Healthy Oocyte Donors Research Articles

Neighborhood deprivation in relation to ovarian reserve and outcomes of ovarian stimulation among oocyte donors

ObjectiveTo study the relation of neighborhood deprivation index (NDI) and markers of ovarian reserve and outcomes of controlled ovarian stimulation among young, healthy oocyte donors. DesignRetrospective cohort study Subjects547 oocyte donors who underwent 905 oocyte retrieval cycles (2008-2020) at a private fertility center in Sandy Springs, Georgia, USA. ExposureNDI was calculated using principal component analysis applied to census-level measures of poverty, employment, household composition, and public assistance, which was then standardized and linked to donor information based on donor residence. Main Outcome MeasuresMarkers of ovarian reserve, including antral follicle count (AFC) and anti-Müllerian hormone (AMH) levels, and outcomes of controlled ovarian stimulation including number of total and mature oocytes retrieved and ovarian sensitivity index (OSI) (defined as the number of oocytes retrieved ÷ total gonadotropin dose × 1,000). Multivariable generalized estimating equations with Poisson and normal distribution were used to model the relationship between NDI and outcome measures adjusting for age, BMI, and year of retrieval. ResultsThe mean (standard deviation) age of donors was 25.0 (2.8) years and 29% of the donors were racial/ethnic minorities. There were no associations between donor NDI and ovarian reserve markers. For every interquartile range increase in NDI, there was a reduction of -1.5% (95% CI -5.3, 2.4%) in total oocytes retrieved although the effect estimate was imprecise. Associations of NDI with number of mature oocytes retrieved and OSI were in a similar direction. We observed evidence for effect modification of the NDI and OSI association by donor race. There was a suggestive positive association between NDI and OSI in Black donors but no association in White donors. Conclusion and relevanceIn this cohort of young, healthy, racially diverse oocyte donors, we found little evidence of associations between NDI and markers of ovarian reserve or outcomes of ovarian stimulation.

Transcriptome analysis of adenomyosis eutopic endometrium reveals molecular mechanisms involved in adenomyosis-related implantation failure and pregnancy disorders

BackgroundWomen with adenomyosis are characterized by having defective decidualization, impaired endometrial receptivity and/or embryo-maternal communication, and implantation failure. However, the molecular mechanisms underlying adenomyosis-related infertility remain unknown, mainly because of the restricted accessibility and the difficult preservation of endometrial tissue in vitro. We have recently shown that adenomyosis patient-derived endometrial organoids, maintain disease-specific features while differentiated into mid-secretory and gestational endometrial phase, overcoming these research barriers and providing a robust platform to study adenomyosis pathogenesis and the associated molecular dysregulation related to implantation and pregnancy disorders. For this reason, we aim to characterize the dysregulated mechanisms in the mid-secretory and gestational endometrium of patients with adenomyosis by RNA-sequencing.MethodsEndometrial organoids were derived from endometrial biopsies collected in the proliferative phase of women with adenomyosis (ADENO) or healthy oocyte donors (CONTROL) (n = 15/group) and differentiated into mid-secretory (-SECorg) and gestational (-GESTorg) phases in vitro. Following RNA-sequencing, the significantly differentially expressed genes (DEGs) (FDR < 0.05) were identified and selected for subsequent functional enrichment analysis and QIAGEN Ingenuity Pathway Analysis (IPA). Statistical differences in gene expression were evaluated with the Student’s t-test or Wilcoxon test.ResultsWe identified 1,430 DEGs in ADENO-SECorg and 1,999 DEGs in ADENO-GESTorg. In ADENO-SECorg, upregulated genes included OLFM1, FXYD5, and RUNX2, which are involved in impaired endometrial receptivity and implantation failure, while downregulated genes included RRM2, SOSTDC1, and CHAC2 implicated in recurrent implantation failure. In ADENO-GESTorg, upregulated CXCL14 and CYP24A1 and downregulated PGR were related to pregnancy loss. IPA predicted a significant inhibition of ID1 signaling, histamine degradation, and activation of HMGB1 and Senescence pathways, which are related to implantation failure. Alternatively, IPA predicted an inhibition of D-myo-inositol biosynthesis and VEGF signaling, and upregulation of Rho pathway, which are related to pregnancy loss and preeclampsia.ConclusionsIdentifying dysregulated molecular mechanisms in mid-secretory and gestational endometrium of adenomyosis women contributes to the understanding of adenomyosis-related implantation failure and/or pregnancy disorders revealing potential therapeutic targets. Following experimental validation of our transcriptomic and in silico findings, our differentiated adenomyosis patient-derived organoids have the potential to provide a reliable platform for drug discovery, development, and personalized drug screening for affected patients.

Ambient traffic related air pollution in relation to ovarian reserve and oocyte quality in young, healthy oocyte donors

Studies in mice and older, subfertile women have found that air pollution exposure may compromise female reproduction. Our objective was to evaluate the effects of air pollution on ovarian reserve and outcomes of ovarian stimulation among young, healthy females. We included 472 oocyte donors who underwent 781 ovarian stimulation cycles at a fertility clinic in Atlanta, Georgia, USA (2008–2019). Antral follicle count (AFC) was assessed with transvaginal ultrasonography and total and mature oocyte count was assessed following oocyte retrieval. Ovarian sensitivity index (OSI) was calculated as the total number of oocytes divided by total gonadotrophin dose × 1000. Daily ambient exposure to nitric oxide (NOx), carbon monoxide (CO), and particulate matter ≤ 2.5 (PM2.5) was estimated using a fused regional + line-source model for near-surface releases at a 250 m resolution based on residential address. Generalized estimating equations were used to evaluate the associations of an interquartile range (IQR) increase in pollutant exposure with outcomes adjusted for donor characteristics, census-level poverty, and meteorological factors. The median (IQR) age among oocyte donors was 25.0 (5.0) years, and 31% of the donors were racial/ethnic minorities. The median (IQR) exposure to NOx, CO, and PM2.5 in the 3 months prior to stimulation was 37.7 (32.0) ppb, 612 (317) ppb, and 9.8 (2.9) µg/m3, respectively. Ambient air pollution exposure in the 3 months before AFC was not associated with AFC. An IQR increase in PM2.5 in the 3 months before AFC and during stimulation was associated with −7.5% (95% CI −14.1, −0.4) and −6.4% (95% CI −11.0, −1.6) fewer mature oocytes, and a −1.9 (95% CI −3.2, −0.5) and −1.0 (95% CI −1.8, −0.2) lower OSI, respectively. Our results suggest that lowering the current 24-h PM2.5 standard in the US to 25 µg/m3 may still not adequately protect against the reprotoxic effects of short-term PM2.5 exposure.

Changes in the immunophenotype of endometrium during implantation window receptivity formation in healthy fertile women

IntroductionEndometrial receptivity is largely determined by the immunophenotype of endometrium, especially uterine NK-cells (uNK). Immune component is directly involved in the formation of favourable microenvironment for the blastocyst implantation and placenta formation, but the way it changes during the maturation of endometrial tissue in healthy fertile women is still underexplored. MethodsThe endometrium was collected from 47 healthy oocyte donors after controlled ovarian stimulation: 23 women on the day of oocytes retrieval (OR) and 24 women on the term of implantation window (IW). The OR group was analysed, published previously and used as a comparison group to show the dynamic of changes.Isolated endometrial lymphocytes and peripheral blood samples were stained with monoclonal antibodies and analysed according to the three-color flow cytometry protocol. ResultsThe proportion of NK-cells (CD3−CD56+) in endometrium grew significantly in the implantation window compared to the oocytes retrieval day. NK-cells acquired a more differentiated phenotype from the day of OR until IW: the expression of CD8 and CD158a significantly increased, while the expression of HLA-DR significantly decreased. Significant correlations between peripheral blood and endometrial NK-cells were found in CD8 expression during OR and IW, CD335(p46)neg and CD335(p46)++ subsets during IW term. DiscussionImmunophenotype of receptive endometrium forms due to the accumulation of uNK-cells, which actively proliferate, become mature, differentiative, and ready to meet the embryo.Endometrial immunophenotype is peculiar and specific but not autonomic and isolated.Differentiation (CD8 on NK-cells), and activity (p46 on NK-cells) of peripheral blood lymphocytes is reflected in endometrial lymphocytes profile, and therefore the research of peripheral blood immunophenotype is relevant.

Ambient air pollution in relation to ovarian reserve and oocyte quality in young, healthy oocyte donors

Ambient air pollution in relation to ovarian reserve and oocyte quality in young, healthy oocyte donors

Human blastocysts uptake extracellular vesicles secreted by endometrial cells containing miRNAs related to implantation.

Are the extracellular vesicles (EVs) secreted by the maternal endometrium uptaken by human embryos and is their miRNA cargo involved in implantation and embryo development? Data suggest that EVs secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. Successful implantation is dependent on coordination between maternal endometrium and embryo, and EVs role in the required cell-to-cell crosstalk has recently been established. In this regard, our group previously showed that protein cargo of EVs secreted by primary human endometrial epithelial cells (pHEECs) is implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development. However, little is known about the regulation of these biological processes through EVs secreted by the endometrium at a transcriptomic level. A prospective descriptive study was performed. Endometrial biopsies were collected from healthy oocyte donors with confirmed fertility on the day of oocyte retrieval, 36 h after the LH surge. pHEECs were isolated from endometrial biopsies (n = 8 in each pool) and cultured in vitro. Subsequently, conditioned medium was collected and EVs were isolated and characterized. Uptake of EVs by human blastocysts and miRNA cargo of these EVs (n = 3 pools) was analyzed. EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs were fluorescently labeled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of EVs was performed using miRNA sequencing, target genes of the most expressed miRNA were annotated, and functional enrichment analysis was performed. EVs measured 100-300 nm in diameter, a concentration of 1.78 × 1011 ± 4.12 × 1010 (SD) particles/ml and expressed intraluminal protein markers Heat shock protein 70 (HSP70) and Tumor Susceptibility Gene 101 (TSG101), in addition to CD9 and CD81 transmembrane proteins. Human blastocysts efficiently internalized fluorescent EVs within 1-2 h, and more pronounced internalization was observed in the hatched pole of the embryos. miRNA-seq analysis featured 149 annotated miRNAs, of which 37 were deemed most relevant. The latter had 6592 reported gene targets, that in turn, have functional implications in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization, and gene expression. Among the relevant miRNAs contained in these EVs, we highlight hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p, and hsa-let-7a-5p as master regulators of the biological processes. This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. This study defines potential biomarkers of endometrial receptivity and embryo competence that could be useful diagnostic and therapeutic targets for implantation success, as well as open insight further investigations to elucidate the molecular mechanisms implicated in a successful implantation. This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), the Health Institute Carlos III awarded to E.J.-B. (FI19/00110) and awarded to H.F. by the Miguel Servet Program 'Fondo Social Europeo «El FSE invierte en tu futuro»' (CP20/00120), and Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139). The authors have no conflicts of interest to disclose. N/A.

P-539 Modelling embryo implantation in vitro using 3D ‘instant’ assembloids and human blastocysts

Abstract Study question Can embryo implantation be recapitulated in vitro using human blastocysts and a new 3D model of the human endometrium representing the architecture of the tissue? Summary answer We present an innovative implantation model consisting of blastocysts attaching on endometrial assembloids containing stromal cells, gland-like epithelial organoids, and overlaid with an epithelial monolayer. What is known already A successful pregnancy denotes the implantation of an embryo to the receptive, luminal lining of the uterus, the endometrium. The tissue transforms into the semi-permanent decidua which provides an optimal microenvironment and nourishes a healthy embryo. The transformation of the tissue is driven by a differentiation process, known as decidualisation, which accounts for the change of tissue-resident stromal cells to specialised decidual cells or senescent decidual cells. Simultaneously, endometrial glands of epithelial origin become secretory and provide histotrophic nutrition to the implanting embryo. Perturbations in this process of decidualisation may also result in an implantation failure or a pregnancy loss. Study design, size, duration Endometrial 'instant' assembloids, consisting of gland organoids and primary stromal cells in collagen hydrogels, were established from freshly isolated cells from endometrial biopsies. Additional cell types were incorporated including immune and endothelial cells creating a layer of epithelial cells recapitulating the luminal epithelium. Hormonal stimulated assembloids were further co-cultured with human blastocysts at 37 °C, 5% O2 and 6% CO2. Attachment of the blastocysts to the luminal epithelium was assessed upon overnight co-culture. Participants/materials, setting, methods Endometrial biopsies (n = 3) were obtained from consenting hormonally stimulated, healthy oocyte donors. After informed consent, human embryos (n = 14) were donated to research after 5 years of cryopreservation. Decidualisation was monitored by RT-qPCR analysis using epithelial and stromal cells from undifferentiated or decidualised assembloids. Attachment of the embryos (n = 11) to the model was assessed by disturbance of the liquid and this was further confirmed by immunofluorescence antibody labelling. Main results and the role of chance 'Instant' assembloids mimic the endometrium morphologically and functionally since hormonal stimulation results in the induction of decidual stromal (PRL, SCARA5 and DIO2) and epithelial (PAEP and SPP1) marker genes. The presence of immune and endothelial cells was confirmed with the induction of IL2RB and VWF respectively. Immunofluorescent antibody labelling confirmed the similarity in morphology of the model to the human endometrium. Aiming to mimic implantation, assembloids underwent a 3-day long differentiation in a chemically defined medium, 8-bromo-cAMP, estradiol, and a progestin, that induced in vitro decidualisation. Following the 3-day treatment, instant assembloids were overlaid with single epithelial cells, isolated from instant assembloids which had undergone prolonged decidualisation treatment to imitate the physiology of the endometrium. Following a 24-hour long incubation period, human hatched day 6 blastocysts were positioned on the top epithelial layer of the instant assembloid. Immunofluorescent antibody labelling using makers for the epiblast (NANOG), primitive endoderm (GATA4) and epithelial cells (E-cadherin) were used to visualise the co-culture. Our findings signify the newly established model as the most advanced method to study embryo implantation in vitro. The uniqueness of the model is owned by the preservation of endometrial cell types and the potential to model disease through patient specificity. Limitations, reasons for caution The proposed system is an innovative method to recapitulate apposition and adhesion during implantation. Our results are preliminary and further experiments will be performed to increase our repeats and therefore reliability. Moreover, due to the complex nature of the co-culture system, imaging remains a challenge that requires further optimisation. Wider implications of the findings The embryo-assembloid co-culture system may provide a useful tool to aid our understanding of the mechanisms of implantation. Future work will include the labelling of the embryos with a nuclear dye to enable tracking using time-lapse microscopy. We further aim to expand our protocol to study the process of invasion. Trial registration number N/A

Increased cytotoxic natural killer cells in the endometrium alone cannot be considered the immunological cause of recurrent miscarriage

To study the distribution and gene expression of endometrial immune cell populations, especially natural killer (NK) subsets, between assisted reproductive technology patients and healthy donors and explore a possible relationship of these results with patients' killer cell immunoglobulin-like receptor (KIR) genotypes and KIR-human antigen leukocyte-C (HLA-C) binding. Prospective observational cohort study. Clinic and university laboratories. Participants included 39 women with recurrent miscarriages who had undergone invitro fertilization cycles with donated oocytes and 21 healthy oocyte donors with proven fertility. Endometrial biopsy samples were collected from both patients and donors, and the KIR genotypes of the assisted reproductive technology patients were analyzed. Endometrial gene expression (cluster of differentiation [CD] antigens and anti-inflammatory and proinflammatory interleukins) and the number and percentage of regulatory T and NK cell populations in patients and donors were determined. Subsequently, the results obtained were categorized in the group of patients by KIR genotype. Killer cell immunoglobulin-like receptor-HLA-C binding was also examined in patients, considering their KIRs. A higher percentage of CD56dimCD16+ NK cells were observed in patients than those in healthy donors. Nevertheless, when categorizing patients by KIR genotype and comparing the KIR AA (35.9%), AB (43.6%), and BB (20.5%) groups, no statistically significant difference was observed in either endometrial gene expression or any of the immune cell populations analyzed. Finally, no differences in binding between KIR and HLA-C molecules were registered among these 3 sets of patients. The reported increase in the number of NK cells with a cytotoxic profile in the endometrium of women with a history of recurrent miscarriages cannot alone explain these events because no relationship is observed between such cellular increase and the KIR genotypes, which individually, and in combination with the different HLA-C alleles, have also been associated, by previous studies, with negative reproductive outcomes. 1405-MAD-025-JG.

Sperm DNA integrity does play a crucial role for embryo development after ICSI, notably when good-quality oocytes from young donors are used

Based on the inconsistent literature published thus far involving infertile patients, whether intracytoplasmic sperm injection (ICSI) allows overcoming total fertilization failure due to sperm DNA fragmentation is still unclear. Related to this, female factors, which may have a significant impact on assisted reproduction outcomes, can mask male infertility. In this scenario, evaluating ICSI outcomes following cycles using healthy donor gametes could shed light on this realm, as it would avoid the influence of (un)known confounding factors present in infertile individuals. The present work, therefore, aimed to address whether single- and double-stranded sperm DNA fragmentation leads to impaired ICSI outcomes in double gamete donation cycles. The study also compared these double-gamete donation cycles to cycles in which only sperm were donated and oocytes were obtained from infertile patients. Two cohorts were included: (a) the Donor-Donor (DD) cohort, which included 27 semen donor samples used in 49 ICSI cycles with young healthy oocyte donors; and (b) the Donor-Infertile (DI) cohort, which involved 34 semen donor samples used in 57 ICSI cycles with oocytes from patients. Single- and double-stranded sperm DNA breaks were determined with alkaline and neutral Comet assays, respectively; ICSI was conducted following standard protocols and embryos were monitored through time-lapse microscopy. In the DD cohort, the percentage of sperm with high overall DNA damage correlated with fertilization rates (Rs = − 0.666; P < 0.001) and with the percentage of blastocysts per injected oocyte (Rs = − 0.414; P = 0.040). In addition, sperm DNA damage delayed the first embryo division (Rs = 0.421; P = 0.036), and development from the 8-cell to the morula stage (Rs = 0.424; P = 0.034). In contrast, double-stranded DNA breaks had no effect in this cohort. As far as the DI cohort is concerned, while overall sperm DNA damage was not found to be correlated to fertilization or blastocyst rates, pronuclei formation following ICSI was delayed when the incidence of double-stranded DNA breaks was high (Rs = 0.485; P = 0.005). In conclusion, this study, which is the first involving double donation cycles (i.e., a donor-donor cohort), supports that sperm DNA damage has a detrimental impact on fertilization rates after ICSI, and delays embryo development. Moreover, the use of oocytes from infertile individuals is suggested to hide the male-factor effect.

Luteinizing hormone receptor promotes angiogenesis in ovarian endothelial cells of Macaca fascicularis and Homo sapiens†.

Angiogenesis within the ovarian follicle is an important component of ovulation. New capillary growth is initiated by the ovulatory surge of luteinizing hormone (LH), and angiogenesis is well underway at the time of follicle rupture. LH-stimulated follicular production of vascular growth factors has been shown to promote new capillary formation in the ovulatory follicle. The possibility that LH acts directly on ovarian endothelial cells to promote ovulatory angiogenesis has not been addressed. For these studies, ovaries containing ovulatory follicles were obtained from cynomolgus macaques and used for histological examination of ovarian vascular endothelial cells, and monkey ovarian microvascular endothelial cells (mOMECs) were enriched from ovulatory follicles for in vitro studies. mOMECs expressed LHCGR mRNA and protein, and immunostaining confirmed LHCGR protein in endothelial cells of ovulatory follicles in vivo. Human chorionic gonadotropin (hCG), a ligand for LHCGR, increased mOMEC proliferation, migration and capillary-like sprout formation in vitro. Treatment of mOMECs with hCG increased cAMP, a common intracellular signal generated by LHCGR activation. The cAMP analog dibutyryl cAMP increased mOMEC proliferation in the absence of hCG. Both the protein kinase A (PKA) inhibitor H89 and the phospholipase C (PLC) inhibitor U73122 blocked hCG-stimulated mOMEC proliferation, suggesting that multiple G-proteins may mediate LHCGR action. Human ovarian microvascular endothelial cells (hOMECs) enriched from ovarian aspirates obtained from healthy oocyte donors also expressed LHCGR. hOMECs also migrated and proliferated in response to hCG. Overall, these findings indicate that the LH surge may directly activate ovarian endothelial cells to stimulate angiogenesis of the ovulatory follicle.

Distress response in granulosa cells of women affected by PCOS with or without insulin resistance.

In this study, we investigated whether metabolic dysfunction in women with Polycystic ovarian syndrome (PCOS) induces granulosa cell (GC) stress and activates in the endoplamatic reticulum and the mitochondria (UPRer and UPRmt, respectively). Women who were diagnosed with PCOS (based on the Rotterdam criteria), were divided into two groups, PCOS with insulin resistance (PCOS-IR; n = 20) and PCOS with no insulin resistance (PCOS-nIR; n = 20), and compared to healthy oocyte donors (CONT; n = 20). Insulin resistance (IR) was assessed on the results of homeostasis model assessment (HOMA) that determines IR using the concentration of fasting plasma glucose and fasting insuline. Expression of UPRer genes (i.e., IRE1, ATF4, ATF6, XBP1, BIP, and CHOP), and UPRmt genes (i.e., HSP60, HSP10, CLPP, and HSP40) was assessed in cumulus GCs by qRT-PCR. We found that several genes involved in UPRer and UPRmt were overexpressed in the GCs of PCOS-IR and PCOS-nIR compared to CONT. IRE1, ATF4 and XBP1, that are activated by ER stress, were significantly overexpressed in PCOS-IR compared to CONT. BIP and CHOP were overexpressed in PCOS groups compared to CONT. HSP10 and HSP40 were upregulated in PCOS-IR and PCOS-nIR groups compared to the CONT. HSP60 and CLPP showed no statistical different expression in PCOS-IR and PCOS-nIR compared to CONT group. Our findings suggest that the GCs of women with PCOS (with or without IR) are metabolically distressed and upregulate UPRer and UPRmt genes. Our study contributes to the understanding of the molecular mechanisms underlying the pathological changes that occur in the follicular microenvironment of women with PCOS.

Embryo Quality May Be Associated With Serum Inhibin B Levels but Not With Serum or Follicular Fluid Levels of Other Components of the Activin–Follistatin–Inhibin Axis

ObjectiveWe investigated the potential associations of embryo quality with serum and/or follicular fluid (FF) concentrations of the molecules of the activin–follistatin–inhibin (AFI) axis and antimüllerian hormone and aimed to identify molecules that could predict a positive assisted reproductive technology (ART) outcome. MethodsIn this cross-sectional study, we measured AFI hormone and antimüllerian hormone levels in the serum and FF of follicles (n = 101) obtained from healthy oocyte donors who underwent an assisted reproductive technology course (n = 32). After egg retrieval, embryos were characterized as good or bad quality according to the European Society of Human Reproduction and Embryology criteria. Women were divided into 3 groups (<50%; 50%-66.7%; and >66.7%) according to the percentage of good quality embryos obtained. ResultsThere was no difference between good and bad quality embryos in any of the molecules measured in FF. Moreover, there was no difference in the parameters measured in the serum among women according to the percentage of good quality embryos (ie, suitable for transfer or freezing) except for inhibin B, which tended to increase along with a good quality embryo rate (55.6 ± 7.9 vs 95.3 ± 14.3 vs 113.9 ± 36.9; P = .045). ConclusionsAmong the molecules of the AFI axis, only serum but not FF inhibin B levels were marginally associated with good quality embryo rates.

Proteomic analysis of extracellular vesicles secreted by primary human epithelial endometrial cells reveals key proteins related to embryo implantation

BackgroundSuccessful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women.MethodsIshikawa cells and pHEECs were in vitro cultured and hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography–tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting.ResultsUltracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers.ConclusionsEV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success.

Comparative characteristics of patients with infertility when applying melatonin in complex preparation for assisted reproductive technologies

Оbjective of the study was to conduct a retrospective comparative characterization of patients with infertility who took or did not take melatonin with assisted reproductive technologies (ART).Materials and methods. In our study, we examined 89 women. The first (control) group included 13 healthy women oocyte donors who got pregnant on their own and gave birth to their own healthy children, the second group - 33 patients with infertility, who took 3 mg of the preparation "Vita-melatonin" produced by "Kyiv Vitamin Plant" at the same time before bedtime, two weeks before and during ovulation stimulation, the third group - 43 patients with infertility who did not take melatonin preparation before and during ovulation stimulation. There were no women who worked night shifts among the patients. Medical documentation of women of the control group and those with infertility, data of gynecological, ultrasound examination, hormones blood were analyzed. Ultrasound examination of the pelvic organs was performed on all patients with the device "Mindray DC-80 X-Insight", and measurements were performed using a transvaginal sensor. The thickness and structure of the endometrium were evaluated, and the number of antral follicles (NAF) ranging in size from 2 to 10 mm was counted in each ovary. All patients were tested for serum levels of the anti-mullerian hormone (AMG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), prolactin (PRL), progesterone (P), thyroid-stimulating hormone (TSH), triiodothyronine (T4).Results. The average age of women in the first (control) group was 27.08 ± 12.38 years, the second (taking melatonin) - 33.12 ± 8.18 years, the third (not taking melatonin) - 30.95 ± 7.07 years &gt; 0.05), i. e. the age of the patients of the examined groups was equal. It should be noted that in the studied patients of both groups, the occurrence of primary infertility exceeded secondary infertility 2.7 times in the second group (p &lt; 0.05) and 1.7 times in the third (p &lt; 0.05). Infertility factors such as reduced ovarian reserve, habitual miscarriage and infertility of unknown origin were more common in patients of the second group, and endometriosis, tubal factor and male factor in the third, although the difference was not significant. The available extragenital pathology did not differ in the patients of the examined groups. The number of antral follicles was significantly higher in both ovaries of women in the control group compared with patients of the second and third groups. While the thickness of the endometrium did not differ significantly in groups, although in women of the control group it was slightly less. Regarding the study of hormonal status, it should be noted that we did not find a significant difference in the levels of hormones in the blood of women we examined. Exceptionally, there was a significant difference (p &lt; 0.001) in progesterone content between the second (0.62 ± 0.052 nmol/l) and third (181.63 ± 13.87 nmol/l) groups. Also, the patients of the third group had significantly (p &lt; 0.05) higher levels of FSH in blood (8.25 ± 0.63 mUn/ml) compared with the control group (4.93 ± 0.69 mUn/ml).Conclusions. The examined women in the control group, as well as infertility patients who received melatonin two weeks before the expected menstruation and during ovulation stimulation, and infertility patients who did not receive this preparation in similar programs, did not differ in age, occurrence of primary and secondary infertility, the factor that led to infertility, concomitant extragenital pathology, ovarian reserve and hormone levels of the reproductive panel. That is, they were equal in our study.

The level of melatonin in the blood and follicular fluid in women with infertility in the program of assisted reproductive technologies and the effectiveness of its application

Objective was to investigate the level of melatonin in the blood and follicular fluid in women treated with infertility by ART method and evaluate the effectiveness of melatonin in their preparation for programs.Material and methods. 89 women were examined. The first (control) group included 13 healthy women oocyte donors who gave birth to their own healthy children, the second group - 33 women with infertility, who two weeks before and during ovulation stimulation were taken simultaneously at the same time before bedtime 3 mg of the drug "Vita-melatonin" produced by "Kyiv Vitamin Plant", the third group - 43 women with infertility who did not take the drug melatonin during ovulation stimulation. ELISA (Germany) reagent kits were used to determine melatonin levels. Melatonin levels were determined in blood plasma, and follicular fluid obtained during the puncture at 9:00 am.Results. The level of melatonin in the blood of female donor oocytes was 130.85 ± 16.91 pg/ml. This rate in the blood of women who used the drug melatonin before and during ovulation stimulation was significantly higher than in the blood of women who did not take the drug (respectively, 143.06 ± 14.87 pg/ml and 123.40 ± 12.65 pg / ml, p &lt;0.05), and in the follicular fluid there was an inverse relationship: the level of melatonin in women of the first group was 97.15 ± 8.69 pg / ml, the second group - 39.46 ± 4.52 pg/ml, which is significantly less (p &lt;0.05), the third group - 62.34 ± 3.94 pg / ml, which is almost twice more (p &lt;0.05) compared with women who took melatonin, but less (p &lt;0.05) compared with the first group. The frequency of pregnancy on transfer in patients of the first group was 80.0 ± 11.01%, in women of the second group probably less - 60.6 ± 8.25% (p &lt;0.05), but also probably higher compared to women in the third group - 45.0 ± 7.62% (p &lt;0.05). A similar pattern was observed by us on the onset of pregnancy in general: in the first group - 100.0%, in the second - 68.9 ± 8.05% (p &lt;0.05), in the third - 60.5 ± 7.47% (p&gt; 0.05).Conclusions. The low level of melatonin in the follicular fluid obtained by us in women who underwent ovulation stimulation in ART programs in the treatment of infertility requires further scientific interpretation. At the same time, the best results of pregnancy in women in ART programs who took melatonin in preparation are encouraging.

P–136 Factors predicting clinical outcomes of 511 recipients of vitrified oocyte donation from an UK-regulated egg bank

Abstract Study question Do established donor and recipient clinical markers predict recipient clinical pregnancy and live birth rates (LBRs) in a vitrified oocyte donation programme? Summary answer Recipient BMI and previous miscarriages predicted cumulative LBR. Likelihood of clinical pregnancy and LBR was higher in recipients of donors aged 23–29 than donors 18–22. What is known already The influence of age on ovarian reserve underlies the upper limit of 35 years for UK donors. However, recent evidence suggests that oocyte aneuploidy rates follow an inverse U-shaped curve in relation to a woman’s age. Conflicting evidence exists regarding the impact of other donor-related factors including BMI, AMH, oocyte yield and prior reproductive history on recipient outcomes. Moreover, the effect of recipient age, BMI, and reproductive history on oocyte donation outcome remains unclear. Study design, size, duration Retrospective cohort study of 325 altruistic oocyte donors matched to a total of 511 recipients. Only first donations taking place between January 2017 and December 2019 were included. Participants/materials, setting, methods All oocyte donors were altruistic volunteers aged 18–35 with no prior infertility diagnosis. Donor and recipient screening for suitability and safety was carried out according to the Human Fertilisation Embryology Authority guidelines. Backward stepwise logistic regression was used to identify donor, recipient and embryology parameters predictive of recipient primary outcomes defined as clinical pregnancy and live birth, either cumulative or after the first embryo transfer (ET). Main results and the role of chance A total of 705 fresh and frozen/thawed ETs were performed, of which 76% were elective single embryo transfers (eSETs) of blastocysts (96.5%), resulting in a cumulative clinical pregnancy and LBR of 83.5% and 70.5% respectively after 3 ETs. Recipient BMI and previous miscarriages were predictors of cumulative LBR (p &amp;lt; 0.05). The ratio of transferrable embryos per oocytes received/fertilised and the number of ETs needed to achieve the intended primary outcome were predictors of cumulative clinical pregnancy and LBR (p &amp;lt; 0.05). Donor age 18–22 was associated with lower incidence of recipient clinical pregnancy and live birth after the first ET, as compared to donor age 23–29 (p &amp;lt; 0.05). Limitations, reasons for caution The present study included only healthy oocyte donors, thus conclusions may not apply to subfertile or less healthy women. Male factors were not accounted for. Wider implications of the findings: We demonstrate the efficacy of vitrified oocyte donation treatment and identify recipient BMI, previous miscarriages and embryology parameters as predictors of cumulative LBR. Additionally, the choice of donors aged 18–22 instead of older donors is found not to be advantageous for increasing the chance of clinical pregnancy and live birth. Trial registration number Not applicable

Nitrite and Nitrate Levels in Follicular Fluid From Human Oocyte Donors Are Related to Ovarian Response and Embryo Quality.

Nitric oxide, a key regulatory molecule in the follicular fluid, has been suggested as a possible biomarker to predict ovarian response in stimulated cycles and the potential of the retrieved oocytes for developing high-quality embryos. Nevertheless, a consensus on whether or not nitric oxide can help in this context has not been reached. We simultaneously measured the oxidation products of nitric oxide, nitrite, and nitrate, via high-performance liquid chromatography (HPLC)-UV in follicular fluid samples from 72 oocyte donors. We found no associations of follicular fluid nitrite, nitrate, total nitric oxide, or nitrate/nitrite ratio with total or metaphase II (MII) oocyte yield. However, nitrite and nitrate levels were related to the yield of MII oocytes when this outcome was expressed as a proportion of all oocytes retrieved. The adjusted MII proportion in the lowest and highest nitrite levels were 68% (58–77%) and 79% (70–85%), respectively (p, linear trend = 0.02), whereas the adjusted MII proportion in extreme tertiles of nitrate levels were 79% (70–85%) and 68% (57–77%) (p, linear trend = 0.03). In addition, nitrate levels showed a suggestive inverse correlation with embryos with maximum or high potential of implantation (p = 0.07). These results suggest that the follicular fluid concentrations of nitrite and nitrate may be a useful tool in predicting how healthy oocyte donors respond to superovulation and the implantation potential of the embryos produced from their oocytes.

Female infertility is associated with an altered expression of the neurokinin B/neurokinin B receptor and kisspeptin/kisspeptin receptor systems in ovarian granulosa and cumulus cells

To analyze and compare the expression profile of TAC3, TACR3, KISS1, and KISS1R in mural granulosa and cumulus cells from healthy oocyte donors and patients with different infertility etiologies, including advanced maternal age, endometriosis, and low ovarian response. Genetic association study. Private fertility clinic and public research laboratory. Healthy oocyte donors and infertile women undergoing invitro fertilization (IVF) treatment. IVF. Gene expression levels of KISS1, KISS1R, TAC3, and TACR3 in human mural granulosa and cumulus cells. Infertile women showed statistically significantly altered expression levels of KISS1 (-2.57 ± 2.30 vs. -1.37 ± 2.11), TAC3 (-1.21 ± 1.40 vs. -1.49 ± 1.98), and TACR3 (-0.77 ± 1.36 vs. -0.03 ± 0.56) when compared with healthy oocyte donors. Advanced maternal age patients, endometriosis patients, and low responders showed specific and altered expression profiles in comparison with oocyte donors. Abnormal expression levels of KISS1/KISS1R and TAC3/TACR3 systems in granulosa cells might be involved in the decreased fertility associated to advanced maternal age, endometriosis, and low ovarian response.

Aberrant DNA methylation profiling affecting the endometrial receptivity in recurrent implantation failure patients undergoing in vitro fertilization.

DNA methylation profile in mid-secretory phase of endometrium is reported to be varied from other phases in natural menstrual cycle. Therefore, we intended to study the impairment in endometrial receptivity by performing whole-genome methylation and gene expression profiling in endometrium of recurrent implantation failure patients (RIF) during IVF under controlled ovarian stimulation (COS). Endometrial biopsies were collected from IVF-RIF patients (cases, n=6) and healthy fertile oocyte donors (controls, n=6) undergoing COS after 6/7th day of human chorionic gonadotropin administration. The whole-genome methylation and gene expression microarray were performed and analysed by GenomeStudio software (P<.05 by Illumina Custom Model), whereas the enrichment analysis was performed using "Database for Annotation, Visualization and Integrated Discovery" (DAVID, V6.8). Significant differentially methylated genes were correlated with dys-regulated genes using Pearson's correlation. Differential methylation in RIF patients revealed 448 CpG sites. The enrichment analysis showed aberrant methylation in genes involved in immunological response and G protein activity. Methylation in NLRP2 gene in inflammatory pathway had significant negative correlation with gene expression (P=.008), whereas SERPINA5 gene that is already known to be involved in endometrial receptivity was observed to be hypomethylated in promoter region with highest delta beta value and up-regulated in gene expression analysis. The aberrant methylation of genes involved in immunological functions and G protein activation was found to be prevalent which might suggest a role in endometrial receptivity. However, the findings need to be further validated on a larger cohort of IVF-RIF patients.

In-vitro development of embryos derived from vitrified-warmed oocytes is delayed compared with embryos derived from fresh oocytes: a time-lapse sibling oocyte study.

Is there a difference in blastocyst formation between fresh and vitrified-warmed sibling oocytes and can this difference be attributed to changes in embryo morphokinetics? Between February 2016 and December 2017, 472 metaphase II (MII) oocytes in 67 donor-recipient cycles from 27 different healthy anonymous oocyte donors were allocated for fresh transfer (FSHO) (n = 220) to a synchronous recipient (n = 36) or vitrified (VITO) (n = 252) to be warmed and transferred to another recipient (n = 31). Embryos derived from the FSHO and their sibling VITO were analysed for morphokinetic development using time-lapse imaging, blastocyst formation and clinical outcome. Time-lapse analysis showed an overall delay in cleavage rate from the time of pronuclei disappearance up to the time of blastulation in the VITO compared with their sibling FSHO. Twelve morphokinetic variables were significantly different between the groups. On Day 5 significantly more FSHO embryos developed to blastocyst (expansion 1-6) and reached the full blastocyst stage (expansion 3-6) compared with the VITO embryos [53.2% (84/158) versus 40.0% (64/160); P = 0.0244 and 48.1% (76/158) versus 31.3% (50/160); P = 0.0028, respectively]. The embryo utilization rate was similar in both groups at the time of cryopreservation; 51.3% (FSHO) versus 45.0% (VITO) (P = 0.3124). The pregnancy rate per cycle was 47.2% (17/36) in FSHO patients and 48.4% (15/31) in VITO patients (P = 1). Limitations in this study: non-randomized, small study size and not powered to detect differences in clinical outcomes. Timing of development is altered and blastocyst formation is delayed in embryos derived from vitrified-warmed donor oocytes compared with their fresh sibling counterparts. Although preliminary results suggest that the clinical impact of this delay may be limited, this needs further investigation in larger randomized studies.