GDF9His209GlnfsTer6/S428T and GDF9Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement.

BACKGROUND: Antral follicles consist of an oocyte cumulus complex surrounding by somatic cells, including mural granulosa cells as the inner layer and theca cells as the outsider layer. The communications between oocytes and granulosa cells have been extensively explored in in vitro studies, however, the role of oocyte-derived factor GDF9 on in vivo antral follicle development remains elusive due to lack of an appropriate animal model. Clinically, the phenotype of GDF9 variants needs to be determined. METHODS: Whole-exome sequencing (WES) was performed on two unrelated infertile women characterized by an early rise of estradiol level and defect in follicle enlargement. Besides, WES data on 1,039 women undergoing ART treatment were collected. A Gdf9Q308X/S415T mouse model was generated based on the variant found in one of the patients. RESULTS: Two probands with bi-allelic GDF9 variants (GDF9His209GlnfsTer6/S428T, GDF9Q321X/S428T) and eight GDF9S428T heterozygotes with normal ovarian response were identified. In vitro experiments confirmed that these variants caused reduction of GDF9 secretion, and/or alleviation in BMP15 binding. Gdf9Q308X/S415T mouse model was constructed, which recapitulated the phenotypes in probands with abnormal estrogen secretion and defected follicle enlargement. Further experiments in mouse model showed an earlier expression of STAR in small antral follicles and decreased proliferative capacity in large antral follicles. In addition, RNA sequencing of granulosa cells revealed the transcriptomic profiles related to defective follicle enlargement in the Gdf9Q308X/S415T group. One of the downregulated genes, P4HA2 (a collagen related gene), was found to be stimulated by GDF9 protein, which partly explained the phenotype of defective follicle enlargement. CONCLUSIONS: GDF9 bi-allelic variants contributed to the defect in antral follicle development. Oocyte itself participated in the regulation of follicle development through GDF9 paracrine effect, highlighting the essential role of oocyte-derived factors on ovarian response.

Mitochondrial Unfolded Protein Response Gene Clpp Is Required for Oocyte Function and Female Fertility.

Mitochondrial unfolded protein stress response (mtUPR) plays a critical role in regulating cellular and metabolic stress response and helps maintain protein homeostasis. Caseinolytic peptidase P (CLPP) is one of the key regulators of mtUPR and promotes unfolded protein degradation. Previous studies demonstrated that global deletion of Clpp resulted in female infertility, whereas no impairment was found in the mouse model with targeted deletion of Clpp in cumulus/granulosa cells. These results suggest the need to delineate the function of Clpp in oocytes. In this study, we aimed to further explore the role of mtUPR in female reproductive competence and senescence using a mouse model. Oocyte-specific targeted deletion of Clpp in mice resulted in female subfertility associated with metabolic and functional abnormalities in oocytes, thus highlighting the importance of CLPP-mediated protein homeostasis in oocyte competence and reproductive function.

Disruption of mitochondrial unfolded protein response results in telomere shortening in mouse oocytes and somatic cells.

Caseinolytic peptidase P (CLPP) plays a central role in mitochondrial unfolded protein response (mtUPR) by promoting the breakdown of misfolded proteins and setting in motion a cascade of reactions to re-establish protein homeostasis. Global germline deletion of Clpp in mice results in female infertility and accelerated follicular depletion. Telomeres are tandem repeats of 5'-TTAGGG-3' sequences found at the ends of the chromosomes. Telomeres are essential for maintaining chromosome stability during somatic cell division and their shortening is associated with cellular senescence and aging. In this study, we asked whether the infertility and ovarian aging phenotype caused by global germline deletion of Clpp is associated with somatic aging, and tested telomere length in tissues of young and aging mice. We found that impaired mtUPR caused by the lack of CLPP is associated with accelerated telomere shortening in both oocytes and somatic cells of aging mice. In addition, expression of several genes that maintain telomere integrity was decreased, and double-strand DNA breaks were increased in telomeric regions. Our results highlight how impaired mtUPR can affect telomere integrity and demonstrate a link between loss of mitochondrial protein hemostasis, infertility, and somatic aging.

A Proteomic Analysis of Human Follicular Fluid: Proteomic Profile Associated with Embryo Quality.

Embryo selection is a key point of in vitro fertilization (IVF). The most commonly used method for embryo selection is morphological assessment. However, it is sometimes inaccurate. Follicular fluid (FF) contains a complex mixture of proteins that are essential for follicle development and oocyte maturation. Analyzing human FF proteomic profiles and identifying predictive biomarkers might be helpful for evaluating embryo quality. A total of 22 human FF samples were collected from 19 infertile women who underwent IVF/intracytoplasmic sperm injection (ICSI) treatment between October 2021 and November 2021. FFs were grouped into two categories on the basis of the day 3 embryo quality, grade I or II in the hqFF group and grade III in the nhqFF group. FF was analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The key differentially expressed proteins (DEPs) were validated by parallel reaction monitoring (PRM) and enzyme-linked immunosorbent assay (ELISA). Differentially expressed proteins were further analyzed using DAVID software. A total of 558 proteins were identified, of which 50 proteins were differentially expressed in the hqFF versus nhqFF group, including 32 upregulated proteins (> 1.20-fold, P < 0.05) and 18 downregulated proteins (< 0.67-fold, P < 0.05). Bioinformatics analyses showed that the upregulated DEPs were enriched in components of the coagulation and complement systems and negative regulation of peptidase activity, while the downregulated DEPs were enriched in molecular function of extracellular matrix structural and constituent collagen binding. Our results suggested that a number of protein biomarkers in FF were associated with embryo quality. It may help develop an effective and noninvasive method for embryo selection.

An unbiased approach of molecular characterization of the endometrium: toward defining endometrial-based infertility.

Infertility is a complex condition affecting millions of couples worldwide. The current definition of infertility, based on clinical criteria, fails to account for the molecular and cellular changes that may occur during the development of infertility. Recent advancements in sequencing technology and single-cell analysis offer new opportunities to gain a deeper understanding of these changes. The endometrium has a potential role in infertility and has been extensively studied to identify gene expression profiles associated with (impaired) endometrial receptivity. However, limited overlap among studies hampers the identification of relevant downstream pathways that could play a role in the development of endometrial-related infertility. To address these challenges, we propose sequencing the endometrial transcriptome of healthy and infertile women at the single-cell level to consistently identify molecular signatures. Establishing consensus on physiological patterns in endometrial samples can aid in identifying deviations in infertile patients. A similar strategy has been used with great success in cancer research. However, large collaborative initiatives, international uniform protocols of sample collection and processing are crucial to ensure reliability and reproducibility. Overall, the proposed approach holds promise for an objective and accurate classification of endometrial-based infertility and has the potential to improve diagnosis and treatment outcomes.

NLRP14 deficiency causes female infertility with oocyte maturation defects and early embryonic arrest by impairing cytoplasmic UHRF1 abundance.

Oocyte maturation is vital to attain full competence required for fertilization and embryogenesis. NLRP14 is preferentially expressed in mammalian oocytes and early embryos. Yet, the role and molecular mechanism of NLRP14 in oocyte maturation and early embryogenesis are poorly understood, and whether NLRP14 deficiency accounts for human infertility is unknown. Here, we found that maternal loss of Nlrp14 resulted in sterility with oocyte maturation defects and early embryonic arrest (EEA). Nlrp14 ablation compromised oocyte competence due to impaired cytoplasmic and nuclear maturation. Importantly, we revealed that NLRP14 maintained cytoplasmic UHRF1 abundance by protecting it from proteasome-dependent degradation and anchoring it from nuclear translocation in the oocyte. Furthermore, we identified compound heterozygous NLRP14 variants in women affected by infertility with EEA, which interrupted the NLRP14-UHRF1 interaction and decreased UHRF1 levels. Our data demonstrate NLRP14 as a cytoplasm-specific regulator of UHRF1 during oocyte maturation, providing insights into genetic diagnosis for female infertility.

Local production of 17ß-oestradiol in the endometrium during the implantation window: a pilot study.

Abstract: Sex steroids are converted to bioactive metabolites and vice versa by endometrial steroid-metabolising enzymes. Studies indicate that alterations in this metabolism might affect endometrial receptivity. This pilot study determined whether the endometrial formation and inactivation of 17ß-oestradiol differed between the supposedly embryo-receptive endometrium and non-receptive endometrium of women undergoing IVF/intracytoplasmic sperm injection (ICSI). Endometrial biopsies were obtained from IVF/ICSI patients 5-8 days after ovulation in a natural cycle, prior to their second IVF/ICSI cycle with fresh embryo transfer (ET). Endometrial biopsies from patients who achieved clinical pregnancy after fresh ET (n = 15) were compared with endometrial biopsies from patients that did not conceive after fresh ET (n = 15). Formation of 17ß-oestradiol (oxidative 17ß-hydroxysteroid dehydrogenases (HSDs)), oestrone (reductive HSD17Bs) and inhibition of HSD17B1 activity were determined by high-performance liquid chromatography. The endometrial transcriptome was profiled using RNA sequencing followed by principal component analysis and differentially expressed gene analysis. The false discovery rate-adjusted P < 0.05 and log fold change >0.5 were selected as the screening threshold. Formation and inactivation of 17ß-oestradiol resulted similar between groups. Inhibition of HSD17B1 activity was significantly higher in the non-pregnant group when only primary infertile women (n = 12) were considered (27.1%, n = 5 vs 16.2%, n = 7, P = 0.04). Gene expression analysis confirmed the presence of HSD17B1 (encoding HSD17B1), HSD17B2 (encoding HSD17B2) and 33 of 46 analysed steroid metabolising enzymes in the endometrium. In the primary infertile subgroup (n = 10) 12 DEGs were found including LINC02349 which has been linked to implantation. However, the exact relationship between steroid-metabolising enzyme activity, expression and implantation outcome requires further investigation in larger, well-defined patient groups. Lay summary: Sex hormones are produced and broken down by enzymes that can be found in the endometrium (the inner lining of the womb). This enzyme activity might influence the chances of becoming pregnant. We compared (i) enzyme activity in the endometrium of 15 women who did and 15 women who did not become pregnant in their second in vitro fertilisation attempt, (ii) how enzyme activity can be blocked by an inhibitor, and (iii) differences in gene expression (the process by which instructions in our DNA are converted into a product). Enzyme activity was similar between groups. We found that in women who have never been pregnant in the past, inhibition of enzyme activity was higher and found differences in a gene that has been linked to the implantation of the embryo, but future studies should be performed in larger, well-defined patient groups to confirm these findings.

Autologous non-invasively derived stem cells mitochondria transfer shows therapeutic advantages in human embryo quality rescue.

BACKGROUND: The decline in the quantity and quality of mitochondria are closely associated with infertility, particularly in advanced maternal age. Transferring autologous mitochondria into the oocytes of infertile females represents an innovative and viable strategy for treating infertility, with no concerns regarding ethical considerations. As the donor cells of mitochondria, stem cells have biological advantages but research and evidence in this area are quite scarce. METHODS: To screen out suitable human autologous ooplasmic mitochondrial donor cells, we performed comprehensive assessment of mitochondrial physiology, function and metabolic capacity on a varity of autologous adipose, marrow, and urine-derived mesenchymal stromal cells (ADSC, BMSC and USC) and ovarian germline granulosa cells (GC). Further, to explore the biosafety, effect and mechanism of stem cell-derived mitochondria transfer on human early embryo development, randomized in-vitro basic studies were performed in both of the young and aged oocytes from infertile females. RESULTS: Compared with other types of mesenchymal stromal cells, USC demonstrated a non-fused spherical mitochondrial morphology and low oxidative stress status which resembled the oocyte stage. Moreover, USC mitochondrial content, activity and function were all higher than other cell types and less affected by age, and it also exhibited a biphasic metabolic pattern similar to the pre-implantation stage of embryonic development. After the biosafety identification of the USC mitochondrial genome, early embryos after USC mitochondrial transfer showed improvements in mitochondrial content, activity, and cytoplasmic Ca2+ levels. Further, aging embryos also showed improvements in embryonic morphological indicators, euploidy rates, and oxidative stress status. CONCLUSION: Autologous non-invasively derived USC mitochondria transfer may be an effective strategy to improve embryonic development and metabolism, especially in infertile females with advanced age or repeated pregnancy failure. It provides evidence and possibility for the autologous treatment of infertile females without invasive and ethical concerns.

Novel variants in TUBB8 gene cause multiple phenotypic abnormalities in human oocytes and early embryos.

BACKGROUND: The genotype-phenotype relationships between TUBB8 variants and female infertility are difficult to clearly define due to the complex inheritance patterns and the highly heterogeneous phenotypes. This study aims to identify novel TUBB8 variants and relevant phenotypes in more infertile females. METHODS: A total of 35 females with primary infertility were recruited from two reproductive centers and investigated for identifying variants in TUBB8. Pedigree analysis, in-silico analysis and molecular remodeling were performed to assess their clinical significance. The effects of the variants on human oocytes and embryos as well as HeLa cells were analyzed by morphological observations, immunostaining and Western blot. RESULTS: We totally identified five novel variants (p.G13R, p.Y50C, p.T136I, p.F265V and p.T366A) and five previously reported variants (p.I4L, p.L42V, p.Q134*, p.V255M and p.V349I) in TUBB8 from 9 unrelated females with primary infertility. These variants were rare and highly conserved among different species, and were inherited in autosomal dominant/recessive patterns, or occurred de novo. In vitro functional assays in HeLa cells revealed that exogenous expression of mutant TUBB8 proteins caused different degrees of microtubule structural disruption. The existence of these pathogenic TUBB8 variants finally induced oocyte maturation arrest or morphological abnormalities, fertilization failure, cleavage failure, embryonic development defects and implantation failure in the affected females. CONCLUSION: These findings enriched the variant spectrum of TUBB8 gene and could contribute to optimize genetic counselling and clinical management of females with primary infertility.

METTL3-regulated m6A modification impairs the decidualization of endometrial stromal cells by regulating YTHDF2-mediated degradation of FOXO1 mRNA in endometriosis-related infertility.

BACKGROUND: Endometriosis-related infertility is a common worldwide reproductive health concern. Despite ongoing research, the causes of infertility remain unclear. Evidence suggests that epigenetic regulation is crucial in reproduction. However, the role of N6-methyladenosine (m6A) modification of RNA in endometriosis-related infertility requires further investigation. METHODS: We examined the expression of m6A and methyltransferase-like 3 (METTL3) in endometrial samples taken from normal fertile women in the proliferative phase (the NP group) or the mid-secretory phase (the NS group) or from women with endometriosis-related infertility at the mid-secretory phase (the ES group). We treated primary endometrial stromal cells (ESCs) with medroxyprogesterone acetate and 8-Bromo-cyclic adenosine monophosphate for in vitro decidualization and detected the expression of m6A, METTL3, and decidual markers. We analyzed the expression of m6A, METTL3, and forkhead box O1 (FOXO1) in ESCs from normal fertile women (the ND group) or women with endometriosis-related infertility (the ED group). We also assessed the expression of m6A, METTL3, and decidual markers, as well as the embryo adhesion rate, upon METTL3 overexpression or knockdown. Additionally, we investigated the role of METTL3 in embryo implantation in vivo by applying mice with endometriosis. Furthermore, we performed RNA stability assays, RNA immunoprecipitation (RIP), and methylated RIP assays to explore the mechanisms underlying the regulation of FOXO1 by METTL3-mediated m6A. RESULTS: The expression of m6A and METTL3 was reduced only in the NS group; the NP and ES groups demonstrated increased m6A and METTL3 levels. m6A and METTL3 levels decreased in ESCs with prolonged decidual treatment. Compared to the ND group, m6A and METTL3 levels in the ED group increased after decidual treatment, whereas the expression of FOXO1 decreased. METTL3 overexpression suppressed the expression of decidual markers and embryo implantation in vitro; METTL3 knockdown exhibited the opposite effect. Inhibition of METTL3 promoted embryo implantation in vivo. Furthermore, we observed that METTL3-mediated m6A regulated the degradation of FOXO1 mRNA through YTHDF2, a m6A binding protein. CONCLUSIONS: METTL3-regulated m6A promotes YTHDF2-mediated decay of FOXO1 mRNA, thereby affecting cellular decidualization and embryo implantation. These findings provide novel insights into the development of therapies for women with endometriosis-related infertility.

Exosomal miR-205-5p Improves Endometrial Receptivity by Upregulating E-Cadherin Expression through ZEB1 Inhibition.

Endometrial receptivity is a complex process that prepares the uterine endometrium for embryo implantation; insufficient endometrial receptivity is one of the causes of implantation failure. Here, we analyzed the microRNA expression profiles of exosomes derived from both receptive (RL95-2) and non-receptive (AN3-CA) endometrial epithelial cell (EEC) lines to identify exosomal miRNAs closely linked to endometrial receptivity. Among the 466 differentially expressed miRNAs, miR-205-5p was the most highly expressed in exosomes secreted from receptive RL95-2 cells. miR-205-5p, enriched at the adhesive junction, was closely related to endometrial receptivity. ZEB1, a transcriptional repressor of E-cadherin associated with endometrial receptivity, was identified as a direct target of miR-205-5p. miR-205-5p expression was significantly lower in the endometrial tissues of infertile women than in that of non-infertile women. In vivo, miR-205-5p expression was upregulated in the post-ovulatory phase, and its inhibitor reduced embryo implantation. Furthermore, administration of genetically modified exosomes overexpressing miR-205-5p mimics upregulated E-cadherin expression by targeting ZEB1 and improved spheroid attachment of non-receptive AN3-CA cells. These results suggest that the miR-205-5p/ZEB1/E-cadherin axis plays an important role in regulating endometrial receptivity. Thus, the use of exosomes harboring miR-205-5p mimics can be considered a potential therapeutic approach for improving embryo implantation.

Down-regulation of the FTO gene in follicular fluid of infertile women with ovarian endometriosis.

OBJECTIVE: To evaluate FTO concentrations in follicular fluid (FF) of women with ovarian endometriosis (OE) and controls women without OE undergoing in vitro fertilization-embryo transfer (IVF-ET). METHODS: FTO concentrations in FF were measured in 74 patients (37 in the control group and 37 in the OE group) by ELISA. We measured the expression of FTO in GCs of 40 patients (19 in the control group and 21 in the OE group) by RT-qPCR. The level of m6A in GCs was measured in 20 patients (10 in the control group and 10 in the OE group) by colorimetry. RESULTS: Compared with the control group, FTO concentrations in FF (6.92 ± 0.44 vs. 5.67 ± 0.40 ng/ml) (p <.05) and FTO mRNA level in GCs of OE group were decreased significantly (p <.05), and the level of m6A was increased (0.21 ± 0.01 vs. 0.17 ± 0.03 ng) (p >.05). CONCLUSIONS: The FTO concentrations in FF of infertility women with OE are decreased, which may be related to the impaired oocyte quality in endometriosis patients.

Endometrial receptivity change: ultrasound evaluation on ovulation day and transplantation day during the natural frozen embryo transfer cycle.

Objective: To obtain quantitative and comprehensive results of the changes in comprehensive ER indicators from ovulation day to transplantation day by ultrasonography during the natural frozen-thawed embryo transfer cycle (FET). Methods: This is a prospective analysis of 230 infertile women undergoing their first FET cycles from April 2019 to July 2021. To evaluate ER, ultrasound scans were performed on the days of ovulation and embryo transfer for all included patients. All included patients were divided into a pregnancy group and a nonpregnancy group according to whether clinical pregnancy was achieved. The ER changes from ovulation day to transplantation day in the overall study population (n=230), pregnancy group (n=158) and nonpregnancy group (n=72) were analyzed. Results: In the overall population, type C was predominant on ovulation day, but type B was the most common on transplantation day (P<0.001). From ovulation day to transplantation day, endometrial thickness was significantly increased (11.26 ± 2.14 vs. 11.89 ± 2.08 mm, P<0.001), but endometrial volume (4.26 ± 1.75 vs. 4.03 ± 1.62 ml, P<0.001), endometrial VI (1.34 ± 1.64 vs. 0.95 ± 1.99, P<0.001), VFI (0.47 ± 0.72 vs. 0.40 ± 1.03, P<0.001), subendometrial VI (5.04 ± 3.89 vs. 3.29 ± 2.92, P<0.001), FI (34.07 ± 4.61 vs. 33.41 ± 5.30, p=0.004), VFI (2.07 ± 2.65 vs. 1.19 ± 1.19, P<0.001) and frequency of endometrial peristalsis (2.90 ± 1.44 vs. 1.40 ± 1.41, P<0.001) were significantly decreased. In the pregnancy group, the changes in all ultrasound parameters were in the same direction as those in the overall population. In the nonpregnancy group, except for endometrial volume and VI, which showed no difference, other ultrasound parameters showed the same direction of change as those in the overall population. No significant difference was found in the pregnancy probability among the different absolute change groups. Conclusion: During a natural cycle, the morphology of the endometrium changes mostly from type C to type B, the endometrial thickness increases, and the volume decreases. The blood supply of the endometrium, the subendometrial 5 mm and the frequency of peristalsis decrease from ovulation day to transplantation day. Compared with the nonpregnancy group, the pregnancy group tended to have more obvious decreases in endometrial volume and blood flow perfusion. However, these endometrial changes do not mean that pregnancy is bound to occur. endometrial receptivity, in vitro fertilization, frozen-thawed embryo transfer, natural cycle, ultrasound evaluation, ovulation day, transplantation day.

Oxidative stress and female infertility: the role of follicular fluid soluble receptor of advanced glycation end-products (sRAGE) in women with endometriosis.

Oxidative Stress (OS) relates to the pathophysiology of endometriosis by activation of the inflammation process in the ovary, abdomen, peritoneum and endometrium. Advanced Glycation end-products (AGEs) cause oxidative damage to the follicles of the ovary. This study aims to investigate the correlation of follicular fluid soluble receptor of AGEs (FF sRAGE) with fertility-related parameters in infertile women with endometriosis. From January 2012 to July 2015 twenty-four women diagnosed with mild to moderate endometriosis aged 28-38 years underwent assisted reproduction. sRAGE levels measured in FF were related to lifestyle factors, sociodemographic characteristics, gynaecological and obstetric parameters, hormonal status and fertilization. sRAGE was inversely associated with BMI (r = -0.503, p = 0.012). No significant association of sRAGE with age (p = 0.714) or alcohol consumption (p = 0.882) was found. Pearson's r correlation coefficient revealed that sRAGE was positively associated with serum AMH (r = 0.518, p = 0.009), FF AMH (r = 0.630, p = 0.001), number of follicles >15mm (r = 0.601, p = 0.002), total number of follicles aspirated (r = 0.698, p < 0.001), total number of MII oocytes obtained, (r = 0.757, p < 0.001) and the number of embryos with good embryo scoring (suitable for ET) (r = 0.522, p = 0.009). It seems that measurement of FF RAGE might be a useful predictive marker for IVF success in infertile women with endometriosis undergoing assisted reproduction.

Single-cell multi-omics sequencing reveals chromosome copy number inconsistency between trophectoderm and inner cell mass in human reconstituted embryos after spindle transfer.

STUDY QUESTION: Is the chromosome copy number of the trophectoderm (TE) of a human reconstituted embryos after spindle transfer (ST) representative of the inner cell mass (ICM)? SUMMARY ANSWER: Single-cell multi-omics sequencing revealed that ST blastocysts have a higher proportion of cell lineages exhibiting intermediate mosaicism than conventional ICSI blastocysts, and that the TE of ST blastocysts does not represent the chromosome copy number of ICM. WHAT IS KNOWN ALREADY: Preimplantation genetic testing for aneuploidy (PGT-A) assumes that TE biopsies are representative of the ICM, but the TE and ICM originate from different cell lineages, and concordance between TE and ICM is not well-studied, especially in ST embryos. STUDY DESIGN, SIZE, DURATION: We recruited 30 infertile women who received treatment at our clinic and obtained 45 usable blastocysts (22 from conventional ICSI and 23 reconstituted embryos after ST). We performed single-cell multi-omics sequencing on all blastocysts to predict and verify copy number variations (CNVs) in each cell. We determined the chromosome copy number of each embryo by analysing the proportion of abnormal cells in each blastocyst. We used the Bland-Altman concordance and the Kappa test to evaluate the concordance between TE and ICM in the both groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted at a public tertiary hospital in China, where all the embryo operations, including oocytes retrieval, ST, and ICSI, were performed in the embryo laboratory. We utilized single-cell multi-omics sequencing technology at the Biomedical Pioneering Innovation Center, School of Life Sciences, Peking University, to analyse the blastocysts. Transcriptome sequencing was used to predict the CNV of each cell through bioinformatics analysis, and the results were validated using the DNA methylation library of each cell to confirm chromosomal normalcy. We conducted statistical analysis and graphical plotting using R 4.2.1, SPSS 27, and GraphPad Prism 9.3. MAIN RESULTS AND THE ROLE OF CHANCE: Mean age of the volunteers, the blastocyst morphology, and the developmental ratewere similar in ST and ICSI groups. The blastocysts in the ST group had some additional chromosomal types that were prone to variations beyond those enriched in the blastocysts of the ICSI group. Finally, both Bland-Altman concordance test and kappa concordancetest showed good chromosomal concordance between TE and ICM in the ICSI blastocysts (kappa = 0.659, P < 0.05), but not in ST blastocysts (P = 1.000), suggesting that the TE in reconstituted embryos is not representative of ICM. Gene functional annotation (GO and KEGG analyses) suggests that there may be new or additional pathways for CNV generation in ST embryos compared to ICSI embryos. LIMITATIONS, REASONS FOR CAUTION: This study was mainly limited by the small sample size and the limitations of single-cell multi-omics sequencing technology. To select eligible single cells, some cells of the embryos were eliminated or not labelled, resulting in a loss of information about them. The findings of this study are innovative and exploratory. A larger sample size of human embryos (especially ST embryos) and more accurate molecular genetics techniques for detecting CNV in single cells are needed to validate our results. WIDER IMPLICATIONS OF THE FINDINGS: Our study justifies the routine clinical use of PGT-A in ICSI blastocysts, as we found that the TE is a good substitute for ICM in predicting chromosomal abnormalities. While PGT-A is not entirely accurate, our data demonstrate good clinical feasibility. This trial was able to provide correct genetic counselling to patients regarding the reliability of PGT-A. Regarding ST blastocysts, the increased mosaicism rate and the inability of the TE to represent the chromosomal copy number of the ICM are both biological characteristics that differentiate them from ICSI blastocysts. Currently, ST is not used clinically on a large scale to produce blastocysts. However, if ST becomes more widely used in the future, our study will be the first to demonstrate that the use of PGT-A in ST blastocysts may not be as accurate as PGT-A for ICSI blastocysts. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the National Key R&D Program of China (2018YFA0107601) and the National Key R&D Program of China (2018YFC1003003). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Increased serine synthesis in cumulus cells of young infertile women with diminished ovarian reserve.

STUDY QUESTION: What are the differences in gene expression of cumulus cells (CCs) between young women with diminished ovarian reserve (DOR) and those of similar age with normal ovarian reserve (NOR)? SUMMARY ANSWER: Gene expression and metabolome profiling analysis demonstrate that the de novo serine synthesis pathway (SSP) is increased in the CCs of young women with DOR. WHAT IS KNOWN ALREADY: The incidence of DOR has risen, tending to present at younger ages. Its mechanisms and aetiologies are still poorly understood. Abnormal metabolism is present in luteinized CCs of patients with DOR. Previous studies have revealed that mitochondrial dysfunction and impaired oxidative phosphorylation in CCs are related to DOR in women of advanced age. The pathogenic mechanisms likely differ between young women with DOR and cases associated with advanced maternal age. Several studies have examined amino acid metabolism in the follicle, with a focus on embryo development, but less information is available about CCs. The physiological significance of de novo serine synthesis in follicles and oocytes remains largely unknown. STUDY DESIGN, SIZE, DURATION: CC samples were obtained from 107 young infertile women (age <38 years) undergoing ICSI, from July 2017 to June 2019, including 54 patients with DOR and 53 patients with NOR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte development data were analysed retrospectively. Comprehensive genome-wide transcriptomics of CCs was performed. Differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to categorize the functions of the DEGs and identify significantly enriched pathways. The transcript and protein levels of key enzymes involved in serine synthesis were verified in additional samples using quantitative real-time PCR (qRT-PCR) (n = 10) and capillary western blotting (n = 36). Targeted metabolomics of amino acids in CC extracts was performed by ultrahigh-performance liquid MS (UHPLC-MS/MS). MAIN RESULTS AND THE ROLE OF CHANCE: The number of oocytes (2.4 ± 2.2 versus 12.1 ± 5.3) and metaphase II oocytes (2.1 ± 2.0 versus 9.9 ± 4.9) retrieved was significantly decreased in the DOR versus the NOR group, respectively (P < 0.0001). The rates of fertilization (80.7% versus 78.8%), viable embryos (73.7% versus 72.5%), and high-quality embryos (42.8% versus 49.0%) did not differ between the DOR and NOR groups, respectively (P > 0.05). A total of 95 DEGs were found by transcriptome sequencing. GO and KEGG analyses demonstrated that the DEGs were linked to amino acid metabolism and suggested significantly higher activity of the de novo SSP in the CCs of young women with DOR. Further qRT-PCR and capillary western blotting revealed that key enzymes (PHGDH, PSAT1, PSPH, and SHMT2) involved in de novo serine synthesis were upregulated, and UHPLC-MS/MS analysis showed increases in serine and glycine (a downstream product of serine) levels in the CCs of young patients with DOR. Our data clearly demonstrate that the de novo SSP, which diverts 3-phosphoglycerate from glycolysis to serine synthesis, was upregulated in young DOR CCs. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Regarding the reproductive capacity of young patients DOR, the pregnancy outcomes were not analysed. The sample size was limited, and only women undergoing ICSI were examined since this was a prerequisite for the acquisition of CCs, which may cause selection bias. The exact mechanisms by which the SSP in CCs regulates ovarian reserve still require further study. WIDER IMPLICATIONS OF THE FINDINGS: Our research presents new evidence that alterations of the SSP in CCs of young infertile women are associated with DOR. We believe this is a significant contribution to the field, which should be key for understanding the cause and mechanisms of ovarian hypofunction in young women. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Ministry of Science and Technology of China (2018YFC1005001) and National Natural Science Foundation of China (31601197). There were no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Increased Expression of TGF-ß1 Contributes to the Downregulation of Progesterone Receptor Expression in the Eutopic Endometrium of Infertile Women with Minimal/Mild Endometriosis.

Endometriosis is a hormone-dependent disease associated with impaired immunoregulation. In our recent study, we have characterized the trascriptomic transformation of eutopic endometrium from patients with minimal/mild endometriosis and controls across the menstrual cycle. However, the regulatory mechanism of altered immune microenvironment in eutopic endometrial stromal cells (ESCs) remains unclear. Here, we want to explore the regulation of immune cell to progesterone resistance and endometrial receptivity in the eutopic ESCs by cytokine (TGF-ß1), and to understand the effect of TGF-ß1 on the decidualization of the eutopic ESCs. Primary culture of eutopic ESCs was performed to explore the effects of TGF-ß1 on the expression of Smad and progesterone receptor (PR) and the in vitro decidualization. Additionally, co-immunoprecipitation (Co-IP) was used to explore the direct interaction between Smad and PR. We found an attenuate expression of PRB protein (p=0.026) after using TGF-ß1 in eutopic ESCs, although the difference of PRA before and after treatment was not significant (p=0.678). Similarly, the results of qRT-PCR showed that the mRNA level of PR (p<0.001), PRB (p=0.003) and HOXA10 (p<0.001) decreased significantly after TGF-ß1 treatment, but that increased (p<0.023, for all) after SB431542 treatment in the eutopic ESCs. Moreover, TGF-ß1 has a negative effect on the in vitro decidualization of eutopic ESCs (p=0.003). And the group with treatment of both TGF-ß1 and SB435142 in eutopic ESCs showed significant decidual-like changes with increased prolactin level (p=0.01). We did not observe any physical interaction between the PR and p-Smad3/Smad3 proteins by using Co-IP. By activating TGF-ß/Smad signaling in eutopic ESCs, elevated TGF-ß1 from CD45+ immune cells could attenuate expression of PR, and further decrease endometrial receptivity.

Autoimmune thyroid disease disrupts immune homeostasis in the endometrium of unexplained infertility women-a single-cell RNA transcriptome study during the implantation window.

Objective: Autoimmune thyroid disease (AITD) is known to be associated with unexplained infertility in women. Although the presence of antithyroid antibodies have been speculated to be a marker of an immune imbalance that might lead to implantation failure, its underlying mechanism influencing the endometrial receptivity remains to be elucidated. In this study, we used single-cell RNA sequencing (scRNA-seq) to dissect immune microenvironment in endometrium of AITD patients during window of implantation (WOI). Methods: We collected CD45+ immune cell populations of endometrium samples of unexplained infertile women with AITD (n=3), as well as samples of AITD- controls (n=3). The cells were then processed with 10X Genomics Chromium for further analysis. Results: We characterized 28 distinct immune cell subtypes totally, and uncovered differences in the composition and gene expression patterns between AITD patients and controls. The proportions of T CD4+, cNK, ILC3, T CD8+ GZMK+, T CD8+ Cytotoxic and ILC3 CD3E - cells were increased, and CD366+ uNK1 was decreased in AITD+ patients. And the abnormal expression of GNLY and chemokines was observed in AITD patients. In addition, uNK and T CD8+ Cytotoxic cells showed lower cytotoxicity but activation of immune response. Genes enriched in cell adhesion of ILC3 and Tregs were downregulated, while the number of ILC3 and Tregs were increased. Conclusion: Immune imbalance exists in endometrium during WOI, which may impact embryo implantation.

Bilirubin Concentration in Follicular Fluid Is Increased in Infertile Females, Correlates with Decreased Antioxidant Levels and Increased Nitric Oxide Metabolites, and Negatively Affects Outcome Measures of In Vitro Fertilization.

In a previous study, we showed that various low-molecular-weight compounds in follicular fluid (FF) samples of control fertile females (CFF) have different concentrations compared to those found in FF of infertile females (IF), before and after their categorization into different subgroups, according to their clinical diagnosis of infertility. Using the same FF samples of this previous study, we here analyzed the FF concentrations of free and bound bilirubin and compared the results obtained in CFF, IF and the different subgroups of IF (endometriosis, EM, polycystic ovary syndrome, PCOS, age-related reduced ovarian reserve, AR-ROR, reduced ovarian reserve, ROR, genetic infertility, GI and unexplained infertility, UI). The results clearly indicated that CFF had lower values of free, bound and total bilirubin compared to the respective values measured in pooled IF. These differences were observed even when IF were categorized into EM, PCOS, AR-ROR, ROR, GI and UI, with EM and PCOS showing the highest values of free, bound and total bilirubin among the six subgroups. Using previous results of ascorbic acid, GSH and nitrite + nitrate measured in the same FF samples of the same FF donors, we found that total bilirubin in FF increased as a function of decreased values of ascorbic acid and GSH, and increased concentrations of nitrite + nitrate. The values of total bilirubin negatively correlated with the clinical parameters of fertilization procedures (number of retrieved oocytes, mature oocytes, fertilized oocytes, blastocysts, high-quality blastocysts) and with clinical pregnancies and birth rates. Bilirubin concentrations in FF were not linked to those found in serum samples of FF donors, thereby strongly suggesting that its over production was due to higher activity of heme oxygenase-1 (HO-1), the key enzyme responsible for bilirubin formation, in granulosa cells, or cumulus cells or oocytes of IF and ultimately leading to bilirubin accumulation in FF. Since increased activity of HO-1 is one of the main enzymatic intracellular mechanisms of defense towards external insults (oxidative/nitrosative stress, inflammation), and since we found correlations among bilirubin and oxidative/nitrosative stress in these FF samples, it may reasonably be supposed that bilirubin increase in FF of IF is the result of protracted exposures to the aforementioned insults evidently playing relevant roles in female infertility.

Cervical fluid pH, electrolytes and osmolarity changes and expression of ion transporters (ENaC, CFTR and AQP) in cervix of women with primary unexplained infertility.

BACKGROUND: Unexplained infertility could arise from a defect in the cervix. However, the contribution of abnormal cervical fluid microenvironment to this problem still needs to be identified. Therefore, this study identifies the changes in the cervical fluid microenvironment, i.e., pH, electrolytes and osmolarity as well as expression of ion transporters in the cervix including ENaC, CFTR and AQP in fertile women and in women suffering from primary unexplained infertility. METHODS: Fertile women and women with unexplained infertility but having regular 28-day menstrual cycles were chosen in this study, Day-22 serum progesterone levels were determined. In the meantime, serum FSH and LH levels were determined on day 2 while, cervical flushing was performed at day 14 to analyse changes in the cervical fluid pH, osmolarity, Na+ and Cl- levels. Meanwhile, cells retrieved from cervical fluid were subjected to mRNA expression and protein distribution analysis for CFTR, AQP and ENaC by qPCR and immunofluorescence, respectively. RESULTS: No significant changes in serum progesterone, FSH and LH levels were observed between the two groups. However, cervical fluid pH, osmolarity, Na+ and Cl- levels were significantly lower in primary unexplained infertile group when compared to fertile group. Expression of CFTR and AQP (AQP 1, AQP 2, AQP 5 and AQP 7) in endocervical cells was lower and expression of ß-ENaC was higher in primary unexplained infertile women (p < 0.05 when compared to fertile group). CONCLUSIONS: Alterations in the cervical fluid microenvironment linked to the defective ion transporter expression in the cervix might contribute towards the unfavourable condition that accounts for unexplained infertility in women.