Healthy Oocytes Research Articles

Comprehensive atlas of mitochondrial distribution and dynamics during oocyte maturation in mouse models

BackgroundOocytes, the largest cells in mammals, harbor numerous mitochondria within their cytoplasm. These highly dynamic organelles are crucial for providing energy resources and serving as central regulators during oogenesis. Mitochondrial dynamics ensure proper energy distribution for various cellular processes involved in oocyte maturation. Previous studies have used alterations in mitochondrial distribution as a biomarker to assess the oocyte health. However, there are discrepancies between studies regarding mitochondrial distribution profiles in healthy oocytes. Consequently, a comprehensive mitochondrial distribution profile in oocytes during maturation has not been fully characterized. Additionally, there is a lack of objective, quantitative methods to evaluate alterations in mitochondrial distribution profiles in oocytes.MethodsThis study aims to provide an in-depth overview of mitochondrial distribution profiles in mouse oocytes at different maturation stages: germinal vesicle (GV) stage, metaphase I (MI), and mature metaphase II (MII). Freshly collected mouse GV, MI and MII oocytes were stained with MitoTracker Red. Confocal microscopy was used to obtain images of mitochondrial distribution profiles in these oocytes. Using the Imaris software, we reconstructed three-dimensional (3D) surface renderings of each oocyte and quantitatively illustrated the mitochondrial distribution profiles.ResultsAt the GV stage, mitochondria in oocytes were evenly distributed throughout the ooplasm. As oocytes progressed to MI and MII stages, mitochondria aggregated and formed clusters, the mean size of mitochondrial clusters and the proportions of clustered mitochondria increased along with the maturation of oocytes.ConclusionsOur findings reveal that mitochondria in mouse oocytes are highly dynamic, undergoing significant reorganizations during oocyte maturation. We for the first time provided comprehensive mitochondrial distribution profiles in mouse oocytes at the GV, MI and MII stages. These mitochondrial distribution profiles were further quantitatively evaluated. Our methods provide an objective and standardized approach for evaluating alterations in mitochondrial dynamics, which can be used as biomarkers to monitor oocyte conditions during maturation.

Single-cell RNA sequencing of human oocytes reveals a differential transcriptomic profile associated with agar-like zona pellucida

BackgroundAgar-like zona pellucida (ZP) is the most common type of abnormal ZP, and is one of the causes of low fertility or infertility. However, the molecular mechanism of agar-like ZP is unclear. Single-cell RNA-sequencing (scRNA-seq) analysis was used to assess the cellular and molecular landscape of oocytes with agar-like ZP.MethodsHuman metaphase I (MI) oocytes were collected from four patients with agar-like ZP and four healthy donors. Total RNA was isolated, cDNA was synthesized, and libraries were generated and subsequently sequenced on a HiSeq 2500 instrument. The scRNA-seq data were analyzed with R software.ResultsWe identified 1320 genes that were differentially expressed between agar-like ZP oocytes and healthy donor oocytes. Gene Ontology term enrichment results showed that the genes downregulated in agar-like ZP oocytes were significantly related to extracellular matrix organization, while the genes upregulated in agar-like ZP oocytes were significantly related to the regulation of response to DNA damage stimulus. The Kyoto Encyclopedia of Genes and Genomes enrichment results showed that genes were enriched in the ECM-receptor interaction pathway and focal adhesion pathway. Other signaling pathways important in oocyte development were also enriched, such as PI3K-Akt. Differential expression analysis identified UBC, TLR4, RELA, ANXA5, CAV1, KPNA2, CCNA2, ACTA2, FYN and ITGB3 as genetic markers of oocytes with agar-like ZP.ConclusionsOur findings suggest that agar-like ZP oocytes exhibit significant downregulation of genes involved in the ECM-receptor interaction signaling pathway and focal adhesion pathway, which could lead to aberrant ZP formation, while the upregulated genes were significantly related to regulation of the response to DNA damage stimulus. Agar-like ZP formation may interfere with the normal exchange of signals between oocytes and perivitelline granulosa cells, thereby preventing cumulus cells from participating in oocyte DNA damage repair and causing MI arrest.

Ovarian tissue cryopreservation by vitrification: The first Vietnam experience

Ovarian Tissue Cryopreservation (OTC) is a promising technique for reproductive preservation in women with cancer. Although this technique has been successfully applied in many Invitro Fertilization (IVF) centers worldwide, OTC has not been recently developed in Vietnam, and there are no publications of OTC. The study of OTC by vitrification at IVFMD, My Duc Hospital, was conducted from 2018 to 2023 with trial registration number NCT04666376. Ovarian tissues from each patient were divided into 3 groups: group 1, which consisted on fresh tissues as a control group; group 2, which consisted of vitrified tissues using the Ova Kit Type M (Kitazato, Japan); and group 3, which consisted of vitrified tissues using IVFMD medium. The findings showed that the rates of viable follicles and follicles with both healthy oocytes and CGs in groups 2 and 3 were significantly lower than in group 1, at [80.3% in group 1, 70% in group 2, and 65% in group 3] and [71.3% in group 1, 59.4% in group 2, and 58.3% in group 3] respectively (P < 0.05). The results aligned with previous findings due to the negative effects of cryopreservation. Moreover, it was illustrated that there was no change in the quality of tissue between the commercial medium and IVFMD’s medium in OTC.

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.

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.

What importance do donors and recipients attribute to the nuclear DNA-related genetic heritage of oocyte donation?

How do oocyte donors and recipients perceive the genetic link related to the transfer of nuclear DNA between donors and offspring? Whether they are donors or recipients, individuals attach great importance to the transmission of their genetic heritage, since 94.5% would opt for the pronuclear transfer method to preserve this genetic link in the context of oocyte donation. Since 1983, the use of oocyte donation has increased worldwide. Performed in France since the late 1980s and initially offered to women with premature ovarian insufficiency, its indications have progressively expanded and now it is proposed in many indications to prevent the transmission of genetically inherited diseases. This has resulted in an increase in the waiting time for access to oocyte donation due to the difficulty in recruiting oocyte donors in French ART centres. Several articles have discussed how to fairly distribute donor oocytes to couples, but few have interviewed women in the general population to record their feelings about oocyte donation, as either the donor or recipient and the importance given to the genetic link between the oocyte donors and the children born. Mitochondrial replacement therapy (MRT) is a technique originally developed for women at risk of transmitting a mitochondrial DNA mutation. Recently, MRT has been considered for embryo arrest and oocyte rejuvenation as it could help females to reproduce with their own genetic material through the transfer of their oocyte nucleus into a healthy donor oocyte cytoplasm. We conducted an opinion survey from January 2021 to December 2021, during which 1956 women completed the questionnaire. Thirteen participants were excluded from the analysis due to incomplete responses to all the questions. Consequently, 1943 women were included in the study. We specifically developed a questionnaire for this study, which was created and distributed using the Drag'n Survey® software. The questionnaire consisted of 21 items presented alongside a video created with whiteboard animation software. The aim was to analyse whether certain factors, such as age, education level, marital status, number of children, use of ART for pregnancy, video viewing, and knowledge about oocyte donation, were associated with feelings towards oocyte donation, by using a univariate conditional logistic regression model. This statistical method was also used to assess whether women would be more inclined to consider oocyte donation with the pronuclear transfer technique rather than the whole oocyte donation. All parameters found to be statistically significant in the univariate analysis were subsequently tested in a multivariate model using logistic regression. Most women were concerned about the biological genetic contribution of the donated oocyte (94.8%). The most common reason for a women's reluctance to donate their oocytes was their unwillingness to pass on their genetic material (33.3%). Nearly 70% of women who were initially hesitant to donate their oocytes indicated that they would reconsider their decision if the oocyte donation was conducted using donated cytoplasm and the pronuclear transfer technique. Concomitantly, >75% of the respondents mentioned that it would be easier to receive a cytoplasm donation. The largest proportion of the population surveyed (94.5%) expressed their support for its legalization. In this study, a substantial portion of the responses came from individuals with medical or paramedical backgrounds, potentially introducing a recruitment bias among potential donors. The rate of missing responses to the question regarding the desire to become an oocyte donor was 13.6%, while the question about becoming an oocyte cytoplasm donor had a missing response rate of 23%. These missing responses may introduce a bias in the interpretation of the data. This study was the first to demonstrate that, for the French population studied, the combination of oocyte cytoplasm donation with pronuclear transfer could offer a promising approach to enhance the acceptance of oocyte donation for both the donor and the recipient. No external funding was used for this study. The authors have no conflicts of interest. N/A.

How great thou ART: biomechanical properties of oocytes and embryos as indicators of quality in assisted reproductive technologies.

Assisted Reproductive Technologies (ART) have revolutionized infertility treatment and animal breeding, but their success largely depends on selecting high-quality oocytes for fertilization and embryos for transfer. During preimplantation development, embryos undergo complex morphogenetic processes, such as compaction and cavitation, driven by cellular forces dependent on cytoskeletal dynamics and cell-cell interactions. These processes are pivotal in dictating an embryo's capacity to implant and progress to full-term development. Hence, a comprehensive grasp of the biomechanical attributes characterizing healthy oocytes and embryos is essential for selecting those with higher developmental potential. Various noninvasive techniques have emerged as valuable tools for assessing biomechanical properties without disturbing the oocyte or embryo physiological state, including morphokinetics, analysis of cytoplasmic movement velocity, or quantification of cortical tension and elasticity using microaspiration. By shedding light on the cytoskeletal processes involved in chromosome segregation, cytokinesis, cellular trafficking, and cell adhesion, underlying oogenesis, and embryonic development, this review explores the significance of embryo biomechanics in ART and its potential implications for improving clinical IVF outcomes, offering valuable insights and research directions to enhance oocyte and embryo selection procedures.

Investigating Ovulation Induction Outcomes in Patients with Decreased Ovarian Reserve Treated with Double Stimulation during The Follicular and Luteal Phases Compared to The Conventional Antagonist Cycle: A Randomized Clinical Trial.

It is difficult to obtain healthy oocytes in poor ovarian responders with conventional treatment methods. Thus, the need to investigate new methods is essential. This study aims to investigate ovulation induction outcomes in patients with decreased ovarian reserve (DOR) in two groups treated with double stimulation (DuoStim) during the follicular and luteal phases in comparison with the antagonist cycle. This was a randomised clinical trial that enrolled the patients with reduced ovarian reserve. The patients referred for in vitro fertilization (IVF) at Molud Infertility Clinic, Ali Ebn Abitalib (AS) Hospital, Zahedan, Iran from 2020 to 2021. Participants were randomly divided into two groups, those who underwent treatment with DuoStim during the follicular and luteal phase (case group) and those who received the conventional antagonist cycle (control group). The mean number of metaphase II (MII) eggs was 7.7 ± 3.1 in the case group and 6.1 ± 3.9 in the control group (P=0.063). The mean total number of retrieved eggs in the case group was 9.2 ± 3.7 and in the control group, it was 6.9 ± 4.4 (P=0.023). The mean number of embryos obtained in the case group was 6.5 ± 3.9; in the control group, it was 4.7 ± 2.8 (P=0.016). The DuoStim method can effectively play a role in increasing the total number of retrieved eggs and embryos (registration number: IRCT20120817010617N8).

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.

Does Mitochondrial Transfer An Alternative to Egg Donation?

Objective&#x0D; To evaluate the utility of pronuclear transfer using healthy oocytes from donors in poor prognosis patients.&#x0D; Design&#x0D; Retrospective study.&#x0D; Subjects&#x0D; A total of 29 patients with no blastocyst development or total fertilization failure were included.&#x0D; Main Outcome Measures&#x0D; Our primary outcome was the blastocyst rate. Secondary outcomes measures included euplody rate, clinical pregnacy rate and live birth rate.&#x0D; Results&#x0D; On day 5/6 of blastocyst development, an average blastocyst rate of 49.2% was obtained. The average rate of euploid blastocysts obtained after PGT-A was 69.5%. All patients underwent frozen embryo transfer. Single blastocyst transfer was performed in all patients, and the clinical pregnancy rate was 48.3%. Of these patients, 14 had a live birth, and the live birth rate was 48.3%. No genetic or morphologic abnormalities have been detected in the babies born. The 29 patients were grouped based on their age, as &lt;35 (n=2), 35-27 (n=6), 38-40 (n=9), 41-42 (n=6) and &gt;42 (n=6). Based on these age groups, blastocyst formation rates were 47.8%, 50.0%, 45.0%, 50.0% and 66.7%, respectively; euploidy rates were 63.6%, 61.1%, 66.7%, 100.0% and 83.3%, respectively; clinical pregnancy and live birth rates were 100.0%, 50.0%, 44.4%, 50.0% and 33.3%, respectively.&#x0D; Conclusions&#x0D; The results of this study show that pronuclear transfer can result in a high blastocyst formation rate, euploidy rate and live birth rate. This was shown among patients across all age groups, particularly in older patients who often show diminished IVF success rates.

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.

Establishment of oxidative stress biomarkers in oocytes from healthy and bleached scleractinian corals

Corals have a symbiotic relationship with photosynthetic dinoflagellates associated with the host's tissue, those responsible for most of their daily energy gain. Warming ocean water breaks down symbiosis, leading to a phenomenon known as bleaching. Without their primary nutritional source, corals depend on heterotrophy to survive, which can stagnate the reproductive cycle. There are corals, however, that manage to maintain gametogenesis even when bleached. However, the physiology of these gametes in such a situation is unknown. Our study is the first to evaluate the effects of bleaching on the oocytes of a scleractinian coral, as well as the strategies of these cells to maintain the balance of the antioxidant system and cellular homeostasis. We evaluated and quantified markers of oxidative balance in oocytes released from colonies of Mussismilia harttii with different physiological conditions: bleached and healthy. Healthy and bleached coral oocytes were collected considering a post-spawning time curve (0, 5 and 10 h) and frozen to evaluate the relationship between oxidative balance markers and the physiological conditions of coral colonies. Total protein levels, the activity of the antioxidants superoxide dismutase (SOD) and catalase (CAT), and the levels of lipoperoxidation (TBARS), a marker of lipid damage, were measured. The oocytes presented a significant difference between 0 h and 5 h after spawning for all the parameters regardless of the colony's health. Health status modulated SOD activity and TBARS levels, with oocytes from the bleached colony suffering the most lipid damage. These organisms seem to preserve the quality of female gametic cells even 10 h after spawning in both colonies, suggesting a robust antioxidant system capable of prolonging their lifespan and, possibly, their fertilization capability. This response may be related to an intensification of heterotrophy, ensuring nutritional support and thus reproductive effort and quality of gametes even in bleached corals.

Inflammatory placental lesions are specifically observed in healthy oocyte donation pregnancies with extreme fetal-maternal incompatibility

IntroductionOocyte donation (OD) pregnancy is a risk factor for pre-eclampsia (PE). Due to a higher extent of fetal-maternal human leukocyte antigens (HLA) mismatching in OD pregnancies compared to naturally conceived (NC) and in vitro fertilization (IVF) pregnancies, the immune response in OD placentas is probably divergent and affects clinical outcomes. We hypothesized that placental pathology varies among diverse pregnancy conditions and is related to fetal-maternal HLA incompatibility. MethodsPlacental lesions were scored in four patient groups: OD-PE (n = 16), OD-healthy (n = 37), NC-PE (n = 45), and IVF-healthy (n = 17). All combinations were genotyped for HLA-A, -B, -C, -DR, and -DQ to calculate fetal-maternal HLA mismatches. Placentas showing chronic deciduitis with plasma cells were immunofluorescently stained with CD138 and the anti-inflammatory cytokine interleukin-10 (IL-10). ResultsThe distribution and severity of placental lesions varied among groups. The OD-healthy group had the highest inflammation score and greatest extent of chronic deciduitis with plasma cells (p < 0.05). However, the majority of CD138+ plasma cells (90%) in OD-healthy group expressed IL-10, in contrast to the OD-PE group (58%). The OD-healthy group was separated into semi-allogeneic (≤5 HLA mismatches) and fully allogeneic (>5 mismatches) subgroups. The elevated inflammatory pathology score and chronic deciduitis with plasma cells were found more often in the HLA-class-I fully allogeneic OD-healthy group than the IVF-healthy group (p < 0.05). DiscussionPlacental inflammatory lesions are most often present in uncomplicated OD pregnancies. Immune cells that infiltrate these lesions might play an immunosuppressive role to protect OD pregnancies from complications when facing a higher extent of fetal-maternal HLA mismatching.

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

LSM14B coordinates protein component expression in the P-body and controls oocyte maturation

The generation of mature and healthy oocytes is the most critical event in the entire female reproductive process, and the mechanisms regulating this process remain to be studied. Here, we demonstrate that Smith-like (LSM) family member 14B (LSM14B) regulates oocyte maturation, and the loss of LSM14B in mouse ovaries leads to abnormal oocyte MII arrest and female infertility. Next, we find the aberrant transcriptional activation, indicated by abnormal non-surrounded nucleolus and surrounded nucleolus oocyte proportions, and abnormal chromosome assembly and segregation in Lsm14b-deficient mouse oocytes. The global transcriptome analysis suggests that many transcripts involved in cytoplasmic processing body (P-body) function are altered in Lsm14b-deficient mouse oocytes. Deletion of Lsm14b results in the expression and/or localization changes of P-body components (such as LSM14A, DCP1A, and 4E-T). Notably, DDX6, a key component of the P-body, is downregulated and accumulates in the nuclei in Lsm14b-deficient mouse oocytes. Taken together, our data suggest that LSM14B links mouse oocyte maturation to female fertility through the regulation of the P-body.

Heat Stress as a Barrier to Successful Reproduction and Potential Alleviation Strategies in Cattle.

In recent decades, the adverse effects of global warming on all living beings have been unanimously recognized across the world. A high environmental temperature that increases the respiration and rectal temperature of cattle is called heat stress (HS), and it can affect both male and female reproductive functions. For successful reproduction and fertilization, mature and healthy oocytes are crucial; however, HS reduces the developmental competence of oocytes, which compromises reproduction. HS disturbs the hormonal balance that plays a crucial role in successful reproduction, particularly in reducing the luteinizing hormone and progesterone levels, which leads to severe problems such as poor follicle development with a poor-quality oocyte and problems related to maturity, silent estrus, abnormal or weak embryo development, and pregnancy loss, resulting in a declining reproduction rate and losses for the cattle industry. Lactating cattle are particularly susceptible to HS and, hence, their reproduction rate is substantially reduced. Additionally, bulls are also affected by HS; during summer, semen quality and sperm motility decline, leading to compromised reproduction. In summer, the conception rate is reduced by 20-30% worldwide. Although various techniques, such as the provision of water sprinklers, shade, and air conditioning, are used during summer, these methods are insufficient to recover the normal reproduction rate and, therefore, special attention is needed to improve reproductive efficiency and minimize the detrimental effect of HS on cattle during summer. The application of advanced reproductive technologies such as the production of embryos in vitro, cryopreservation during the hot season, embryo transfer, and timed artificial insemination may minimize the detrimental effects of HS on livestock reproduction and recover the losses in the cattle industry.

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-108 Does DNA Fragmentation.level significantly impact clinical outcomes in patients undertaking Assisted Reproduction with using donor oocytes?

Abstract Study question Does an abnormal Sperm DNA Fragmentation Index significantly impact clinical outcomes in patients undergoing Assisted Reproduction with ICSI using donor oocytes? Summary answer No statistical difference was seen in pregnancy or miscarriage rates between high or low DNA fragmentation indexes with oocyte donation cycles using intracytoplasmic sperm injection. What is known already High levels of sperm DNA fragmentation are proposed to have an adverse impact on reproduction. While subject to debate, generally accepted ranges of &amp;gt; 30% considered high and associated with poorer outcomes, with levels &amp;lt;15% considered normal. Impact in the intermediate range if 15-30% is less understood. Potential interventions for elevated DFI include ICSI, lifestyle changes, antioxidant therapy or more controversially TESE. It is thought that high quality oocytes have the potential to repair sperm DNA damage- thus the use of younger donors with higher fertility in oocyte donation cycles may improve outcome and overcome a high DFI. Study design, size, duration A retrospective cohort analysis was performed on our database of donor oocyte FET treatment between January 2016 and December 2020, with 1370 cycles identified Screening to identify those couples where pre- treatment sperm DFI was performed as part of the treatment workup, and DFI was analysed in 9% of these cycle (n = 124). Clinical outcomes were recorded including positive HCG, clinical pregnancy rate, miscarriage rate and ongoing pregnancy rate at 12/40 gestation, and compared between groups. Participants/materials, setting, methods Oocyte donation cycles with baseline Sperm DFI measurements in a tertiary academic centre were identified. Donor treatments were chosen to reduce the impact of female factor variation on results. 49 cases had normal DFI(&amp;lt;15%), and were compared to 21 patients in the high group ( &amp;gt; 30). 54 patients were on the intermediate range (15-30%) and were excluded from the analysis. Pregnancy and miscarriage rates were compared between high and low groups to assess impact of DFI. Main results and the role of chance Elevated DFI has been proposed to have an abnormal impact on assisted reproduction outcomes. Other studies have suggested that ICSI may compensate for this, or that healthy oocytes may be able to negate any detrimental impact from the sperm. With autologous gamete treatments, large variations in the expected prognosis are due to female characteristics such as age, ovarian reserve or gynaecological disorders such as endometriosis or PCOS. This use of younger egg donors with normal AMH and the exclusion of any identifiable pelvic abnormality allows for some standardisation of the female cohort, therefore allowing a more direct assessment of the impact of any male factor abnormality on the outcome. Positive HCG rates per embryo transfer were the same between DFI groups. Normal 53.1% (26/49) vs High 47.65% (10/21) p = 0.68 Similarly there was no difference in Clinical Pregnancy Rates based on DFI level Normal 44.9% (22/49) vs High 47.7% (10/21) p = 0.83 Miscarriage rates per positive HCG test were again not impacted by male sperm DFI: Normal 30.7% (8/26) vs High 33.3 (3/10) p = 0.72 The study data demonstrates that in treatment cycles using a combination of ICSI and good quality oocytes, the clinical outcomes do not appear to be impacted by raised DFI Limitations, reasons for caution We previously demonstrated that fertilisation and blastulation rates were unaffected by DFI. The study was limited by a relatively small sample size, as DFI analysis was not routinely measured before treatment. Larger multi-centre studies to increase the study population and statistical power would be useful for confirmation of the findings. Wider implications of the findings Our data suggests that biochemical, clinical and ongoing pregnancy rates, and also miscarriage rate, were not adversely impacted by high DFI in donor oocyte cycles.This could be attributed to the ability of a high quality oocyte to repair sperm DNA damage when combined with ICSI for sperm selection and fertilisation. Trial registration number Not applicable

P-250 The exclusive role of sperm DNA fragmentation on the clinical outcome of a sibling donor oocyte cohort

Abstract Study question Can sperm chromatin fragmentation (SCF) overwhelm the oocyte repair mechanism of healthy, young oocytes and impair embryo development and clinical outcome? Summary answer Elevated SCF, with its double-stranded DNA (dsDNA) component, remarkably impairs clinical outcome, despite oocyte repair mechanisms present in a sibling donor oocyte cohort. What is known already Despite a normal semen analysis, approximately 10-20% of males of reproductive age still have a subtle infertility. Therefore, conventional semen analysis is insufficient to identify the specific issues related to sperm function and its embryo developmental competence. Indeed, SCF with its dsDNA break component, is known to be associated with poor clinical outcome such as poor embryo development and pregnancy loss, by possibly increasing structural chromosomal abnormalities. Although a healthy oocyte may address certain levels of SCF, these oocyte repair mechanisms are incapable of repairing dsDNA breaks. Study design, size, duration Thirty-one couples were divided according to the degree and type of SCF, whether normal or abnormal, and shared the same sibling donor oocytes. The male partners in both groups had comparable semen parameters. Total SCF and dsDNA fragmentation were assessed in all men. Fertilization, implantation, clinical pregnancy (CPR; +FHB), delivery, and pregnancy loss rates were compared between the two cohorts. Participants/materials, setting, methods A total of 31 infertile couples underwent ICSI cycles utilizing split sibling donor oocytes due to advanced maternal age or premature ovarian insufficiency. Oocytes from the same donor were equally allocated among 2 couples. Semen analyses were performed according to WHO 6th edition. Overall SCF was assessed by TUNEL with a ≤ 15% normal threshold. Double-strand DNA (dsDNA) fragmentation was assessed by neutral comet assay with ≤3% considered normal. Main results and the role of chance A total of 18 male partners (44.3 ± 7yrs) with a normal SCF of 8.9 ± 2% had their spermatozoa injected into sibling donor oocytes, while 13 male partners with abnormal SCF at 25.4% ± 11 (P&amp;lt;0.001) had their spermatozoa injected into the remaining half. In the latter, the average dsDNA fragmentation rate was 3.6%, while in the former it was 0.3% (P&amp;lt;0.001). Sibling donor oocytes injected with normal SCF resulted in a fertilization of 87.4% (146/167), implantation of 56.4% (22/39), +bHCG of 69.0% (20/29), CPR of 62.1% (18/29), and delivery rate of 51.7% (15/29). The other half of the sibling oocytes, injected with spermatozoa with elevated SCF, resulted in a fertilization of 70.8% (97/137) (P&amp;lt;0.001), implantation of 15.4% (4/26) (P&amp;lt;0.01), +bHCG of 42.1% (8/19), CPR of 21.1% (4/19) (P&amp;lt;0.01), and delivery rate of 10.5% (2/19) (P&amp;lt;0.01). The pregnancy loss in cycles utilizing the oocyte cohort injected with spermatozoa with normal SCF was 8.7% (2/23), while the pregnancy loss in those injected with spermatozoa with elevated SCF was 50.0% (2/4). Limitations, reasons for caution This preliminary study indicates that SCF and dsDNA fragmentation negatively affects clinical outcome, overwhelming oocyte repair mechanisms. Nonetheless, the study has a limited number of observations and needs to be validated in a larger cohort. Wider implications of the findings SCF, with its dsDNA fragmentation component, seems to contribute a higher incidence of chromosomal abnormalities. The compounded aneuploidy of male and female gametes may explain the compromised embryo development and higher pregnancy loss rate observed in infertile couples with apparently normal semen parameters and younger oocytes. Trial registration number not applicable