Cumulus matrix selection leads to isolation of spermatozoa with better motility, morphology, and lower DNA fragmentation.

The objective of this study was to compare the motility, morphology, and levels of DNA fragmentation of spermatozoa subjected to conventional swim-up or cumulus matrix (CM) sperm selection. Semen samples were collected from 60 normozoospermic men at a private hospital between December 2021 and March 2022. After liquefaction, semen samples were separated into two portions - one part was subjected to conventional swim-up preparation and the remaining spermatozoa were subjected to CM selection. The CM was obtained by mechanical isolation from healthy donor oocytes. Semen analysis and evaluation of sperm were performed according to WHO 6th Edition Laboratory Manual and Kruger's strict criteria, respectively. Sperm DNA fragmentation (SDF, %) of the two preparations was evaluated using Halosperm G2 detection kit (Halotech, Madrid, Spain). Wilcoxon rank-sum test was used to compare the characteristics of spermatozoa obtained by the two preparations. Spermatozoa selected by CM showed significantly better rapidly progressive motility (43.5% vs. 30.6%, respectively, p<0.001), higher percentage of morphologically normal forms (14.0% vs. 9.0%, respectively, p<0.05), and lower levels of SDF (26.0% vs. 45.0%, p<0.05) in comparison to those prepared by conventional swim-up. Moreover, the incidence of multiple sperm defects was considerably lower in the samples that underwent CM selection compared to those that did not (30.0% vs. 49.0%, respectively, p<0.05).The selection by CM significantly increases sperm motility and reduces morphologically abnormal spermatozoa and DNA fragmentation rates in comparison to the conventional swim-up preparation. The application of this selection technique may increase chances of successful IVF outcomes.

Sperm selection by zona adhesion improves assisted reproductive treatment outcomes.

BACKGROUND: Zona pellucida is one of the main selective barriers for the spermatozoa before reaching the oocyte. Using native zona in the sperm selection prior to Intracytoplasmic Sperm Injection (ICSI) has been proven effective but inconvenient approach in In Vitro Fertilisation (IVF) laboratory. The application of autologous solubilised zonae pellucidae in the sperm selection prior to ICSI has not been studied yet. OBJECTIVES: To compare the assisted reproductive treatment (ART) outcomes (implantation, pregnancy, live birth, and miscarriage rates) after ICSI performed with spermatozoa selected on their ability to adhere to immobilised solubilised zonae pellucidae and conventionally selected spermatozoa. MATERIALS AND METHODS: In total, 500 couples fulfilled the inclusion criteria and 368 of them were included in the study. After random allocation, 192 couples had spermatozoa selected by sperm-zona adhesion for ICSI (study group) and 176 patients underwent standard ICSI (control group). In the study group, patients' own zonae were acid solubilised and immobilised on petri dishes. The partner's motile spermatozoa were placed in the dishes and the adhered spermatozoa were used for ICSI. For the control group, the conventional sperm selection by morphological criteria was applied prior ICSI. All women underwent frozen ET with euploid embryos. Chi square test was used to compare the data. RESULTS: The sperm selection by zona adhesion resulted in significantly higher implantation rate (50.4% vs. 37.0%, p = 0.003), clinical pregnancy rate (43.8% vs. 33.3%, p = 0.018) and live birth rate (38.0% vs. 25.9%, p = 0.004) and significantly lower incidence of miscarriage (11.3% vs. 22.2%, p = 0.044) in comparison to the conventional method of the sperm selection. DISCUSSION AND CONCLUSION: The application of solubilised zonae pellucidae in the sperm selection for ICSI benefits ART outcomes in couples with unexplained infertility. Moreover, sperm-zona selection significantly reduces the risk of miscarriages.

Pathomorphological Diagnostic Criteria for Focal Cortical Dysplasias and Other Common Epileptogenic Lesions-Review of the Literature.

Focal cortical dysplasia (FCD) represents a heterogeneous group of morphological changes in the brain tissue that can predispose the development of pharmacoresistant epilepsy (recurring, unprovoked seizures which cannot be managed with medications). This group of neurological disorders affects not only the cerebral cortex but also the subjacent white matter. This work reviews the literature describing the morphological substrate of pharmacoresistant epilepsy. All illustrations presented in this study are obtained from brain biopsies from refractory epilepsy patients investigated by the authors. Regarding classification, there are three main FCD types, all of which involve cortical dyslamination. The 2022 revision of the International League Against Epilepsy (ILAE) FCD classification includes new histologically defined pathological entities: mild malformation of cortical development (mMCD), mild malformation of cortical development with oligodendroglial hyperplasia in frontal lobe epilepsy (MOGHE), and "no FCD on histopathology". Although the pathomorphological characteristics of the various forms of focal cortical dysplasias are well known, their aetiologic and pathogenetic features remain elusive. The identification of genetic variants in FCD opens an avenue for novel treatment strategies, which are of particular utility in cases where total resection of the epileptogenic area is impossible.

Endometrial immune cell ratios and implantation success in patients with recurrent implantation failure.

The objective of this study was to compare the endometrial immune cells quantities and ratios during the mid-luteal phase between women with recurrent implantation failure (RIF) with successful and unsuccessful embryo implantation. For this purpose, endometrial biopsies from 116 women aged between 29 and 46 with history of RIF undergoing Assisted Reproductive Technology (ART) without endometrial pathologies were immunohistochemically stained for CD3 + T-cells, CD4 + T-helpers, CD8 + T-killers, CD14 + monocytes, CD68 + macrophages, CD56 + NK cells and CD79α+ B-cells. Endometrial immune cells quantities and ratios were compared based on the embryo implantation outcome in the subsequent embryo transfer cycle. Spearman correlation analysis and Mann-Whitney U test were used to analyse the obtained data. Patients who experienced successful implantation at the subsequent cycle had significantly lower percentage of CD3 + T cells, and higher ratios of CD4 + /CD8 + , CD4 + /CD3 + and CD68 + /CD3 + than the patients who experienced another failure in implantation. In addition, the ratios of CD3 + /CD14 + , CD79α+ /CD14 + and CD56 + /CD14 + were significantly lower in the successful implantation group than that in the unsuccessful one. A cut off value of CD68 + /CD3 + ratio higher than 0.85 (AUC 0.67, 95% CI 0.56-0.79), CD4 + /CD3 + ratio higher than 0.19 (AUC 0.67, 95% CI 0.56-0.79) and CD4 + /CD8 + ratio higher than 0.43 (AUC 0.62, 95% CI 0.50-0.73) could be predictive factors for successful implantation in RIF patients. Knowledge on the immune cell composition could assist in the evaluation of the endometrial receptivity in RIF patients.

NOTCH1- and CD117-positive stem cells in human endometrium and their implications for successful implantation.

OBJECTIVE: To investigate the quantity of 2 stem cell types in the endometrial stroma of women undergoing in vitro fertilization and their association with steroid hormone signaling and implantation success after embryo transfer. DESIGN: Prospective cohort study. SETTING: Private hospital. PATIENT(S): A total of 109 patients undergoing in vitro fertilization. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Immunohistochemistry staining of endometrial biopsies taken during the midluteal phase using antibodies against NOTCH1 and CD117 was performed. The percentage of endometrial stromal cells positive for these markers was determined. The link of these stem cell percentages with the serum progesterone and estradiol levels and the endometrial expression of their respective receptors were assessed. After embryo transfer, the quantity of stained cells for each marker was also compared according to implantation outcome. RESULT(S): The percentage of NOTCH1+ stromal cells ranged from 0.003%-2.112% (median, 0.062%) and was significantly higher than that of CD117+ cells, which ranged from 0.000%-0.210% (median, 0.020%) (Z = -7.035). The percentage of NOTCH1+ stem cells showed no difference between the studied serum hormone level groups and no relationship with the expression of their receptors in the endometrium. In contrast, the number of CD117+ cells significantly differed between patients with high and low levels of serum progesterone (cutoff, 14.9 ng/mL) and estradiol (cutoff, 135.6 pg/mL). Furthermore, the quantity of CD117+ stem cells was positively correlated with the progesterone receptor (R = 0.277) and estradiol receptor (R= 0.318) expression levels in the endometrium. Although the quantity of NOTCH1+ cells did not differ between the 2 implantation groups, the median percentage of CD117+ cells was significantly higher in patients with successful implantation than in those with unsuccessful implantation (0.03% vs. 0.01%, respectively). The cutoff value for the percentage of CD117+ cells predicting successful implantation was 0.018% (area under the curve, 0.66; 95% confidence interval, 0.56-0.77; sensitivity, 63.1%; specificity, 61.4%). CONCLUSION(S): This study indicates that the quantity of certain stem cell types (CD117+), but not others (NOTCH1+), in the functional endometrium is associated with implantation success and sex hormone signaling during the midluteal phase. These findings highlight the role of CD117+ cells in preparing the endometrium for embryo implantation, and their quantity may be an indirect indicator of endometrial receptivity.

Vertical Transmission of SARS-CoV-2 Infection and Miscarriage in the Second Trimester: Report of an Immunohistochemically Proven Case.

It is an acknowledged fact that SARS-CoV-2 exhibits tropism for the human placenta. A possible mechanism of SARS-CoV-2 entry into host cells is via angiotensin-converting enzyme 2 (ACE2) receptors, which are expressed in trophoblasts, endothelial cells, and macrophages. The present study describes a case of spontaneous miscarriage in the 20th gestational week after maternal SARS-CoV-2 infection. The placenta and various fetal organs were examined for structural alterations and expression of the viral nucleocapsid protein and several immune cell markers via immunohistochemistry (IHC). Histopathological examination of the placenta revealed acute chorioamnionitis, acute subamnionic placentitis, multiple intervillous thrombi, increased fibrinoid deposition, and necrotic changes of the chorionic villi. Immunohistochemistry confirmed the presence of SARS-CoV-2 nucleocapsid protein regions predominantly in the syncytiotrophoblast. Staining of the placental tissue for different markers helped elucidate the distribution of immune cells. Pathomorphological examination of the fetal organs demonstrated changes in microcirculation with the presence of sludge phenomenon and diapedesis haemorrhages, mostly in the lungs, brain, and myocardium. IHC staining of fetal organs revealed expression of SARS-CoV-2 nucleocapsid protein, which was detected to the highest extent in the brain, lungs, and liver. The findings of the present report support the hypothesis of possible vertical transmission of SARS-CoV-2 from mother to fetus.

Hysteroscopy and female infertility: a fresh look to a busy corner.

Hysteroscopy has evolved from the traditional art of examining the uterine cavity for diagnostic purposes to an invaluable modality to concomitantly diagnose and (see and) treat a multitude of intrauterine pathologies, especially in the field and clinics specialising in female reproduction. This article reviews the literature on the most common cervical, endometrial, uterine and tubal pathologies such as chronic endometritis, endometrial polyps, adenomyosis, endometriosis, endometrial atrophy, adhesions, endometrial hyperplasia, cancer, and uterine malformations. The aim is to determine the efficiency of hysteroscopy compared with other available techniques as a diagnostic and treatment tool and its association with the success of in vitro fertilisation procedures. Although hysteroscopy requires an experienced operator for optimal results and is still an invasive procedure, it has the unique advantage of combining great diagnostic and treatment opportunities before and after ART procedures. In conclusion, hysteroscopy should be recommended as a first-line procedure in all cases with female infertility, and a special effort should be made for its implementation in the development of new high-tech procedures for identification and treatment infertility-associated conditions.

Decreased number of p16-positive senescent cells in human endometrium as a marker of miscarriage.

PURPOSE: The aim of this study was to evaluate whether the number of p16-positive cells in the functional layer of the endometrium could be a useful biomarker to identify women with recurrent implantation failure (RIF) undergoing in vitro fertilization (IVF) at risk of miscarriage. METHODS: Immunohistochemical staining was performed in 311 endometrial biopsies taken during mid-luteal phase using antibody against p16INK4A. The percentage of p16-positive cells was determined in luminal, glandular and stromal endometrial cells. After embryo transfer, women were divided into the following groups: unsuccessful embryo implantation (n = 151), miscarriage (n = 66) and live birth (n = 94). The percentage of p16-positive cells in all endometrial compartments was compared among these groups. RESULTS: We found that the percentages of p16-positive glandular and luminal epithelial endometrial cells were significantly higher in patients with live births compared to women with miscarriage (9.3% vs. 2.9%, P < 0.001; and 35.2% vs. 11.7%, P = 0.001, respectively). This tendency was not confirmed in thе stroma. The cut-off values with p16-positive luminal cells lower than 12.5% and p16-positive glandular cells lower than 3.2% could be predictive factors for miscarriage (AUC 0.80 and 0.79; sensitivity 71.3% and 74.5%; specificity 74.2% and 71.2%, respectively). CONCLUSION: A decreased number of senescent p16-positive cells could be involved in the implantation failures and aetiology of recurrent miscarriage. Women with history of RIF with reduced populations of p16-positive cells in the endometrial glandular and luminal epithelium may be at greater risk for unsuccessful implantation and miscarriage. The percentage of p16-positive luminal epithelial cells may be clinically useful as a biomarker of miscarriage.

Re-introduction of Griffon Vulture (Gyps fulvus) in the Eastern Balkan Mountains, Bulgaria - completion of the establishment phase 2010-2020.

The current study presents and analyses the results from the recently completed 11-year-establishment phase, following the start of the local re-introduction of the Griffon Vulture (Gyps fulvus) in Kotlenska Planina SPA and Sinite Kamani Nature Park in the Eastern Balkan Mountains of Bulgaria in the period 2010-2020. As a result of the re-introduction efforts and release of 153 individuals, the Griffon Vulture has been successfully reproducing again in the Eastern Balkan Mountains since 2016, after more than 40-50 years of absence. At 2020, the local population consists of some 80 local and up to 80-115 birds, together with sojourn individuals. Amongst them, 23-25 breeding pairs, located in five different colonies and two more frequently used roosting sites. The current average productivity remains relatively low: 0.41 fledglings/territorial pair and fledging success of 0.61 fledglings/breeding pair between 2016 and 2020, but shows a trend to increase with time and the growing experience of the young locally re-introduced population. The mortality confirmed between 2010-2021 accounts for 33%, mostly due to electrocution as a post-release effect in the first six months following their release. Our data show that the newly established population in the Eastern Balkan Mountains mostly forages on feeding sites, having a comparatively small 95% home range: 281.88 ± 91 km2 and 50% core area: 6.6 ± 2.28 km2 (range 4.7-8.5 km2). We, therefore, consider the establishment phase of the re-introduction of Griffon Vulture in this particular site as successfully completed, but management should continue. Furthermore, the area of the Eastern Balkan Mountains can currently be regarded as a "source" for the species within the source-sink population regulation concept in the national and Balkan context.

Sperm morphology and DNA fragmentation after zona pellucida selection.

Sperm DNA fragmentation (SDF) and sperm morphological defects can negatively affect ART outcomes. Consequently, there is a need for additional semen processing technique that accounts for sperm DNA status and morphology prior to ICSI. The objective was to evaluate the efficacy of an additional zona pellucida adhesion-based sperm selection for obtaining sperm populations with a high percentage of normal morphology and DNA integrity as compared to native semen and routine swim-up preparation. Semen samples from 78 normozoospermic men were subjected to swim up and placed in petri dishes coated with 48 acid-solubilized zonae pellucidae. Sperm DNA fragmentation and morphology were assessed in the native semen, the swim-up samples, and the zona-adhered spermatozoa from each patient. The mean sperm DNA fragmentation of the zona-selected spermatozoa (3.5 ± 0.7%) was significantly lower than the swim-up samples (15.3 ± 5.2%) (P < 0.001) and native semen (24.9 ± 7.1%) (P < 0.001). All of the samples had lower levels of DNA damage after additional selection by zona pellucida adhesion. Significantly higher percentage of sperm with normal morphology was observed after zona-adhesion selection (11.4 ± 3.9%) when compared to the swim-up samples (8.9 ± 4.3%) (P < 0.001) or the native semen (5.3 ± 3.2%) (P < 0.001). In 94% of the samples, the percentage of spermatozoa with normal morphology increased after the additional zona selection. This study demonstrates that sperm selection by additional zona-adhesion technique yields a significantly higher percentage of spermatozoa with normal morphology as well as a significantly decreased level of DNA fragmentation when compared to the native semen and the swim-up-only prepared samples. LAY SUMMARY: High level of DNA folding known as sperm DNA fragmentation (SDF) inside each sperm and defects in the shape, size, and structure of the sperm can negatively affect assisted reproduction treatment (ART) outcomes. Consequently, there is a need for additional semen processing techniques that account for sperm quality prior to ART. Our team designed a simple technique using proteins from the coat around the egg (zona pellucida) to enhance sperm selection procedures based on natural sperm-egg interactions. Using this technique in combination with the most common techniques used in ART yields a significantly higher percentage of sperm with normal shape, size, and structure and a decreased level of DNA fragmentation. This sperm zona-selection technique would be beneficial if introduced in the ART practice to yield sperm with higher fertilization potential.

Unbalanced human embryos secrete more hyperglycosylated human chorionic gonadotrophin (hCG-H) than balanced ones.

PURPOSE: The aim of this study was to compare the levels of hyperglycosylated human chorionic gonadotropin (hCG-H) secreted from balanced and unbalanced human embryos. METHODS: Single-step culture media samples from 155 good quality embryos, derived from 90 good prognosis patients undergoing intracytoplasmic sperm injection (ICSI), were collected on the fifth day of embryo cultivation. All embryos were tested by next-generation sequencing (NGS) technique. The hCG-H levels in the culture media were evaluated by ELISA kit (Cusabio Biotech, CBS-E15803h) according to the manufacturer's instructions. Statistical analysis was performed using SPSS v.21 (IBM Corp., Armonk, NY, USA). RESULTS: The NGS analysis revealed that 36% of the embryos (n = 56) were balanced, and 64% of the embryos were unbalanced (n = 99). The presence of hCG-H was confirmed in all embryo culture media samples but was absent in the negative control. In addition, hCG-H concentration was significantly higher in the culture media from unbalanced embryos compared with the balanced ones (0.72 ± 0.30 mIU/ml vs. 0.62 ± 0.12 mIU/ml, p = 0.02, respectively). Furthermore, the mean levels of hCG-H were significantly increased in the samples from embryos with multiple abnormalities. Finally, the highest levels of hCG-H were expressed from embryos with monosomy of chromosome 11 (1.28 ± 0.04 mIU/ml) and those with trisomies of chromosomes 21 (2.23 mIU/ml) and 4 (1.02 ± 0.35 mIU/ml). CONCLUSION: Our results suggest that chromosomal aberrations in human embryos are associated with an increased secretion of hCG-H. However, hCG-H concentration in embryo culture media as a single biomarker is not sufficient for an accurate selection of balanced embryos.

Impact of sperm characteristics on time-lapse embryo morphokinetic parameters and clinical outcome of conventional in vitro fertilization.

BACKGROUND: Sperm abnormalities may negatively affect embryo development. OBJECTIVES: To determine the influence of sperm abnormalities (morphology, motility, DNA fragmentation) on embryo morphokinetic variables and clinical outcome of conventional IVF. MATERIALS AND METHODS: Participants were 86 couples undergoing in vitro fertilization (IVF). Sperm morphology was evaluated according to the strict criteria proposed by Kruger/Tygerberg. CASA system was applied for sperm motility assessment. Sperm DNA fragmentation was assessed by the chromatin structure assay (SCSA). Morphokinetic parameters were determined in 223 embryos obtained from conventional IVF only and cultured in a single-step medium using time-lapse imaging technology. RESULTS: Time-lapse variables from the initial embryo development, such as time of pronuclei fading (tPNf) and time for two cells (t2), were those more strongly related with abnormalities of sperm motility, morphology, and DNA fragmentation. Sperm morphological abnormalities rather than sperm motility were more closely associated with embryo morphokinetics. Sperm head defects were mainly correlated with the last stages of embryonic development (t9 to tHB), sperm midpiece defects with intermediate cleaving embryos (t5-t9), and sperm tail defects with the initial stages of embryonic development (tPNa-t4). Excess residual cytoplasm was positively correlated with all embryo morphokinetic parameters except t2 and tM. Absence of acrosomes, pinheads, coiled tails, and multiple sperm morphological defects correlated negatively with time-lapse embryo morphokinetic variables. DISCUSSION: A large number of sperm-related variables, including frequency of specific morphological defects, morphological indexes, DNA fragmentation and motility, and time-lapse embryo variables, such as time intervals based mainly of 15 time points were recorded. CONCLUSION: There were strong associations between specific sperm defects of the head, midpiece, and tail with certain stages of embryonic development from observation of pronuclei to the hatched blastocyst. Coiled tail, cumulative head defects, and multiple abnormalities index (MAI) were associated both with embryo morphokinetics and the implantation success.

Successful Pregnancy following Mixed Double Embryo Transfer in a Patient with Variable Window of Implantation.

The process of embryo implantation is carried out during the receptive stage of the endometrium in the midluteal phase of the menstrual cycle, known as window of implantation (WOI). It has been assumed that the WOI is not a constant variable in all women and the determination of its displacement is of crucial importance, especially for patients with recurrent implantation failure (RIF). Furthermore, in rare cases it could have different duration and position in the menstrual cycle even in the same woman but during different periods. Here, we report a 37-year-old woman with RIF, who was previously classified as idiopathic but has now been diagnosed as having a variable WOI. This interpretation was done after the performance of immunohistochemical and histomorphological analyses of endometrial biopsies taken in the midluteal phase during three sequential menstrual cycles. In order to solve the problem with pinpointing a variable WOI, a specific type of embryo transfer, called mixed double embryo transfer (MDET), was done where one Day 3 and one Day 5 good quality embryos were transferred simultaneously 7 days after ovulation. A viable single pregnancy was confirmed by ultrasound scan and a healthy girl was born. This case showed a unique approach in overcoming the problem in RIF patients with variable WOI.

Vulture mortality resulting from illegal poisoning in the southern Balkan Peninsula.

A study was carried out to determine the incidents of illegal poisoning of griffon vulture (Gyps fulvus), Egyptian vulture (Neophron percnopterus), black vulture (Aegypius monachus), and bearded vulture (Gypaetus barbatus) in the southern Balkan Peninsula between 1982 and 2017. A total of 38 poisoning cases affecting 224 vultures were analyzed to identify their causes and the primary target species for poisoning. Nine different compounds were used in these incidents and the most frequently applied were strychnine, carbamate, and organophosphoros compounds. The poison used to kill gray wolf had the most significant collateral damage to the vulture populations in comparison to the other investigated reasons. It was the primary cause of 60% of all registered vulture poisoning events in the southern Balkan Peninsula during the last 36 years. Establishing permanent feeding sites for vultures in areas with wolves appears to be an effective way to minimize the risk of poisoning. There is a pressing need for the development of an appropriate conservation practice taking into consideration relationships among the main and casual target species for poisoning as an essential element in conjunction with the human activities.

Mixed double-embryo transfer: A promising approach for patients with repeated implantation failure.

OBJECTIVE: The purpose of this study was to evaluate the efficacy of frozen mixed double-embryo transfer (MDET; the simultaneous transfer of day 3 and day 5 embryos) in comparison with frozen blastocyst double-embryo transfer (BDET; transfer of two day 5 blastocysts) in patients with repeated implantation failure (RIF). METHODS: A total of 104 women with RIF who underwent frozen MDET (n=48) or BDET (n=56) with excellent-quality embryos were included in this retrospective analysis. All frozen embryo transfers were performed in natural cycles. The main outcome measures were the implantation rate, clinical pregnancy rate, multiple pregnancy rate, and miscarriage rate. These measures were compared between the patients who underwent MDET or BDET using the chi-square test or the Fisher exact test, as appropriate. RESULTS: The implantation and clinical pregnancy rates were significantly higher in patients who underwent MDET than in those who underwent BDET (60.4% vs. 39.3%, p=0.03 and 52.1% vs. 30.4%, p=0.05, respectively). A significantly lower miscarriage rate was observed in the MDET group (6.9% vs. 10.7%, p=0.05). In addition, the multiple pregnancy rate was slightly, but not significantly, higher in the MDET group (27.1% vs. 25.0%). CONCLUSION: MDET was found to be significantly superior to double blastocyst transfer. It could be regarded as an appropriate approach to improve in vitro fertilization success rates in RIF patients.