correlation (r = 0.563), whereas serum and FF concentrations of PAPP-A were independent of each other. The total amount of Inhibin A in FF correlated well with concentrations of Inhibin A in paired serum samples (r = 0.858), whereas the correlation between the total amount of FF PAPP-A and PAPP-A serum concentrations remains poor (r = 0.215). CONCLUSION(S): These observations suggest that at the day of oocyte retrieval, FF is a major source of serum Inhibin A but not of serum PAPP-A.

The cultivation of human granulosa cells.

The major functions of granulosa cells (GCs) include the production of steroids, as well as a myriad of growth factors to interact with the oocyte during its development within the ovarian follicle. Also FSH stimulates GCs to convert androgens (coming from the thecal cells) to estradiol by aromatase. However, after ovulation the GCs produce progesterone that may maintain a potential pregnancy. Experiments with human GCs are mainly focused on the purification of GCs from ovarian follicular fluid followed by FACS analysis or short-term cultivation. The aim of our study was to cultivate GCs for a long period, to characterize their morphology and phenotype. Moreover, we have cultivated GCs under gonadotropin stimulation in order to simulate different pathological mechanisms during folliculogenesis (e.g. ovarian hyperstimulation syndrome). GCs were harvested from women undergoing in vitro fertilization. Complex oocyte-cumulus oophorus was dissociated by hyaluronidase. The best condition for transport of GCs was optimized as short transport in follicular fluid at 37 degrees C. GCs expansion medium consisted of DMEM/F12, 2% FCS, ascorbic acid, dexamethasone, L-glutamine, gentamycine, penicillin, streptomycin and growth factors (EGF, bFGF). GCs transported in follicular fluid and cultivated in 2% FCS containing DMEM/F12 medium supplemented with follicular fluid presented increased adhesion, proliferation, viability and decreased doubling time. Cell viability was 92% and mean cell doubling time was 52 hrs. We have optimized transport and cultivation protocols for long-term cultivation of GCs.