Reproductive Biology and Endocrinology

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Prediction of pregnancy chances of model A after single embryo transfer using female age and (a) t3-t2, given that t5-t4 is 13 h, and (b) t5-t4 given that t3-t2 is 11 h. The different coloured lines depict different female ages
Prediction of pregnancy chances of model B after single embryo transfer using female age and (a) t2, given that t3-t2 is 11 h and t5-t4 is 13 h; b t3-t2 given that t2 is 25 h and t5-t4 is 13 h and (c) t5-t4 given that t2 is 25 h and t3-t2 is 11 h. The different coloured lines depict different female ages
a Predicted probabilities by model B are plotted against the actual probability in the dataset of clinic A (solid black lines). b Predicted probabilities by model A are plotted against the actual probability in the dataset of clinic B (solid black line). The grey lines represent perfect calibration
Illustration of the predicted probability of pregnancy after transfer of embryos originating from 10 patients of clinic A, where a double embryo transfer (DET) was performed. Patients were selected according to at least a 30% pregnancy chance predicted by our model A (according to morphokinetic parameters and female age), of both embryos. The light blue and dark blue bars represent the individual predicted probability of pregnancy after single embryo transfer (SET) for the first and second embryo. The white dots indicate the predicted probability of a twin pregnancy after transfer of both embryos originating from one patient; the black dots indicate the predicted probability of a singleton pregnancy after DET. Abbreviations: DET, double embryo transfer; SET, single embryo transfer
Background The predictive capability of time-lapse monitoring (TLM) selection algorithms is influenced by patient characteristics, type and quality of data included in the analysis and the used statistical methods. Previous studies excluded DET cycles of which only one embryo implanted, introducing bias into the data. Therefore, we wanted to develop a TLM prediction model that is able to predict pregnancy chances after both single- and double embryo transfer (SET and DET). Methods This is a retrospective study of couples (n = 1770) undergoing an in vitro fertilization cycle at the Erasmus MC, University Medical Centre Rotterdam (clinic A) or the Reinier de Graaf Hospital (clinic B). This resulted in 2058 transferred embryos with time-lapse and pregnancy outcome information. For each dataset a prediction model was established by using the Embryo-Uterus statistical model with the number of gestational sacs as the outcome variable. This process was followed by cross-validation. Results Prediction model A (based on data of clinic A) included female age, t3-t2 and t5-t4, and model B (clinic B) included female age, t2, t3-t2 and t5-t4. Internal validation showed overfitting of model A (calibration slope 0.765 and area under the curve (AUC) 0.60), and minor overfitting of model B (slope 0.915 and AUC 0.65). External validation showed that model A was capable of predicting pregnancy in the dataset of clinic B with an AUC of 0.65 (95% CI: 0.61–0.69; slope 1.223, 95% CI: 0.903–1.561). Model B was less accurate in predicting pregnancy in the dataset of clinic A (AUC 0.60, 95% CI: 0.56–0.65; slope 0.671, 95% CI: 0.422–0.939). Conclusion Our study demonstrates a novel approach to the development of a TLM prediction model by applying the EU statistical model. With further development and validation in clinical practice, our prediction model approach can aid in embryo selection and decision making for SET or DET.
Background Non-obstructive azoospermia (NOA) affects approximately 1% of the male population worldwide. The underlying mechanism and gene transcription remain unclear. This study aims to explore the potential pathogenesis for the detection and management of NOA. Methods Based on four microarray datasets from the Gene Expression Omnibus database, integrated analysis and weighted correlation network analysis (WGCNA) were used to obtain the intersected common differentially expressed genes (DESs). Differential signaling pathways were identified via GO and GSVA-KEGG analyses. We constructed a seventeen-gene signature model using least absolute shrinkage and selection operation (LASSO) regression, and validated its efficacy in another two GEO datasets. Three patients with NOA and three patients with obstructive azoospermia were recruited. The mRNA levels of seven key genes were measured in testicular samples, and the gene expression profile was evaluated in the Human Protein Atlas (HPA) database. Results In total, 388 upregulated and 795 downregulated common DEGs were identified between the NOA and control groups. ATPase activity, tubulin binding, microtubule binding, and metabolism- and immune-associated signaling pathways were significantly enriched. A seventeen-gene signature predictive model was constructed, and receiver operating characteristic (ROC) analysis showed that the area under the curve (AUC) values were 1.000 (training group), 0.901 (testing group), and 0.940 (validation set). The AUCs of seven key genes (REC8, CPS1, DHX57, RRS1, GSTA4, SI, and COX7B) were all > 0.8 in both the testing group and the validation set. The qRT-PCR results showed that consistent with the sequencing data, the mRNA levels of RRS1, GSTA4, and COX7B were upregulated, while CPS1, DHX57, and SI were downregulated in NOA. Four genes (CPS1, DHX57, RRS1, and SI) showed significant differences. Expression data from the HPA database showed the localization characteristics and trajectories of seven key genes in spermatogenic cells, Sertoli cells, and Leydig cells. Conclusions Our findings suggest a novel seventeen-gene signature model with a favorable predictive power, and identify seven key genes with potential as NOA-associated marker genes. Our study provides a new perspective for exploring the underlying pathological mechanism in male infertility.
Participant flow diagram. The rate of live delivery among patients was 32% (16/50) in the G-CSF group, significantly higher than 14% (7/49) in controls (chi-squared test). G-CSF, granulocyte colony-stimulating factor; OPU, oocyte pick-up; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; ET, embryo transfer. a All 3 of these patients conceived with spontaneous ovulation following menstruation after the initial administration of G-CSF
Numbers of retrieved oocytes, fertilized oocytes, and day-2 embryos per successful oocyte retrieval (A), development to day-5 embryos (B), and oocyte developmental competence (C) were compared between G-CSF and control groups. Significantly more fertilized oocytes and day-2 embryos, a higher rate of blastocyst acquisition, and higher embryo quality were obtained in the G-CSF group. Implantation rate per transferred embryo was defined as (number of gestational sacs / number of transferred embryos) × 100%. Serum AMH significantly increased after G-CSF priming; in controls AMH decreased, resulting in higher final concentrations of AMH in the G-CSF group (D). G-CSF, granulocyte colony-stimulating factor; AMH, anti-Müllerian hormone
Background Granulocyte colony-stimulating factor (G-CSF) administration increased ovarian preantral follicles and anti-Müllerian hormone (AMH) in animal models with diminished ovarian reserve. We investigated whether G-CSF priming before treatment with assisted reproductive technology (ART) improved embryo development and pregnancy rate while increasing serum AMH in patients with poor ovarian reserve. Methods In this prospective randomized open-label controlled trial, 100 patients 20 to 42 years old with AMH below 2 ng/mL were randomized to priming or control groups (50 patients each). None had over 1 ART failure, day-3 follicle-stimulating hormone (FSH) above 30 IU/L, uterine anomalies, or a partner with azoospermia. All patients initially underwent conventional infertility treatment for 2 consecutive cycles in which the priming group but not controls received a subcutaneous G-CSF priming injection during the early luteal phase. Each group then underwent 1 cycle of in vitro fertilization/intracytoplasmic sperm injection and fresh embryo transfer (IVF/ICSI-fresh ET), followed by cryopreserved ET if needed until live birth or embryo depletion. AMH was measured before and after priming. Results Fertilization rate, embryonic development, and implantation rate by fresh ET were significantly improved by priming. Clinical and ongoing pregnancy rates by IVF/ICSI-fresh ET were significantly higher with priming (30% and 26% in 47 ART patients; 3 delivered with conventional treatment) than in controls (12% and 10% in 49 ART patients; 1 dropped out). With priming, significantly more patients achieved cryopreservation of redundant blastocysts. The cumulative live birth rate was 32% in 50 patients with priming, significantly higher than 14% in 49 controls (relative risk, 2.8; 95% confidence interval, 1.04–7.7). Infants derived from priming had no congenital anomalies, while infant weights, birth weeks, and Apgar scores were similar between groups. Among 4 variables (age, day-3 FSH, AMH, and priming), logistic regression significantly associated age and priming with cumulative live birth. Priming significantly increased serum AMH. No adverse effects of priming were observed. Conclusion G-CSF priming improved embryonic development and pregnancy rate during ART treatment and increased AMH in patients with poor ovarian reserve. Enhanced preantral follicle growth likely was responsible. Trial registration UMIN registration in Japan (UMIN000013956) on May 14, 2014.
Background: Ovarian tissue transplantation can restore fertility in young cancer survivors, however the detrimental loss of follicles following transplantation of cryopreserved ovarian tissue is hampering the efficiency of the procedure. This study investigates whether needle puncturing prior to transplantation can enhance revascularization and improve follicle survival in xenotransplanted human ovarian cortex. Methods: Cryopreserved human ovarian cortex pieces (N = 36) from 20 women aged 24-36 years were included. During the thawing process, each piece of tissue was cut in halves; one half serving as the untreated control and the other half was punctured approximately 150-200 times with a 29-gauge needle. The cortex pieces were transplanted subcutaneously to immunodeficient mice for 3, 6 and 10 days (N = 8 patients) and for 4 weeks (N = 12 patients). After 3, 6 and 10 days, revascularization of the ovarian xenografts were assessed using immunohistochemical detection of CD31 and gene expression of angiogenic factors (Vegfα, Angptl4, Ang1, and Ang2), and apoptotic factors (BCL2 and BAX) were performed by qPCR. Follicle density and morphology were evaluated in ovarian xenografts after 4 weeks. Results: A significant increase in the CD31 positive area in human ovarian xenografts was evident from day 3 to 10, but no significant differences were observed between the needle and control group. The gene expression of Vegfα was consistently higher in the needle group compared to control at all three time points, but not statistically significant. The expression of Ang1 and Ang2 increased significantly from day 3 to day 10 in the control group (p < 0.001, p = 0.0023), however, in the needle group this increase was not observed from day 6 to 10 (Ang2 p = 0.027). The BAX/BCL2 ratio was similar in the needle and control groups. After 4-weeks xenografting, follicle density (follicles/mm3, mean ± SEM) was higher in the needle group (5.18 ± 2.24) compared to control (2.36 ± 0.67) (p = 0.208), and a significant lower percentage of necrotic follicles was found in the needle group (19%) compared to control (36%) (p = 0.045). Conclusions: Needle puncturing of human ovarian cortex prior to transplantation had no effect on revascularization of ovarian grafts after 3, 6 and 10 days xenotransplantation. However, needle puncturing did affect angiogenic genes and improved follicle morphology.
The general idea of this article
Intergenerational transmission of mtDNA mutations in oocytes
The decline of oocyte quality has profound impacts on fertilization, implantation, embryonic development, and the genetic quality of future generations. One factor that is often ignored but is involved in the decline of oocyte quality is mitochondrial DNA (mtDNA) abnormalities. Abnormalities in mtDNA affect the energy production of mitochondria, the dynamic balance of the mitochondrial network, and the pathogenesis of mtDNA diseases in offspring. In this review, we have detailed the characteristics of mtDNA in oocytes and the maternal inheritance of mtDNA. Next, we summarized the mtDNA abnormalities in oocytes derived from aging, diabetes, obesity, and assisted reproductive technology (ART) in an attempt to further elucidate the possible mechanisms underlying the decline in oocyte health. Because multiple infertility factors are often involved when an individual is infertile, a comprehensive understanding of the individual effects of each infertility-related factor on mtDNA is necessary. Herein, we consider the influence of infertility-related factors on the mtDNA of the oocyte as a collective perspective for the first time, providing a supplementary angle and reference for multi-directional improvement strategies of oocyte quality in the future. In addition, we highlight the importance of studying ART-derived mitochondrial abnormalities during every ART procedure.
Flow chart for the selection of participants in the cohort study
The violin plot of sperm DFI and the studied routine semen parameters stratified by the 4 clusters based on the variables among all participants. Green dots refer to cluster 1 (low-level DFI/high-level semen parameter group); red dots refer to cluster 2 (low-level DFI/median-level semen parameter group); blue dots refer to cluster 3 (low-level DFI/low-level semen parameter); purple dots refer to cluster 4 (high-level DFI/low-level semen parameter)
The forest plots of IVF outcomes in relation to the levels of sperm DFI and the studied routine semen parameters. Abbreviations: DFI, DNA fragmentation index; OR, odds ratio; CI, confidence interval; BMI, body mass index; AMH, Anti-Mullerian hormone; E2, Estradiol; FSH, Follicle-stimulating hormone. Notes: Model 1 was adjusted for duration of attempt to conceive, female age, male age, female BMI, and male BMI. Model 2 was further adjusted for controlled ovulation stimulation protocols, AMH, E2, FSH, endometrial thickness, and numbers of oocytes retrieved
Results of live birth, clinical pregnancy, and β-hCG positive odds ratios (95%CI) across the 4 clusters. Model 1 was adjusted for duration of the attempt to conceive, female age, male age, female BMI, and male BMI; model 2 was additionally adjusted for controlled ovulation stimulation protocols, AMH, E2, FSH, endometrial thickness, and numbers of oocytes retrieved; C1, cluster 1 (low-level DFI/high-level semen parameter group); C2, cluster 2 (low-level DFI/median-level semen parameter group); C3, cluster 3 (low-level DFI/low-level semen parameter); C4, cluster 4 (high-level DFI/low-level semen parameter)
Background Previous studies have demonstrated an association between male sperm quality and assisted reproduction outcomes, focusing on the effects of individual parameters and reaching controversial conclusions. The WHO 6th edition manual highlights a new semen assay, the sperm DNA fragmentation index, for use after routine semen examination. However, the combined effect of the sperm DNA fragmentation index (DFI) and routine semen parameters remains largely unknown. Methods We assessed the combined effect of the sperm DFI and conventional semen parameters on single fresh conventional IVF outcomes for infertile couples from January 1, 2017, to December 31, 2020. IVF outcomes were obtained from the cohort database follow-up records of the Clinical Reproductive Medicine Management System of the Third Affiliated Hospital of Guangzhou Medical University. An unsupervised K-means clustering method was applied to classify participants into several coexposure pattern groups. A multivariate logistic regression model was used for statistical analysis. Results A total of 549 live births among 1258 couples occurred during the follow-up period. A linear exposure–response relationship was observed among the sperm DFI, sperm motility, and IVF outcomes. In multivariable adjustment, increased sperm DFI values and decreased sperm motility and semen concentration levels were associated with reduced odds of favourable IVF outcomes. Four coexposure patterns were generated based on the sperm DFI and the studied semen parameters, as follows: Cluster 1 (low sperm DFI values and high sperm motility and semen concentration levels), Cluster 2 (low sperm DFI values and moderate sperm motility and semen concentration levels), Cluster 3 (low sperm DFI values and low sperm motility and semen concentration levels) and Cluster 4 (high sperm DFI values and low sperm motility and semen concentration levels). Compared with those in Cluster 1, participants in Cluster 3 and Cluster 4 had lower odds of a live birth outcome, with odds ratios (95% confidence intervals [CIs]) of 0.733 (0.537, 0.998) and 0.620 (0.394, 0.967), respectively. Conclusions When combined with low sperm DFI values, there was no significant difference between high or moderate sperm concentration and motility levels, and both were associated with favourable IVF outcomes. Low sperm parameter levels, even when DFI values remain low, may still lead to poor IVF outcomes. Participants with high sperm DFI values and low sperm motility and semen concentration levels had the worst outcomes. Our findings offer a novel perspective for exploring the joint effects of sperm DFI and routine semen parameter values.
Background After the longest time opposing all transfers of embryos by preimplantation genetic testing for aneuploidy (PGT-A) diagnosed as “chromosomal-abnormal,” the field has over recent years slowly been moving toward selective transfers of by PGT-A as “mosaic” diagnosed embryos, but is still rejecting transfers of embryos by PGT-A defined as “aneuploid.” Methods Upon review of the literature, we report published cases of euploid pregnancies following transfers of PGT-A as “aneuploid” diagnosed embryos and add several additional, ongoing cases at our center. Results Among the published cases from our center, we identified seven euploid pregnancies from “aneuploid” embryos, four of which preceded the PGT-A industry’s 2016 switch from binary “euploid” – “aneuploid” reporting to “euploid,” “mosaic,” and “aneuploid” reporting. That those four cases post 2016 PGT-A definition involving “mosaic” embryos, therefore, cannot be ruled out. Since then, we recently established three additional ongoing pregnancies from transfers of “aneuploid” embryos which still await confirmation of euploidy after delivery. A recent fourth pregnancy from the transfer of a trisomy 9 embryo miscarried before a fetal heart. Outside our own center’s experience, the literature revealed only one additional such transfer, involving PGT-A as a “chaotic-aneuploid” diagnosed embryo with six abnormalities, leading to normal euploid delivery. In reviewing the literature, we furthermore demonstrate why current PGT-A reporting that differentiates between “mosaic” and “aneuploid” embryos based on relative percentages of euploid and aneuploid DNA in a single trophectoderm biopsy of on average 5-6 cells, is biologically non-sensical. Conclusion Basic biological evidence and a clinically still very limited experience with transfers of PGT-A as “aneuploid” labeled embryos demonstrate beyond reasonable doubt that at least some “aneuploid” embryos can lead to healthy euploid births. Therefore, this observation establishes beyond reasonable doubt that the rejection of all “aneuploid” embryos from transfer reduces pregnancy and live birth chances for IVF patients. Whether (and to what possible degree) pregnancy and live birth chances differ between “mosaic” and “aneuploid” embryos, remains to be determined. The answer will likely depend on the aneuploidy(ies) of an embryo and to what degree percentages of “mosaicism” in a single, on average 5/6-cell trophectoderm biopsy can reflect the ploidy-status of a complete embryo.
PRISMA flow diagram of screening literatures
Network plot for the included trials comparing different pharmacological treatments for PCOS women. MET, metformin; CC, clomiphene citrate; PIO, pioglitazone; LZ, letrozole; ROS, rosiglitazone; EXE, exenatide; TAM, tamoxifen
Forest plot for outcomes of different pharmacological treatments compared with placebo. OR, odds ratio; CI, credibility interval; NNT, number needed to treat; NNH, number treated to harm; NA, not applicable
The ranking probability histogram for the outcomes. The color of the bar indicates the possibility rank of outcomes and the larger the proportion of dark colors, the higher the ranking
Background Polycystic ovarian syndrome (PCOS) is one of the most common causes of infertility in reproductive-age women. However, the efficacy and optimal therapeutic strategy for reproductive outcomes are still under debate. We conducted a systematic review and network meta-analysis to compare the efficacy of different first-line pharmacological therapies in terms of reproductive outcomes for women with PCOS and infertility. Methods A systematic retrieval of databases was conducted, and randomized clinical trials (RCTs) of pharmacological interventions for infertile PCOS women were included. The primary outcomes were clinical pregnancy and live birth, and the secondary outcomes were miscarriage, ectopic pregnancy and multiple pregnancy. A network meta-analysis based on a Bayesian model was performed to compare the effects of the pharmacological strategies. Results A total of 27 RCTs with 12 interventions were included, and all therapies tended to increase clinical pregnancy, especially pioglitazone (PIO) (log OR 3.14, 95% CI 1.56 ~ 4.70, moderate confidence), clomiphene citrate (CC) + exenatide (EXE) (2.96, 1.07 ~ 4.82, moderate confidence) and CC + metformin (MET) + PIO (2.82, 0.99 ~ 4.60, moderate confidence). Moreover, CC + MET + PIO (2.8, -0.25 ~ 6.06, very low confidence) could increase live birth most when compared to placebo, even without a significant difference. For secondary outcomes, PIO showed a tendency to increase miscarriage (1.44, -1.69 ~ 5.28, very low confidence). MET (-11.25, -33.7 ~ 0.57, low confidence) and LZ + MET (-10.44, -59.56 ~ 42.11, very low confidence) were beneficial for decreasing ectopic pregnancy. MET (0.07, -4.26 ~ 4.34, low confidence) showed a neutral effect in multiple pregnancy. Subgroup analysis demonstrated no significant difference between these medications and placebo in obese participants. Conclusions Most first-line pharmacological treatments were effective in improving clinical pregnancy. CC + MET + PIO should be recommended as the optimal therapeutic strategy to improve pregnancy outcomes. However, none of the above treatments had a beneficial effect on clinical pregnancy in obese PCOS. Trial registration CRD42020183541; 05 July 2020
Pedigree and sequencing results of families 1–3. The pedigree showed core family members: square symbol represented males; circle symbol indicated females; Roman numerals represented generations; and Arabic numerals indicated the position of each individual within the family. A Pedigree and sequencing results of family 1. Sanger sequencing showed that the proband (II1) carried a hemizygous ANOS1 c.1063-2 A > T mutation, while the proband’s mother (I2) carried a heterozygous ANOS1 c.1063-2 A > T mutation. B Pedigree and sequencing results of family 2. NGS showed that the proband (II1) carried a hemizygous ANOS1 c.711 G > T mutation, while Sanger sequencing showed that the proband’s mother (I2) carried a heterozygous ANOS1 c.711 G > T mutation. C Pedigree and sequencing results of family 3. Sanger sequencing showed that the proband (II1) carried a hemizygous ANOS1 c.709 T > A mutation, the proband’s mother (I2) and sister (II2) carried a same heterozygous ANOS1 c.709 T > A mutation, and the proband’s nephew (III1) carried a hemizygote ANOS1 c.709 T > A mutation
Pedigree and sequencing results of families 4 and 5. A Pedigree and sequencing results of family 4. Sanger sequencing showed that the proband (II1) carried a heterozygous FGFR1 c.1835delA mutation, while the proband’s father (I1) and mother (I2) were normal. B Pedigree and CNV-seq results of family 5. CNV-seq results showed that the proband (II1) carried a 2.32 Mb deletion at 8p12-p11.22 (36,140,000–38,460,000), while the proband’s father (I1) and mother (I2) were normal
Results of the ANOS1 mRNA transcripts analysis. A Sanger sequencing results of the RT-PCR products showed existence of both the wild-type (WT) transcript and the mutant transcript in the peripheral blood. The mutant ANOS1 transcript was aberrant splicing with c.1063–1086 deletion. B Schematic structures of anosmin-1 proteins. The domains of anosmin-1 protein were retrieved from the Ensembl database (Transcript ID: ENST00000262648.8). The truncated protein exhibited a mutated FnIII.2 (ΔFnIII.2) domain with amino acid deletion at 355–362
Results of minigene splicing assay. A Schematic diagram of minigene construction. The asterisk indicates the location of the c.1063-2 A > T mutation. The splicing pattern of wild-type (WT, top) and mutant (MUT, bottom) was showed respectively. B Minigene RT-PCR product sequencing results. The WT minigene formed normal mRNA composed of exon A, exon 8 and exon B. The mutant minigene caused a splicing abnormality, resulting in the 24 bp nucleotides deletion of 5′ end of exon 8 (c.1063–1086 deletion)
Chromosome 8p12-p11.22 gene map and the topological structure of FGFR1. A The orange box in the chromosome 8 diagram at the top indicated the region highlighted below. The OMIM morbid genes are listed at the bottom. B Membrane topology structure of full length FGFR1. Each circle represented a residue with the one-letter symbol. The different domains were labeled as indicated. Locations of pathogenic variations were colored as indicated. and the red box marked the amino acids that affected by FRFR1 c.1835delA mutation
Background Kallmann syndrome (KS) is a common type of idiopathic hypogonadotropic hypogonadism. To date, more than 30 genes including ANOS1 and FGFR1 have been identified in different genetic models of KS without affirmatory genotype–phenotype correlation, and novel mutations have been found. Methods A total of 35 unrelated patients with clinical features of disorder of sex development were recruited. Custom-panel sequencing or whole-exome sequencing was performed to detect the pathogenic mutations. Sanger sequencing was performed to verify single-nucleotide variants. Copy number variation-sequencing (CNV-seq) was performed to determine CNVs. The pathogenicity of the identified variant was predicted in silico. mRNA transcript analysis and minigene reporter assay were performed to test the effect of the mutation on splicing. Results ANOS1 gene c.709 T > A and c.711 G > T were evaluated as pathogenic by several commonly used software, and c.1063-2 A > T was verified by transcriptional splicing assay. The c.1063-2 A > T mutation activated a cryptic splice acceptor site downstream of the original splice acceptor site and resulted in an aberrant splicing of the 24-basepair at the 5′ end of exon 8, yielding a new transcript with c.1063–1086 deletion. FRFR1 gene c.1835delA was assessed as pathogenic according to the ACMG guideline. The CNV of del(8)(p12p11.22)chr8:g.36140000_38460000del was judged as pathogenic according to the ACMG & ClinGen technical standards. Conclusions Herein, we identified three novel ANOS1 mutations and two novel FGFR1 variations in Chinese KS families. In silico prediction and functional experiment evaluated the pathogenesis of ANOS1 mutations. FRFR1 c.1835delA mutation and del(8)(p12p11.22)chr8:g.36140000_38460000del were assessed as pathogenic variations. Therefore, our study expands the spectrum of mutations associated with KS and provides diagnostic evidence for patients who carry the same mutation in the future.
Histogram Comparing the Mean Live Birth Rate Percentages from All Ages of Fresh Donor Oocytes versus Cryopreserved Donor Oocytes from 2013-2020
Abstract Mature oocyte cryopreservation (OC) has become increasingly common since the American Society for Reproductive Medicine declared OC to no longer be experimental. Utilization of the open vitrification protocol has led to a marked improvement in the efficacy of oocyte cryopreservation. However, the safety and effectiveness of this cryopreservation method remain controversial. A previous report stated that among all initiated recipient cycles, the live-birth rate among recipients of all ages was significantly higher when using fresh donor oocytes (FDOs) rather than cryopreserved donor oocytes (CDOs). Confounding patient characteristics were noted as possible causes. OC stands as an acceptable elective medical intervention for preserving fertility in women. To further understand the effects of OC on the live birth rate resulting from fresh versus cryopreserved donor oocytes, reported data from the Society for Assisted Reproductive Technology from 2013 to 2020 were analyzed. The mean of the mean live-birth rate in all ages resulting from FDOs was 49.0% (44.6–53.3%) versus 41.0% (39.1–43.2%) for CDOs (difference, 8.0% [95% confidence interval, 5.35–10.57%], p value
Increasing evidence supports that the co-treatment with growth hormone (GH) enhances ovarian response and oocyte quality during controlled ovarian stimulation (COS) in patients with diminished ovarian reserve (DOR). The composition of follicular fluid (FF) plays an essential role in oocyte development and mirrors the communication occurring between the oocyte and follicular microenvironment. However, the effect of GH on the FF metabolome remains unclear. This prospective observational study recruited DOR patients undergoing in vitro fertilization (IVF) cycles with minimal stimulation protocol for COS. Each patient receiving GH co-treatment was matched to a patient without GH co-treatment by propensity score matching. The FF was collected after isolating oocytes and assayed by gas chromatograph-mass spectrometry (GC-MS) metabolomics. The Pearson correlation was performed to evaluate the relationship between the number of oocytes retrieved and the levels of differential metabolites. The KEGG database was used to map differential metabolites onto various metabolic pathways. One hundred thirty-four FF metabolites were identified by GC-MS metabolomics. Twenty-four metabolites, including glutathione, itaconic acid and S-adenosylmethionin (SAM) showed significant differences between the GH and control groups (p-value < 0.05 and q-value < 0.1). In addition, the number of oocytes retrieved was significantly higher in the GH group compared to the control group (3 vs 2, p = 0.04) and correlated with the levels of five differential metabolites. Among them, the levels of antioxidant metabolite itaconic acid were upregulated by GH administration, while SAM levels were downregulated. The co-treatment with GH during COS may improve oocyte development by altering FF metabolite profiles in DOR patients. However, given the downregulation of SAM, a regulator of genomic imprinting, the potential risk of imprinting disturbances should not be neglected.
Flow diagram of the establishment and application of the noninvasive RNA-seq-based endometrial receptivity test
Differential expression analysis and functional enrichment across endometrial receptivity conditions. A Volcano plot for RNA profile of PR, PO and RE samples. The cut-off is set as 0.05 for p-value and 2 for fold-change. B Bubble chart for functional enrichment analysis of DEGs. X-axis: fold of enrichment. Color of bubble: log10 (p-value). Size of bubble: number of genes enriched in the corresponding GO term
Partial predictive markers of the nirsERT. A Inferred source of marker and hub genes for the nirsERT; B Co-expression modules of uterine fluid DEGs generated via WGCNA
Establishment and validation of the nirsERT. A Clustering the training set via LDA using selected predictive markers; B Prediction results of training set samples with a probability threshold of 0.6. Pink dot: samples predicted as post-receptivity. Green triangle: samples predicted as pre-receptivity. Blue square: samples predicted as receptivity
Background Embryo implantation in a receptive endometrium is crucial for successful pregnancy. Endometrial receptivity (ER) prediction tools based on endometrial transcriptome biomarkers by endometrial biopsy have been used to guide successful embryo implantation in in vitro fertilization (IVF) patients. However, no reliable noninvasive ER prediction method has been established, and one is greatly needed. We aimed to identify biomarkers from uterine fluid transcriptomic sequencing data for establishing noninvasive ER prediction tool and to evaluate its clinical application potential in patients undergoing IVF. Methods The non-invasive RNA-seq based endometrial receptivity test (nirsERT) was established by analyzing transcriptomic profile of 144 uterine fluid specimens (LH + 5, LH + 7, and LH + 9) at three different receptive status from 48 IVF patients with normal ER in combination with random forest algorithm. Subsequently, 22 IVF patients who underwent frozen-thaw blastocyst transfer were recruited and analyzed the correlation between the predicted results of nirsERT and pregnancy outcomes. Results A total of 864 ER-associated differentially expressed genes (DEGs) involved in biological processes associated with endometrium-embryo crosstalk, including protein binding, signal reception and transduction, biomacromolecule transport and cell-cell adherens junctions, were selected. Subsequently, a nirsERT model consisting of 87 markers and 3 hub genes was established using a random forest algorithm. 10-fold cross-validation resulted in a mean accuracy of 93.0%. A small cohort (n = 22) retrospective observation shows that 77.8% (14/18) of IVF patients predicted with a normal WOI had successful intrauterine pregnancies, while none of the 3 patients with a displaced WOI had successful pregnancies. One patient failed due to poor sequencing data quality. Conclusions NirsERT based on uterine fluid transcriptome biomarkers can predict the WOI period relatively accurately and may serve as a noninvasive, reliable and same cycle test for ER in reproductive clinics. Trial registration Chinese Clinical Trial Registry: ChiCTR-DDD-17013375. Registered 14 November 2017,
Flowchart of included cycles
Risk of no normal embryos after PGT-A by AMH and Age
Background: While anti-Müllerian hormone (AMH) predicts quantitative IVF outcomes such as oocyte yield, it is not certain whether AMH predicts markers of oocyte quality such as aneuploidy. Methods: Retrospective case-control analysis of the SART-CORS database, 2014-2016, to determine whether anti-Müllerian hormone (AMH) predicts aneuploidy and live birth in IVF cycles utilizing preimplantation genetic testing for aneuploidy (PGT-A). Results: Of 51,273 cycles utilizing PGT-A for all embryos, 10,878 cycles were included in the final analysis; of these, 2,100 cycles resulted in canceled transfer due to lack of normal embryos and 8,778 cycles resulted in primary FET. AMH levels of cycles with ≥ 1 euploid embryo were greater than those of cycles with no normal embryos, stratifying by number of embryos biopsied (1-2, 3-4, 5-6, and ≥ 7), P < 0.017 for each stratum. Adjusting for age and number of embryos biopsied, AMH was a significant independent predictor of ≥ 1 euploid embryo for all age groups: < 35 yrs (aOR 1.074; 95%CI 1.005-1.163), 35-37 years (aOR 1.085; 95%CI 1.018-1.165) and ≥ 38 years (aOR 1.055; 95%CI 1.020-1.093). In comparative model analysis, AMH was superior to age as a predictor of ≥ 1 euploid embryo for age groups < 35 years and 35-37 years, but not ≥ 38 years. Across all cycles, age (aOR 0.945, 95% CI 0.935-0.956) and number of embryos (aOR 1.144, 95%CI 1.127-1.162) were associated with live birth per transfer, but AMH was not (aOR 0.995, 95%CI 0.983-1.008). In the subset of cycles resulting in ≥ 1 euploid embryo for transfer, neither age nor AMH were associated with live birth. Conclusions: Adjusting for age and number of embryos biopsied, AMH independently predicted likelihood of obtaining ≥ 1 euploid embryo for transfer in IVF PGT-A cycles. However, neither age nor AMH were predictive of live birth once a euploid embryo was identified by PGT-A for transfer. This analysis suggests a predictive role of AMH for oocyte quality (aneuploidy risk), but not live birth per transfer once a euploid embryo is identified following PGT-A.
Background Low vitamin D status has been associated with an increased risk for infertility. Recent evidence regarding the efficacy of vitamin D supplementation in improving reproductive outcomes is inconsistent. Therefore, this systematic review was conducted to investigate whether vitamin D supplementation could improve the reproductive outcomes of infertile patients and evaluate how the parameters of vitamin D supplementation affected the clinical pregnancy rate. Methods We searched seven electronic databases (CNKI, Cqvip, Wanfang, PubMed, Medline, Embase, and Cochrane Library) up to March 2022. Randomized and cohort studies were collected to assess the reproductive outcomes difference between the intervention (vitamin D) vs. the control (placebo or none). Mantel-Haenszel random effects models were used. Effects were reported as odds ratio (OR) and their 95% confidence interval (CI). PROSPERO database registration number: CRD42022304018. Results Twelve eligible studies ( n = 2352) were included: 9 randomized controlled trials (RCTs, n = 1677) and 3 cohort studies ( n = 675). Pooled results indicated that infertile women treated with vitamin D had a significantly increased clinical pregnancy rate compared with the control group (OR: 1.70, 95% CI: 1.24–2.34; I ² = 63%, P = 0.001). However, the implantation, biochemical pregnancy, miscarriage, and multiple pregnancy rates had no significant difference (OR: 1.86, 95% CI: 1.00–3.47; I ² = 85%, P = 0.05; OR: 1.49; 0.98–2.26; I ² = 63%, P = 0.06; OR: 0.98, 95% CI: 0.63–1.53; I ² = 0%, P = 0.94 and OR: 3.64, 95% CI: 0.58–11.98; I ² = 68%, P = 0.21). The improvement of clinical pregnancy rate in the intervention group was influenced by the vitamin D level of patients, drug type, the total vitamin D dosage, the duration, administration frequency, and daily dosage of vitamin D supplementation. The infertile women (vitamin D level < 30 ng/mL) treated with the multicomponent drugs including vitamin D (10,000–50,000 IU or 50,000–500,000 IU), or got vitamin D 1000–10,000 IU daily, lasting for 30–60 days could achieve better pregnancy outcome. Conclusion To the best of our knowledge, this is the first meta-analysis systematically investigated that moderate daily dosing of vitamin D supplementation could improve the clinical pregnancy rate of infertile women and reported the effects of vitamin D supplementation parameters on pregnancy outcomes. A larger sample size and high-quality RCTs are necessary to optimize the parameters of vitamin D supplementation to help more infertile patients benefit from this therapy.
Box plot showing serum AMH levels in women with no pregnancy, ectopic pregnancy, clinical pregnancy, and heterotopic pregnancy
Ectopic pregnancy rate in women with different AMH levels. Left: women who underwent fresh embryo transfer cycle; Right: women who resulted in clinical pregnancy. Upper: heterotopic pregnancy was excluded; Lower: heterotopic pregnancy was included as ectopic pregnancy
Risk factors for ectopic pregnancy in women who underwent fresh embryo transfer cycles and women who resulted in pregnancy. Left: heterotopic pregnancy was excluded; Right: heterotopic pregnancy was included as ectopic pregnancy. Model 1: female age, BMI, parity, primary infertility, ectopic pregnancy history, ovulatory dysfunction, tube factor and serum AMH levels ≥ 7 ng/ml were included in the regression analysis. Model 2: female age, BMI, parity, primary infertility, ectopic pregnancy history, ovulatory dysfunction, tube factor, serum AMH levels ≥ 7 ng/ml, Gn doses, the use of ICSI, blastocyst transfer, double embryo transfer, transfer of at least one poor quality embryo, the number of oocyte yeild, estradiol levels, thin endometrium, and the use of GnRH antagonist were included in the regression analysis
Background Ectopic pregnancy is more common amongst assisted reproduction cycles and is a cause of significant maternal morbidity. Few predictive markers exist to help identify and modify risk of ectopic pregnancy in preparing for embryo transfer. The relationship between serum and AMH and ectopic pregnancy rate is unknown. Methods This was a retrospective cohort study investigating women who underwent fresh embryo transfer cycles from January 2017 to December 2019 in Peking University Third Hospital. The primary outcome was ectopic pregnancy. Restricted cubic splines with four knots for AMH concentration (0-3, 3-6, 6-12, 12-max) were used to map out the non-linear relationship between the predicted ectopic pregnancy rate and the serum AMH concentration. Log binomial regression was used to test the crude risk ratio (cRR) and the adjusted risk ratio (aRR) after adjustment for confounders with 95% confidence intervals (CI) to determine the difference across various groups. Results A total of 13,718 cycles in women undergoing fresh embryo transfer were eligible for analysis. The ectopic pregnancy rate was 1.3% per embryo transfer cycle initiated and 3.3% per pregnancy. Serum AMH concentrations were higher amongst women with ectopic pregnancy than in women with a confirmed intrauterine pregnancy or heterotopic pregnancy or who did not become pregnant (Mean levels: 4.0 ng/ml vs 3.2 ng/ml, 1.7 ng/ml, and 2.8 ng/ml). An AMH concentration of 7 ng/ml represented the best cut-off value to predict ectopic pregnancy. The ectopic pregnancy rate was 3.4% per cycle and 7.5% per pregnancy in women with AMH levels ≥ 7 ng/ml; and 1.2% per cycle and 2.9% per pregnancy in women with AMH levels < 7 ng/ml. Serum AMH concentration ≥ 7 ng/ml was associated with an increased risk of ectopic pregnancy in all fresh embryo transfer cycles started (aRR = 2.35 (1.45, 3.58)) as well in women who became pregnant (aRR = 2.23 (1.49, 3.33). Conclusions Baseline AMH concentration ≥ 7 ng/ml is associated with an increased risk of ectopic pregnancy in fresh embryo transfer cycles.
The diagram of study participants. PCOS, polycystic ovary syndrome; PCOM, polycystic ovary morphology
ROC curves for the diagnosis of PCOS based on different BMIs. AUC, area under the curve; BMI, body mass index
Background: This study aimed to evaluate the cut-off value of anti-Müllerian hormone (AMH) combined with body mass index (BMI) in the diagnosis of polycystic ovary syndrome (PCOS) and polycystic ovary morphology (PCOM). Methods: This retrospective study included 15,970 patients: 3775 women with PCOS, 2879 women with PCOM, and 9316 patients as controls. Multivariate logistic regression analysis was used to calculate adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for AMH. We randomly divided the patients into two data sets. In dataset 1, a receiver operating characteristic (ROC) curve was generated to analyze the accuracy of basic AMH levels in diagnosing PCOS and PCOM. The optimal cut-off value was calculated in dataset 1 and validated in dataset 2, expressed as sensitivity and specificity. Results: In the PCOS group, obese patients had the lowest AMH levels, while underweight patients had the highest AMH level (P < 0.001). After adjusting for age, the ratio of luteinizing hormone (LH) and follicle stimulating hormone (FSH), serum testosterone level, and BMI, AMH was an independent predictor of PCOS and PCOM. In the group with BMI < 18.5 kg/m2, the optimistic AMH cut-off value was 5.145 ng/mL with a sensitivity of 84.3% and specificity of 89.1%, whereas in the BMI ≥ 28 kg/m2 group, the optimistic AMH cut-off value was 3.165 ng/mL with a sensitivity of 88.7% and specificity of 74.6%. For the BMI range categories of 18.5-24, 24.0-28 kg/m2, the optimistic AMH cut-off values were 4.345 ng/mL and 4.115 ng/mL, respectively. The tendency that the group with lower weight corresponded to higher AMH cut-off values was also applicable to PCOM. In the same BMI category, patients with PCOM had a lower AMH diagnosis threshold than those with PCOS (< 18.5 kg/m2, 5.145 vs. 4.3 ng/mL; 18.5-24 kg/m2, 4.345 vs. 3.635 ng/mL; 24.0-28 kg/m2, 4.115 vs. 3.73 ng/mL; ≥ 28 kg /m2, 3.165 vs. 3.155 ng/mL). These cut-off values had a good diagnostic efficacy in the validation dataset. Based on different phenotypes and severity of ovulation disorders, the distribution of AMH in PCOS were also significantly different (P < 0.001). Conclusions: AMH is a potential diagnostic indicator of PCOS and is adversely associated with BMI. The AMH cut-off value for diagnosing PCOS was significantly higher than that for PCOM.
A PFOA chemical formula. B PFOA standard curve. C Distribution of PFOA in different groups
GAMM model of the relationship between PFOA concentration in follicular fluid and high-quality embryo rate. The abscissa indicates the PFOA concentration (ng/ml) in follicular fluid, the ordinate indicates the good-quality embryo during IVF. A DOR group. B NOR group. C PCOS group
Partial least squares discriminant analysis (PLS-DA) score from follicle fluid metabolomics profiles comparing high concentration group and low concentration group (A-B). A Positive ESI mode; B Negative ESI mode. Orthogonal partial least squares discriminant analysis (OPLS-DA) score from follicle fluid metabolomics profiles comparing high concentration group and low concentration group (C-D). C Positive ESI mode. D Negative ESI mode
Volcano plot of metabolite components based on the normalized peak intensity between high concentration group and low concentration group. Colored dots indicate metabolites with a VIP value greater than 1, while the gray dots indicate the remaining metabolites. Red for up-regulated and blue for down regulated. The closer to the left and right side and the top point, the more significant the difference
Significance of metabolites in between groups
Owing to its difficulty in degrading and ease of accumulation in the body, perfluorooctanoic acid (PFOA) has a detrimental effect on reproduction. This study aimed to examine the effect of PFOA concentration in follicular fluid during ovulation stimulation on embryo quality and the impact of PFOA exposure on the metabolic components of follicular fluid. This was a single-center prospective study that included 25 patients with diminished ovarian reserve (DOR), 25 with normal ovarian reserve (NOR), and 25 with polycystic ovary syndrome (PCOS). Follicular fluid samples were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry. We demonstrated that the PFOA levels of follicular fluid in the DOR group were higher than those in the NOR group and PCOS group (P < 0.05). PFOA concentration in the PCOS group was negatively correlated with high-quality embryos (P < 0.05). To gain more insight into the impact of PFOA on the metabolic composition of follicular fluid, we classified the DOR group based on the PFOA concentration, for which metabolomic analysis was performed. In the high-concentration PFOA group, there was an increase and a decrease in three and nine metabolites, respectively, compared to that in the low-concentration group. These results suggest that PFOA may alter the metabolic composition of follicular fluid, thus, affecting ovarian reserve function.
Background: Acromegaly is a disease of growth hormone excess that results in enlargement of extremities, abnormal glucose and lipid metabolism, and gonadal disruption. Manifestations of the disease are insidious and typically lead to a diagnostic delay of 7-10 years. Classically the polycystic ovary syndrome (PCOS) phenotype is described in women with irregular menses, clinical or biochemical evidence of androgen excess, and/or multiple ovarian follicles on pelvic ultrasonography. Women with acromegaly may present with some or all of these symptoms. Our objective was to evaluate the prevalence of PCOS in patients with acromegaly and to determine if diagnosis of PCOS results in a delay in diagnosing acromegaly. Methods: Using patient databases at two academic health centers, we identified 97 premenopausal women aged 18-49 years old presenting with acromegaly. Data were collected regarding pelvic sonography and reproductive history, including the diagnosis of PCOS. Patients carrying the diagnosis of PCOS before their diagnosis of acromegaly were identified and the remaining patients were screened using the Rotterdam criteria to identify additional patients meeting the criteria for PCOS prior to their diagnosis of acromegaly. Results: Mean age of the population (n = 97) at the time of diagnosis of acromegaly was 33.4 ± 7.5 years (SD). Thirty-three percent of patients (n = 32) either carried a diagnosis of PCOS or met diagnostic criteria for PCOS before their diagnosis of acromegaly. In the subset of patients in whom data on symptom onset were available, those who met criteria for PCOS were diagnosed with acromegaly a median of 5 years [4, 9] after the onset of symptoms compared to 2 years [0.92, 3] (p = 0.006) in the patients who did not meet criteria for PCOS. Conclusions: Our data demonstrate a high prevalence of signs and symptoms of PCOS in reproductive-aged women with acromegaly and a longer time to diagnosis in women who meet the clinical criteria for PCOS. As screening for acromegaly is relatively simple and done with measurement of a random, non-fasting IGF-1 level that can be drawn at any time during the menstrual cycle, screening patients with PCOS for acromegaly may lessen the delay in diagnosis for reproductive-aged women with this disease.
The Stop GnRH-agonist/GnRH-antagonist protocol
Ovarian stimulation (OS) is one of the key factors in the success of in vitro fertilization-embryo transfer (IVF-ET), by enabling the recruitment of numerous healthy fertilizable oocytes and, thereby, multiple embryos. The Stop GnRH-agonist/GnRH-antagonist (GnRH-ag/GnRH-ant), which offers all the advantages of using long suppressive GnRH-ag, with GnRH-ant, is in my opinion a valuable addition to the armamentarium of OS protocols. It allows cycle programming, better follicular synchronization and offers successful outcome in a variety of challenging cases such as poor responders, Poseidon group 4 poor prognosis patients, those with elevated peak progesterone (P) serum levels, poor embryo quality or repeated IVF failures.
Relationship between IIEF-5 scores and sperm quality (A: sperm progressive motility, B: sperm normal morphology and C: sperm DNA fragmentation)
Comparison among groups of men for the severity of erectile dysfunction and sperm quality (A: sperm progressive motility, B: sperm normal morphology and C: sperm DNA fragmentation). Comparisons were performed after adjustment for age. Graphs for each group considered show the mean and standard deviation of the parameters evaluated
Comparison among groups of men for the severity of erectile dysfunction (ED) and anxiety. Comparison among groups for: GAD-7 scores (panel G); Anxiety state (%) (panel H). Comparisons were performed after adjustment for age. Graphs for each group considered show the mean and standard deviation (panel G) or percentage (panel H) of the parameters evaluated
Comparison among groups of men for the severity of erectile dysfunction (ED) and timed intercourse (%)
Background Erectile dysfunction is a common problem in males of couples experiencing pregnancy loss. Erectile dysfunction in males with couple infertile has been extensively investigated and found to be closely linked with semen quality impairment and psychological distress, but it is less clear if this relation exists in males of couples experiencing pregnancy loss. Method A cross-sectional analysis of 437 men who attended our outpatient clinic between June 2021 and October 2021 for couple pregnancy loss. All subjects underwent a complete physical examination, palpation, inspection of the male genitalia, and semen analysis. Validated assessment tools for erectile dysfunction (the International Index of Sexual Function5 -IIEF-5) and anxiety (the seven-item Generalized Anxiety Disorder Scale- GAD-7) were used. Results Among 437 men of couples with pregnancy loss, we found several relevant sperm parameters confirmed a significant correlation between IIEF-5 scores and sperm parameters, including: sperm progressive motility ( r = 0.1627, p = 0.001), sperm normal morphology ( r = 0.1373, p = 0.004) and sperm DNA fragmentation ( r =—0.1248, p = 0.009). Males with an IIEF-5 scores range between 5–11 presented the worst results in terms of sperm progressive motility ( p = 0.002), normal morphology ( p = 0.001), and SDF levels ( p = 0.003). GAD-7 score, as well as anxiety level, was significantly higher in those males with an IIEF-5 score between 5 and 11 ( p = 0.000). Conclusion Although current evidence does not demonstrate the importance of spermatozoa in the etiology of pregnancy loss, significant correlations have been observed between impaired sperm quality and low IIEF-5 scores. Also, anxiety is more likely to occur in males with sexual dysfunction.
Early embryo development and compaction patterns. Three primary compaction patterns were identified and a fourth categorization was made for embryos that exhibit both cell extrusion and exclusion. 1) CP-F, fully compacted morula; 2) P-exc, partially compacted morula with blastomere excluded from morula development, 3) P-ext, partially compacted morula with blastomere excluded following previous integration into morula
Compaction pattern and proportion of embryos with optimal kinetics of development. This graph shows the percentage of embryos with kinetic timings falling into optimal ranges. Optimal ranges were defined as follows: cc2 (> 5 and ≤ 11.9 h), s2 (≤ 1 h), t5 (45–57 h), cc3 (9.7–21 h), tSB (< 96.2 h) and tEB (≤ 116 h). p < 0.05 was considered statistically significant. (*) marks partial compaction patterns significantly different from CP-F
Comparison of blastocyst formation and biopsy date by compaction pattern
Comparison of morphokinetic parameters by compaction pattern
Background: Compaction is an important marker of embryonic genome activation and marks a critical step in the development to blastocyst. The objective of our study was to determine whether visualization of the embryonic compaction process through time-lapse imaging (TL) can assist in predicting the kinetics of embryo development as well as the likelihood for blastocyst formation, grade, or ploidy. Methods: This study is a retrospective review of prospectively collected datafrom a single academic institution. Couples included were thosewho underwent preimplantation genetic testing for aneuploidy (PGT-A) following in vitro fertilization between Januaryand December 2020. Embryos were cultured in the Embrysocope. Embryo morphokinetic data was prospectively collected and analyzed.TL videos werelater reviewed in detail for compaction pattern. Embryo compaction patterns (CP) were categorized as follows: 1) full compaction (CP-F), 2) partial compaction with cell extrusion (P-ext), 3) partial compactionwith cell exclusion (P-exc) and 4) partial compactionwith both cell extrusion and exclusion (P-both). Assessment of embryo decompaction and re-compaction was evaluated. The association between CP, morphokinetic parameters,blastocyst formation, grade and ploidy were then analyzed. Results: A total of 349 embryos were studied. Amongst embryos which progressed to morula (n = 281), the distribution of compaction patterns were: CP-F 45.6%, P-ext12.5%, P-exc29.5% and P-both 12.5%. Embryos exhibiting a CP-F were more likely to proceed to blastocyst compared with those that demonstrated partial compaction patterns (p = 0.006). When compared to CP-F, partial compaction patterns were significantly associated with poorer ICM and TE grades (P < 0.001). Of the 281 morula, 59.8% (n = 168) demonstrated at least one episode of decompaction and re-compaction. Of the 249 blastocysts formed, 200 were cryopreserved for future use after undergoing PGT-A evaluation. Of those, 42.5% were diagnosed as euploid, 39.0% as aneuploid, 9.0% as mosaic and 9.5% had no result. When compared to CP-F, partialCPs exhibited a significantly greater percentage of mosaic embryos (3.6% v. 15.6%, p = 0.032). Additionally, we found that a greater percentage of embryos demonstrating CP-F exhibited morphokinetics that fell into optimal ranges for embryo development when compared to those with partial compaction patterns. Conclusion: Time-lapse visualization of compaction patterns identified exclusions and/or extrusions as negative indicators of blastocyst formation and blastocyst grade. When compared to full compaction patterns, partial compaction patterns were associated with delayed embryonic development as well as lower rates of optimal kinetic development.
Overall H3K4me3 profile in uterine leiomyoma compared to adjacent myometrium tissues. Based on data extracted from GSE142332. A Principal component analysis of global H3K4me3 status in uterine leiomyomas (ULs; violet) and adjacent myometrium (MM; gray) (n = 19/group). B Heatmap representing the fold-enrichment score of genes with a CHIP-seq H3K4me3 peak in the promotor region after unsupervised clustering of ULs (violet) and MM (gray) (n = 19/group). The color scale ranges from red, for a higher normalized fold-enrichment score, to blue, for lower levels. C Boxplot analysis of the distribution of normalized fold-enrichment score for each peak in ULs (violet) compared to the adjacent MM (gray) samples (n = 19/group), representing the H3K4me3 profile
Selection of differentially expressed genes and description of their H3K4me3 status in ULs compared to MM tissue. A Venn diagram representing common DEGs (FDR-adjusted p-value< 0.01, log2FC > 1 or < − 1) between GSE192354 (n = 28) and GSE142332 (n = 19) datasets. B Principal component analysis of global H3K4me3 profile. C Heatmap based on the fold-enrichment score of selected DEGs shared between GSE192354 and GSE142332, whose promoter region presented a peak after unsupervised clustering of CHIP-seq data from uterine leiomyomas (ULs; violet) and their adjacent myometrium (MM; gray) (n = 19/group). The color scale ranges from red, for a higher normalized fold-enrichment score, to blue, for lower levels. D Boxplot analysis of the distribution of normalized fold-enrichment score for each peak of downregulated and upregulated genes in ULs (violet) compared to their adjacent MM (gray) samples (n = 19/group), representing the H3K4me3 status in each group of genes
Functional enrichment of H3K4me3-mediated differential expressed genes with altered status in UL vs MM. The most significant (A) biological processes and (B) cellular components were obtained following functional enrichment analysis of common DEGs associated with H3K4me3 instabilities in UL vs MM tissues. C Functional implication of selected hypertrimethylated/upregulated oncogenes (blue squares) and hypotrimethylated downregulated genes (yellow squares). The genes that were not been previously related to UL are in bold
Validation of RNA-seq results. mRNA expression levels of (A) CAPN6, (B) NPTX2, (C) SATB2, (D) SHOX2, (E) ST8SIA2, (F) DCX, (G) ABCA8, (H) HOXB4, and (I) KRT19 in a separate cohort of ULs compared to their adjacent MM matched tissues (n = 10). Relative gene expression was analyzed by qRT-PCR, quantified with the ΔΔCt method, and expressed as fold change. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Background: Uterine leiomyomas (UL) are the most common benign tumor in women of reproductive age. Their pathology remains unclear, which hampers the development of safe and effective treatments. Raising evidence suggests epigenetics as a main mechanism involved in tumor development. Histone modification is a key component in the epigenetic regulation of gene expression. Specifically, the histone mark H3K4me3, which promotes gene expression, is altered in many tumors. In this study, we aimed to identify if the histone modification H3K4me3 regulates the expression of genes involved in uterine leiomyoma pathogenesis. Methods: Prospective study integrating RNA-seq (n = 48) and H3K4me3 CHIP-seq (n = 19) data of uterine leiomyomas versus their adjacent myometrium. Differentially expressed genes (FDR < 0.01, log2FC > 1 or < - 1) were selected following DESeq2, edgeR, and limma analysis. Their differential methylation and functional enrichment (FDR < 0.05) were respectively analyzed with limma and ShinyGO. Results: CHIP-seq data showed a global suppression of H3K4me3 in uterine leiomyomas versus their adjacent myometrial tissue (p-value< 2.2e-16). Integrating CHIP-seq and RNA-seq data highlighted that transcription of 696/922 uterine leiomyoma-related differentially expressed genes (DEG) (FDR < 0.01, log2FC > 1 or < - 1) was epigenetically mediated by H3K4me3. Further, 50 genes were differentially trimethylated (FDR < 0.05), including 33 hypertrimethylated/upregulated, and 17 hypotrimethylated/downregulated genes. Functional enrichment analysis of the latter showed dysregulation of neuron-related processes and synapsis-related cellular components in uterine leiomyomas, and a literature review study of these DEG found additional implications with tumorigenesis (i.e. aberrant proliferation, invasion, and dysregulation of Wnt/β-catenin, and TGF-β pathways). Finally, SATB2, DCX, SHOX2, ST8SIA2, CAPN6, and NPTX2 proto-oncogenes were identified among the hypertrimethylated/upregulated DEG, while KRT19, ABCA8, and HOXB4 tumor suppressor genes were identified among hypotrimethylated/downregulated DEG. Conclusions: H3K4me3 instabilities alter the expression of oncogenes and tumor suppressor genes, inducing aberrant proliferation, and dysregulated Wnt/β-catenin, and TGF-β pathways, that ultimately promote uterine leiomyoma progression. The reversal of these histone modifications may be a promising new therapeutic alternative for uterine leiomyoma patients.
Flow chart of the cycle selection in this study. LFR: Low fertilization rate; TFF: Total fertilization failure
Theoretical model diagram of the Bayesian network model. Before the BN structure was established, a theoretical model diagram was developed according to prior knowledge from previous literature and clinical practice on the association between the selected variables. The variables were divided into seven hierarchical layers and were categorized into three subgroups
Directed acyclic graphs of the Bayesian network model. Abbreviations: “In_” refers to “Infertility factor_”; In_Ovulatory_dis: Infertility factor_ovulatory disorder; ART_failure: ART failure history; AFC: Antral Follicle Counting; COH: Ovulating induction protocol
Conditional probabilities for node fertilization failure
Receiver operating characteristic curve of the Bayesian network model. The best threshold points and values were shown in each curve. The specificities and sensitivities at the threshold point were shown in the brackets. LFR: low fertilization rate; TFF: total fertilization failure
Study question To construct prediction models based on the Bayesian network (BN) learning method for the probability of fertilization failure (including low fertilization rate [LRF] and total fertilization failure [TFF]) in assisted reproductive technology (ART) treatment. Summary answer A BN model was developed to predict TFF/LFR. The model showed relatively high calibration in external validation, which could facilitate the identification of risk factors for fertilization disorders and improve the efficiency of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment. What is known already The prediction of TFF/LFR is very complex. Although some studies attempted to construct prediction models for TFF/LRF, most of the reported models were based on limited variables and traditional regression-based models, which are unsuitable for analyzing real-world clinical data. Therefore, none of the reported models have been widely used in routine clinical practice. To date, BN modeling analysis is a prominent and increasingly popular machine learning method that is powerful in dealing with dynamic and complex real-world data. Study design, size, duration A retrospective study was performed with 106,640 fresh embryo IVF/ICSI cycles from 2009 to 2019 in one of China's largest reproductive health centers. Participants/materials, setting, methods A total of 106, 640 cycles were included in this study, including 97,102 controls, 4,339 LFR cases, and 5,199 TFF cases. Twenty-four predictors were initially included, including 13 female-related variables, five male-related variables, and six variables related to IVF/ICSI treatment. BN modeling analysis with tenfold cross-validation was performed to construct the predictive model for TFF/LFR. The receiver operating characteristic (ROC) curves and the corresponding area under the curves (AUCs) were used to evaluate the performance of the BN model. Main results and the role of chance All twenty-four predictors were first organized into seven hierarchical layers in a theoretical BN model, according to prior knowledge from previous literature and clinical practice. A machine-learning BN model was generated based on real-world clinical data, containing a total of eighteen predictors, of which the infertility type, ART method, and number of retrieved oocytes directly influence the probabilities of LFR/TFF. The prediction accuracy of the BN model was 91.7%. The AUC of the TFF versus control groups was 0.779 (95% CI: 0.766-0.791), with a sensitivity of 71.2% and specificity of 70.1%; the AUC of of TFF versus LFR groups was 0.807 (95% CI: 0.790-0.824), with a sensitivity of 49.0% and specificity of 99.0%. Limitations, reason for caution First, our study was based on clinical data from a single center, and the results of this study should be further verified by external data. In addition, some critical data (e.g., the detailed IVF laboratory parameters of the sperm and oocytes used for insemination) were not available in this study, which should be given full consideration when further improving the performance of the BN model. Wider implications of the findings Based on extensive clinical real-world data, we developed a BN model to predict the probabilities of fertilization failures in ART, which provides new clues for clinical decision-making support for clinicians in formulating personalized treatment plans and further improving ART treatment outcomes. Study funding/competing interest(s) Dr. Y. Wang was supported by grants from the Beijing Municipal Science & Technology Commission (Z191100006619086). We declare that there are no conflicts of interest. Trial registration number N/A.
PRISMA 2020 flowchart representing the study selection process
Forest plots representing the risk of cycle normalization in the groups treated with inositols compared to placebo or metformin
Basic characteristics of the included studies
Background Metformin is the gold standard insulin sensitizer, which is widely used to treat insulin resistance in polycystic ovary syndrome (PCOS). However, metformin may induce gastrointestinal side effects. Objective Inositols have long been debated as a potential alternative for metformin in treating PCOS. Therefore, the present systematic review aimed to evaluate the efficacy and safety of inositols in treating PCOS. Methods The present systematic search was performed in CENTRAL, MEDLINE, and Embase from the inception until October 20th, 2021. Eligible randomized controlled trials (RCTs) included women diagnosed with PCOS and compared any inositols with metformin or placebo. Our primary outcome was cycle normalization, whereas secondary outcomes were body mass index (BMI), parameters of carbohydrate metabolism and clinical and laboratory hyperandrogenism. Results are reported as risk ratios or mean differences (MDs) with 95% confidence intervals (CIs). Results Twenty-six RCTs were identified, including data of 1691 patients (806 inositol, 311 with placebo, and 509 metformin groups). In patients treated with inositols, the risk (CI: 1.13; 2.85) of having a regular menstrual cycle was found by 1.79 higher than in the case of placebo. Moreover, the inositols showed non-inferiority compared to metformin in this outcome. In the case of BMI (MD = -0.45; CI: -0.89; -0.02), free testosterone (MD = -0,41, CI: -0.69; -0.13), total testosterone (MD = -20.39, CI: -40.12; -0.66), androstenedione (MD = -0.69, CI: -1,16; -0.22), glucose (MD = -3.14; CI: -5.75; -0.54) levels and AUC insulin (MD = -2081.05, CI: -2745.32; -1416.78) inositol treatment induced greater decrease compared to placebo. Inositol increased sex-hormone-binding globulin significantly compared to placebo (MD = 32.06, CI:1.27; 62.85). Conclusion Inositol is an effective and safe treatment in PCOS. Moreover, inositols showed non-inferiority in most outcomes compared to the gold standard treatment; metformin. Trial registration PROSPERO registration number: CRD42021283275.
A Participants’ job categories. B Frequency of drinking between 2018 and 2021. The horizontal and vertical axes of the stacked bar graph represent years and the frequency, respectively. Blue, yellow, and red parts represent less than once a week, several times a week, and every day groups, respectively. C BMI change between 2015 and 2021. Scatter plots of mean BMI with regression lines for each age group. The regression lines (solid lines) are based on the measured BMI in the pre-pandemic period (2015–2019) and post-pandemic period (2020–2021); dashed lines are extensions of the former. Shaded areas denote 95% confidence intervals
Background Accumulating studies suggest that strict lockdown with enforcement including segregation to control the coronavirus disease 2019 (COVID-19) pandemic is associated with excess weight gain, but the such lockdown was not practiced in Japan. We aimed to compare the age-related weight gain before and after the COVID-19 pandemic in Japan where achieved epidemic control based on individual voluntary action. Methods This multicenter retrospective cohort study used electronic data from annual health checkups for workers from January 2015 to December 2021 at four facilities belonging to the Central Clinic Group, Aichi, Japan. We defined pre-pandemic and post-pandemic periods as January 2015–December 2019 and January 2020–December 2021, respectively. Participants were grouped by sex, age, and body mass index (BMI) stratus as of 2015, and the pre-pandemic and post-pandemic age-related BMI changes in overall individuals and each specific group were compared using a paired t-test. Results The total number of eligible participants was 19,290. During the pre-pandemic period, the mean BMI increased linearly in every group. The mean age-related BMI changes in females’ pre-pandemic and post-pandemic periods were + 0.11 and + 0.02 kg/m²/year, respectively. This significant decrease was also shown in males, + 0.11 in the pre-pandemic and − 0.02 kg/m²/year in the post-pandemic periods. The reduction was consistently observed in all age strata. Furthermore, a significant reduction was also observed in the normal-weight females of reproductive ages aged 15–44 years. Conclusions This is the first report showing that age-related weight gain was reduced after the COVID-19 pandemic in Japan, which could affect the reproductive age of females.
Recovery culture with prolactin (PRL) upregulates the expression of epithelial-to-mesenchymal transition (EMT)- and focal adhesion-related genes in human blastocysts. mRNA expression levels of (A) ezrin (EZR), (B) radixin (RDX), (C) moesin (MSN), (D) transforming growth factor beta 1 (TGFB1), (E) snail family transcriptional repressor 1 (SNAI1), (F) twist family bHLH transcription factor 1 (TWIST1), (G) cadherin 1 (CDH1), (H) cadherin 2 (CDH2), (I) integrin subunit α 5 (ITGA5), (J) integrin subunit beta 1 (ITGB1), (K) integrin subunit alpha V (ITGAV), and (L) integrin subunit β 3 (ITGB3) in embryos cultured on fibronectin-coated dishes for 12 h [PRL non-treated (control), n = 14; PRL 15 min, n = 14; PRL 30 min, n = 14; PRL 60 min, n = 14; PRL 120 min, n = 14]. The expression of each target gene was normalized to that of H2A histone family member Z. N.S., not significant. Error bars represent the standard error of the mean. a–c Different letters indicate a significant difference at P < 0.05
Prolactin (PRL) treatment during recovery culture promotes trophoblast migration and reduces outgrowth degeneration. A Comparison of blastocyst adhesion rates to the fibronectin-coated dishes between the control and PRL-treated blastocysts (control, n = 152; PRL 120 min, n = 152); B comparison of outgrowth area after 96 h of outgrowth culture between the control and PRL-treated blastocysts; C migration distance of trophoblast cells at 48–72 h after outgrowth culture; D rates of outgrowth degeneration after 96 h outgrowth culture. N.S., not significant. Error bars represent the standard error of the mean. *Significant difference at P < 0.05
Stratified blastocyst outgrowth according to blastocyst characteristics. The blastocyst adhesion rate and outgrowth area stratified by blastocyst vitrification day (Day 5 vs. Day 6; a and b, respectively), diameter (< 180 μm vs. ≥180 μm; c and d, respectively), or morphology (Good [Gardner’s criteria: AA, AB, and BA] vs. Poor [Gardner’s criteria: AC, CA, BC, CB, and CC]; e and f, respectively). N.S., not significant. Error bars represent the standard error of the mean. *Significant difference at P < 0.05
Background Human embryos express the prolactin (PRL) receptor at the morula and blastocyst stages. Treatment with PRL from cleavage to the blastocyst stage improves blastocyst outgrowth on fibronectin-coated dishes. However, whether post-warming PRL treatment of blastocysts cultured without PRL could improve outgrowth competence remains unknown. Furthermore, the optimal time for post-warming PRL treatment remains to be ascertained. This study investigated the effects of PRL treatment during recovery culture on human blastocyst outgrowth and its related genes. Methods In total, 374 discarded vitrified blastocysts were randomly allocated to two groups, to be cultured with ( n = 208) or without PRL (control; n = 166) for 120 min for recovery, and then plated on fibronectin-coated dishes. The expression level of PRL-interacting genes, blastocyst adhesion rate, outgrowth area, distance of trophoblast migration, and outgrowth degeneration were examined. Results The mRNA expression of ezrin, radixin, and moesin, which regulate cell adhesion and invasion by controlling actin reorganization during epithelial-to-mesenchymal transition (EMT), was stimulated by PRL treatment for 120 min. The expression of EMT-related genes, transforming growth factor β1, snail1, and twist1 was also promoted following treatment with PRL for 120 min. PRL-treated blastocysts also exhibited augmented expression of cadherin 2 and transcriptional repression of cadherin 1. Higher mRNA expression of integrin-based focal adhesion-related genes, ITGA5 and ITGB1, was observed after treatment with PRL for 120 min than in the non- and shorter-treatment groups. PRL treatment for 120 min did not alter the rate of blastocyst adhesion to fibronectin-coated dishes 96 h after the outgrowth culture assay. However, multiple linear regression analysis revealed that the outgrowth area was significantly increased in PRL-treated blastocysts. The migration distance of trophoblast cells was significantly increased and degeneration rate was significantly decreased after PRL treatment. Furthermore, a more beneficial effect of PRL treatment on blastocyst outgrowth was observed when the blastocysts were vitrified on day 5 than when they were vitrified on day 6. Conclusions Post-warming culture of human vitrified blastocysts with PRL for 120 min promoted trophoblast outgrowth in vitrified human blastocysts. Furthermore, PRL treatment may reduce outgrowth degeneration by increasing resistance to apoptosis during trophoblast migration.
PRISMA 2020 flowchart showing the study selection process
forest plot of associated health conditions
forest plot of lifestyle factors
forest plot of other factors
Introduction Infertility affects one in every six couples in developed countries, and approximately 50% is of male origin. In 2021, sperm DNA fragmentation (SDF) testing became an evidence-based test for fertility evaluations depicting fertility more clearly than standard semen parameters. Therefore, we aimed to summarize the potential prognostic factors of a higher SDF. Methods We conducted a systematic search in three medical databases and included studies investigating any risk factors for SDF values. We calculated mean differences (MD) in SDF with 95% confidence interval (CI) for exposed and non-exposed individuals. Results We included 190 studies in our analysis. In the group of associated health conditions, varicocele (MD = 13.62%, CI: 9.39–17.84) and impaired glucose tolerance (MD = 13.75%, CI: 6.99–20.51) had the most significant increase in SDF. Among malignancies, testicular tumors had the highest impact, with a maximum of MD = 11.3% (CI: 7.84–14.76). Among infections, the overall effects of both Chlamydia and HPV were negligible. Of lifestyle factors, smoking had the most disruptive effect on SDF – an increase of 9.19% (CI: 4.33–14.06). Different periods of sexual abstinence did not show significant variations in SDF values. Age seemed to have a more drastic effect on SDF from age 50 onwards, with a mean difference of 12.58% (CI: 7.31–17.86). Pollution also had a detrimental effect – 9.68% (CI: 6.85–12.52). Conclusion Of the above risk factors, varicocele, impaired glucose tolerance, testicular tumors, smoking, pollution, and paternal age of over 50 were associated with the highest SDF. Trial registration CRD42021282533.
Background COVID-19 infection has been linked with erectile dysfunction, which has also raised apprehensions about the impact of COVID-19 vaccination on male sexual functions. The purpose of this study was to investigate the impact of COVID-19 vaccination on male sexual functions, such as erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall satisfaction. Methods We used International Index of Erectile Function (IIEF) questionnaire for data collection. Mixed methods were adopted for this study, which consisted of Google online form distribution and the distribution of hard copies of the form to those who were not internet friendly. All data were entered in a spreadsheet and scores were assigned to each response according to the standard scores given in the IIEF questionnaire. Fifteen questions, one corresponding to each question in the IIEF questionnaire, were included to assess the impact of COVID-19 vaccination on each sexual function. Results In the first part of analysis, we calculated sexual function scores and men reporting low sexual function scores (~ 15%) were excluded, providing us with 465 individuals for further analysis. Regarding the impact of COVID-19 vaccination on male sexual functions, 71% individuals reported no impact, 3% reported a decline, 2.7% reported an improvement, and 23.3% could not assess the impact. We also performed analysis on the basis of age-groups of the participants and the duration after vaccination, finding that there was no impact irrespective of the age of subjects or the length of period after vaccination. Conclusions COVID-19 vaccination does not affect male sexual functions, including erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall sexual satisfaction.
Features of peritoneal DCs in endometriosis patients. A Representative FACS analysis of peritoneal DCs in endometriosis group (EMS) and control group (CON). B Quantitative analysis of the number of peritoneal DCs in EMS and CON. C Quantitative analysis of the proportion of mDCs in EMS and CON. D Quantitative analysis of the proportion of iDCs in EMS and CON. (*P < 0.05 and ***P < 0.001)
Features of peritoneal DCs in murine endometriosis models. A The flow diagram of the murine endometriosis models experimental design. B FACS analysis of the mDCs and iDCs in murine models. C Representative FACS analysis of dynamic changes of mDCs and iDCs in the development of endometriosis. D Quantitative analysis of the number of peritoneal DCs in the development of endometriosis. E Quantitative analysis of the proportion of mDCs in the development of endometriosis. F Quantitative analysis of the proportion of iDCs in the development of endometriosis
Ectopic lesions in murine endometriosis models. A Typical ectopic endometrial lesions in peritoneal cavity of endometriosis models. B HE staining of the eutopic and ectopic endometrial tissues. C Total number of lesions collected on Day 42 after modeling. D Total volume of lesions collected on Day 42 after modeling. E Total weight of lesions collected on Day 42 after modeling (*P < 0.05)
Background Emerging evidence of immunological dysfunction have been described in endometriosis. Dendritic cells (DCs), one of the main antigen-presenting cells, are specialized in the initiation and modulation of the adaptive immune response. Emerging studies demonstrated both endometrial and circulating differences in DCs populations in women with endometriosis. However, the role and mechanism of peritoneal DCs in endometriosis is still unclear. The present study was undertaken to explore the features of peritoneal DCs in the pathogenesis of endometriosis. This study is beneficial to further clarify the cause of endometriosis and provide a new insight into the medical treatment for endometriosis. Methods The study included 12 women with endometriosis and 11 women without endometriosis. The C57BL6 mouse model of endometriosis was established by intraperitoneal injection of endometrial segments. The peritoneal DCs of endometriosis patients and mouse models were analyzed by fluorescence associated cell sorting (FACS) examination. Results Increased cell density of peritoneal DCs were observed in endometriosis patients. Moreover, the proportion of mature DCs (mDCs, CD80highCD1alow cells) in the peritoneal DCs was lower whereas the proportion of immature DCs (iDCs, CD80lowCD1ahigh cells) was increased in endometriosis patients. Similarly, the cell density of peritoneal DCs in murine models increased immediately after the injection of endometrial tissues and reached the highest level at 14 days. In addition, the proportion of mDCs (CD11chighCD80high cells) in the peritoneal DCs decreased immediately after the injection of endometrial tissues and then increased with the time until 42 days, but still lower than the control group. In contrast, the proportion of iDCs (CD11chighCD80low cells) in the peritoneal DCs showed the opposite dynamic changes. However, after treated with LPS, the mDCs proportion was significantly increased, leading to lower volume and weight of the endometriosis lesions. Conclusions Increased level of peritoneal DCs facilitated the pathogenesis of endometriosis lesions, especially in the early stage of the disease. Furthermore, peritoneal DCs maturation played an important role in the development of endometriosis.
Distribution of patients with sex chromosome DSD according to their karyotype. 47,XXY was the most common observation
A Schematic representation of ZNRF3 protein indicating the known functional domains. The sequence alignment indicating the position and evolutionary conservation of the mutated isoleucine 338 residue, immediately adjacent to the RING finger domain. Previously published variants linked to 46,XY DSD are shown and located within the intracellular domain. B Schematic representation of HHAT protein indicating the position of the mutated p.R312 residue. Other published variants associated with this syndromic form of 46,XY DSD are indicated. C Representation of the SOX8 protein showing the position of the mutated p.T226 residue located in the evolutionary conserved TA1 domain. The only other SOX8 variant known to be associated with 46,XY DSD is the p.E156D mutation located within the HMG-box. Right, the mutated threonine residue is conserved in the SOXE group of proteins. DIM, DNA-dependent dimerization domain; HMG, high mobility group; MBOAT, Membrane Bound O-Acyltransferase domain; TA, transactivation domain; TM, transmembrane domain; SP, signal peptide
Clinical phenotype, hormonal profil and details of potentially pathogenic variants identified by WES in a cohort of individuals with 46,XY DSD
Background :46,XY Differences/Disorders of Sex Development (DSD) are characterized by a broad phenotypic spectrum ranging from typical female to male with undervirilized external genitalia, or more rarely testicular regression with a typical male phenotype. Despite progress in the genetic diagnosis of DSD, most 46,XY DSD cases remain idiopathic. Methods:To determine the genetic causes of 46,XY DSD, we studied 165 patients of Tunisian ancestry, who presented a wide range of DSD phenotypes. Karyotyping, candidate gene sequencing, and whole-exome sequencing (WES) were performed. Results: Cytogenetic abnormalities, including a high frequency of sex chromosomal anomalies (85.4%), explained the phenotype in 30.9% (51/165) of the cohort. Sanger sequencing of candidate genes identified a novel pathogenic variant in the SRY gene in a patient with 46,XY gonadal dysgenesis. An exome screen of a subgroup of 44 patients with 46,XY DSD revealed pathogenic or likely pathogenic variants in 38.6% (17/44) of patients. Conclusion : Rare or novel pathogenic variants were identified in the AR, SRD5A2, ZNRF3, SOX8, SOX9 and HHAT genes. Overall our data indicate a genetic diagnosis rate of 41.2% (68/165) in the group of 46,XY DSD.
Flow cytometric out-gating of ROS sperm particles based on scatter properties and histogram after Triple-Staining. Sperm cells were selected using discontinuous density gradients method and stained with SYTOX Red, MitoSOX Red and DCFH-DA. A FSC/SSC dot plot obtained from a semen sample. A region (P1) is established to exclude debris. B SYTOX Red fluorescence histogram obtained within P1 region. The B1-L and B1-R peaks represent viable and nonviable sperm, respectively. C MitoSOX Red fluorescence distribution of viable sperm, were derived by gating on the B1-L population in panels B. The B2-R region represents ROS positive living sperm. D DCFH-DA fluorescence histogram obtained within B1-L region. The B3-R populations contains hydrogen peroxide positive live sperm events
RCS analysis of the effect of sperm ROS on IVF embryo development in patients with anovulatory infertility (A-C) or normo-ovulatory infertility (D-I) (D and E for unexplained infertility and F for male sterility, G for tubal infertility, H and I for infertility due to endometriosis)
Background The exact role of sperm reactive oxygen species (ROS) in early embryo development has yet to be fully identified, and most of existing research did not differentiate female infertility factors, ignoring the importance of oocyte quality in embryo development and the large differences in oocyte quality in women with infertility of different etiologies. And there has been no relevant report on whether different types of sperm ROS have distinct effects on embryo development. This study aimed to study the impact of selected sperm ROS, namely, sperm mitochondrial ROS (mROS) and hydrogen peroxide, on human embryo development after conventional in vitro fertilization (IVF) cycles in patients with normo-ovulatory infertility vs. anovulatory infertility. Methods This was a prospective investigation including 393 couples underwent IVF cycles, among whom 90 patients had anovulatory infertility and 303 patients had normo-ovulatory infertility in a public university-affiliated in vitro fertilization center. Sperm mROS and hydrogen peroxide testing were performed by flow cytometry and analyzed for their relationship with embryo development indices on days 1–6 after IVF. Multivariate logistic regression analysis was used to control for female potential confounders. The nonlinear effects of sperm ROS on embryo development were analyzed by the Restricted cubic spline (RCS) method. Results 1. Multivariate linear logistic regression analysis showed that high proportion of mROS positive sperm improved the 2PN rate (OR = 1.325, 95% CI: 1.103–1.595), day 3 embryo utilization rate (OR = 1.362, 95% CI: 1.151–1.614) and good-quality day 3 embryo rate (OR = 1.391, 95% CI: 1.089–1.783) in patients with anovulatory infertility. High percentage of sperm mROS and hydrogen peroxide had adverse effects on cleavage-stage embryo and blastocyst development in patients with normo-ovulatory infertility. 2. For patients with polycystic ovarian syndrome (PCOS) anovulatory infertility, there were significant distinct effects on embryo development indices between sperm mROS and hydrogen peroxide, and the increased rate of sperm mROS improved the good-quality day 3 embryo rate (OR = 1.435, 95% CI: 1.045–1.981); however, high percentage of sperm hydrogen peroxide reduced the blastocyst utilization rate (OR = 0.555, 95% CI: 0.353–0.864) and the good-quality blastocyst rate (OR = 0.461, 95% CI: 0.292–0.718). 3. Multivariate RCS analysis revealed that sperm ROS had a nonlinear (such as a parabolic curve) effect on embryo development in patients with anovulatory infertility ( P < 0.05), and either greatly increased or greatly decreased affected cleavage-stage embryo and blastocyst development. The effects of sperm ROS in patients with normo-ovulatory infertility were both linear and nonlinear. Conclusions These findings indicate that contrary effects of sperm mROS on embryo development depending on whether patients treated with IVF cycles had normal ovulation. Regardless of whether the patients ovulated normally, increased sperm hydrogen peroxide rate damaged blastocyst development. It is necessary to evaluate male sperm ROS levels and the female ovulatory state to determine an individualized intervention plan before starting cycles, as this may be beneficial for infertile couples.
Vitamin D metabolic pathway. Figure’s images source: Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. UVB: ultraviolet B (UVB) rays. DBP: Vitamin D Binding Protein
Study flow chart
Quantitative synthesis results
Background There is a growing body of human, animal and in vitro studies on vitamin D (vit D) substitution in endometriosis. The aim of this systematic review is to critically appraise and qualitatively synthesize the results of the available studies that examine the supplementation of vit D for endometriosis treatment. Methods A systematic search of the literature was conducted in four electronic databases (Medline, Cochrane, Scopus, Embase) and grey literature for original research articles on humans, animals and in vitro models published in any language. Results Four human studies, four animal studies and four in vitro studies were included. Quantitative synthesis of human studies showed no significant effect of vit D intake for dysmenorrhea (2 studies, 44 vit D vs 44 placebo, mean -0.71, 95% CI -1.94, 0.51) and non-cyclic pelvic pain (2 studies, 42 vit D vs 38 placebo, mean 0.34, 95% CI -0.02, 0.71). Regarding reproductive outcomes in women with endometriosis after in vitro fertilization, the only available study showed no differences between women taking vit D and women taking placebo. Three of the four included animal studies showed regression of endometriotic implants when treated with vit D. The in vitro studies demonstrated that vit D decreases invasion and proliferation of endometriotic lesions without affecting apoptosis. Conclusions Although in vitro and animal studies suggest regression of the endometriotic implants and decrease of invasion and proliferation after vit D supplementation, this was not reflected in the results of the meta-analysis, which showed no benefit of vit D supplementation in patients with endometriosis and dysmenorrhea or non-cyclic pelvic pain as well as on the outcome of IVF treatment. However, given the heterogeneity and the diversity of the available studies, more research is required to shed light on the role of vit D supplementation in women with endometriosis.
Study flowchart. ABPM: ambulatory blood pressure monitoring; GDM: gestational diabetes mellitus
Prevalence of circadian patters in women with and without HDP. HDP: hypertensive disorders of pregnancy; extreme dipper pattern: subjects with a > 20% fall in nocturnal blood pressure; dipper pattern: subjects with a 10–20% fall in nocturnal blood pressure; non-dipper pattern: subjects with a 0–10% fall in nocturnal blood pressure; riser pattern: subjects with nocturnal blood pressure higher than diurnal blood pressure
Correlation between ABPM and leptin levels in whole population (a); in women with HDP (b). * p < 0.05; ** p < 0.01. HDP: hypertensive disorders of pregnancy; PB: blood pressure; SBP: systolic blood pressure; DBP: diastolic blood pressure
Correlation between ABPM and sFlt-1/PlGF ratio in whole population (a); in women with preeclampsia (b). * p < 0.05; ** p < 0.01. HDP: hypertensive disorders of pregnancy; PB: blood pressure; SBP: systolic blood pressure; DBP: diastolic blood pressure; sFlt-1/PlGF ratio: soluble fms-like tyrosine kinase-1 / placental growth factor ratio
Background The risk of hypertensive disorders of pregnancy (HDP) varies in women with gestational diabetes mellitus (GDM), depending on the degree of insulin resistance and is also influenced by obesity. The aim of this study was to evaluate clinical features, blood pressure (BP) profiles and inflammatory markers, to identify patients with an elevated risk of developing HDP. Methods A total of 146 normotensive pregnant women were studied. We analysed the relationships of BP profiles detected by ambulatory blood pressure monitoring (ABPM) with serum biomarkers and angiogenic factors and their association with the development of HDP. Results Fourteen (9.6%) women developed HDP, of which 11 had GDM and 8 had obesity. Women with HDP had higher values of 24-h and daytime systolic/diastolic BP (113/69 vs. 104/64; 115/72 vs. 106/66 mmHg, respectively; p < 0.05). Higher levels of leptin (10.97 ± 0.82 vs. 10.2 ± 1.11; p = 0.018) andmonocyte chemoattractant protein-1 (MCP-1) (5.24 ± 0.60 vs. 4.9 ± 0.55; p = 0.044) and a higher soluble fms-like tyrosine kinase-1/placental growth factor (sFlt-1/PlGF) ratio (4.37 ± 2.2 vs. 2.2 ± 1.43; p = 0.003) were also observed in the HDP patients. Multivariate analysis showed that a higher sFlt-1/PlGF ratio was associated with an increased risk of developing HDP [OR = 2.02; IC 95%: 1.35–3.05]. Furthermore, higher daytime systolic BP [OR = 1.27; IC 95% 1.00–1.26] and prepregnancy body mass index (BMI) [OR = 1.14; IC 95%: 1.01–1.30] significantly increased the risk of developing HDP. Conclusions Higher daytime systolic BP values, prepregnancy BMI and the sFlt-1/PlGF ratio are useful for identifying normotensive pregnant women with an increased risk of developing HDP.
Identification of studies via databases and register
Extracellular vesicle cargo identified in subjects with endometriosis from different sites, with proposed function
Proposed role of extracellular vesicles (EVs) in the development of endometriosis lesions. EVs derived from the endometrium act on endometrial cells shed during retrograde menstruation, allowing migration, implantation and immunomodulation. EVs from newly implanted ectopic endometrial cells enhance pathogenesis with ongoing immunomodulation and cell proliferation, as well as angiogenesis and fibrosis, leading to the formation of endometriosis lesions
Key characteristics of studies of extracellular vesicles in endometriosis
Endometriosis is a chronic, inflammatory gynaecological disease that can have severe negative impacts on quality of life and fertility, placing burden on patients and the healthcare system. Due to the heterogeneous nature of endometriosis, and the lack of correlation between symptom and surgical disease severity, diagnosis and treatment remain a significant clinical challenge. Extracellular vesicles (EVs) are biologically active particles containing molecular cargo involved in intercellular communication, that can be exploited for diagnostic and therapeutic purposes. We systematically reviewed studies exploring EVs and their role in endometriosis, specifically addressing diagnostic and therapeutic potential and current understanding of pathophysiology. Five databases (Pubmed, Embase, Medline, Web of Science, Google Scholar) were searched for keywords ‘endometriosis’ and either ‘extracellular vesicles’ or ‘exosomes’. There were 28 studies included in the review. Endometrium derived EVs contribute to the development of endometriosis. EVs derived from endometriosis lesions contribute to angiogenesis, immunomodulation and fibrosis. Such EVs can be detected in blood, with early data demonstrating utility in diagnosis and recurrence detection. EV isolation techniques varied between studies and only eight of twenty-eight studies fully characterised EVs according to current recommended standards. Reporting/type of endometriosis was limited across studies. Varied patient population, type of sample and isolation techniques created bias and difficulty in comparing studies. EVs hold promise for improving care for symptomatic patients who have never had surgery, as well as those with recurrent symptoms after previous surgery. We encourage further EV research in endometriosis with the inclusion of rigorous reporting of both the patient population and technical methodology used, with the ultimate goal of achieving clinical utility for diagnosis, prognosis and eventually treatment.
Background High-temperature requirement protease A2 (HtrA2/Omi) is a mitochondrial chaperone that is highly conserved from bacteria to humans. It plays an important role in mitochondrial homeostasis and apoptosis. In this study, we investigated the role of HtrA2 in mouse oocyte maturation. Methods The role of HtrA2 in mouse oocyte maturation was investigated by employing knockdown (KD) or overexpression (OE) of HtrA2 in young or old germinal vesicle (GV) oocytes. We employed immunoblotting, immunostaining, fluorescent intensity quantification to test the HtrA2 knockdown on the GV oocyte maturation progression, spindle assembly checkpoint, mitochondrial distribution, spindle organization, chromosome alignment, actin polymerization, DNA damage and chromosome numbers and acetylated tubulin levels. Results We observed a significant reduction in HtrA2 protein levels in aging germinal vesicle (GV) oocytes. Young oocytes with low levels of HtrA2 due to siRNA knockdown were unable to complete meiosis and were partially blocked at metaphase I (MI). They also displayed significantly more BubR1 on kinetochores, indicating that the spindle assembly checkpoint was triggered at MI. Extrusion of the first polar body (Pb1) was significantly less frequent and oocytes with large polar bodies were observed when HtrA2 was depleted. In addition, HtrA2 knockdown induced meiotic spindle/chromosome disorganization, leading to aneuploidy at metaphase II (MII), possibly due to the elevated level of acetylated tubulin. Importantly, overexpression of HtrA2 partially rescued spindle/chromosome disorganization and reduced the rate of aneuploidy in aging GV oocytes. Conclusions Collectively, our data suggest that HtrA2 is a key regulator of oocyte maturation, and its deficiency with age appears to contribute to reproduction failure in females.
Abstract Objective Nowadays, patients attempting social/elective egg freezing has spread globally. Ovarian stimulation (OS) with high daily gonatotropin doses, are commonly offered to this group of patients, aiming to achieve the maximal oocytes cohort with minimum IVF cycle attempts. We aim to assess the IVF-ET outcome, and specifically the oocyte yield, of patients undergoing two successive IVF cycle attempts for elective egg freezing (EEF), and whether changing the daily gonadotropin dose in the second IVF cycle attempt, affect the outcome. Patients and methods All women admitted to our IVF unit for social/EEF, who underwent 2 consecutive IVF cycle attempts, with only those who used in the first attempt a starting daily gonadotropin dose of 300 IU were included. Ovarian stimulation characteristics, duration of OS, number of retrieved oocytes, number of mature oocytes were assessed and compared between the 1st and the 2nd IVF cycle attempts, and between the different daily gonadotropin doses and the oocyte yields in the 2nd cycle attempt (increase, decrease or no change). Main outcome measures Oocytes and mature oocytes yield in the 2nd as compared to the 1st IVF cycle attempt. Results A reduced oocyte yield in the 2nd cycle attempt was observed in those who highly responded in the 1st attempt, regardless the daily dose in the 2nd cycle attempt (whether it was increased, no change and decreased). Moreover, the proportion of patients with same or more oocytes in the 2nd IVF cycle attempt was significantly lower in patients with high peak E2 levels, compared to those with peak E2 levels 9175 pmol/L), those who achieved a lower oocytes yield in the 2nd IVF cycle attempt had lower basal Day-3 FSH/LH ratio (1.5 + 0.5 vs 1.8 + 0.8, p 9 oocytes and 61.8% of those with 9175 pmol/L) in the 1st attempt, and basal Day-3 FSH/LH ratio
Expression of CD44v3 in the endometrium of women with RIF (A) mRNA levels of CD44s, CD44v3, and CD44v6 in the endometrium of women in the control (n = 18) and RIF (n = 24) groups. B Immunohistochemical staining and (C) semiquantitative analysis of CD44s, CD44v3, and CD44v6 in the control and RIF groups (n = 15 per group). D Representative western blots and (E) densitometric quantification of CD44v3 in the control and RIF groups. (F) ROC curve for determining the expression of CD44v3 in endometrial tissue. Bar = 50 µm. *p < 0.05; **p < 0.01
Expression of CD44v3 in endometrial tissues and primary endometrial cells after E2 and P4 treatments (A)Immunohistochemical staining and (B) semiquantitative analysis of CD44v3 during the proliferative (n = 12) and mid-secretory phases (n = 15) in the control patients. (C) Western blot and (D) statistical analyses of CD44v3 expression in the endometrium during the proliferative and mid-secretory phases in the control group. (E and G) Western blot and (F and H) statistical analyses of CD44v3 expression in primary HESCs and HEECs with or without estrogen (E2) and progesterone (P4) treatment for 72 h. P, proliferative phase; MS, mid-secretory phase. Bar = 50 µm. *p < 0.05.
Changes in cell proliferation and stromal cell decidualization following CD44v3 knockdown/overexpression in T-HESCs (A, C) Western blot and (B, D) densitometric quantification of CD44v3 after the knockdown/overexpression of CD44v3 in T-HESCs. (E) Wound healing analysis and (F) semiquantitative analysis of wound closure in T-HESCs after the knockdown/overexpression of CD44v3. (G) Cell proliferation analyses after the knockdown/overexpression of CD44v3 in T-HESCs. (H) Expression levels of CD44v3, PRL, and IGFBP1 after the knockdown/overexpression of CD44v3 during the in vitro decidualization assay in T-HESCs. *p < 0.05; **p < 0.01
Changes in cell proliferation and stromal cell decidualization following CD44v3 knockdown/overexpression in primary HESCs (A, C) Western blot and (B, D) densitometric quantification of CD44v3 expression after the knockdown/overexpression of CD44v3 in primary HESCs. (E) Wound healing analysis and (F) semiquantitative analysis of wound closure in primary HESCs after the knockdown/overexpression of CD44v3. (G) Cell proliferation after the knockdown/overexpression of CD44v3 in primary HESCs. (H) mRNA expression of CD44v3, PRL, and IGFBP1 after the knockdown/overexpression of CD44v3 during the in vitro decidualization assay in primary HESCs. (I) Western blot and (J, K) densitometric quantification of CD44v3, PRL, and IGFBP1 after the knockdown/overexpression of CD44v3 during the in vitro decidualization assay in primary HESCs. *p < 0.05; **p < 0.01
CD44v3 knockdown in ESCs inhibits trophoblast outgrowth in vitro (A) Representative images of spreading mouse trophoblast (white line) cocultured on negative control and CD44v3 knockdown HESCs, mock, and CD44v3 overexpressing HESCs. (B) Quantification of the outgrowth area. The mean value of the control group was set to 1. *p < 0.05
Background The precise pathogenesis of poor endometrial receptivity in recurrent implantation failure (RIF) remains unclear. This study was aimed at exploring the effects of different CD44 isoforms in the mid-secretory phase endometrium on endometrial receptivity in women with RIF. Methods Mid-secretory phase endometrial tissue samples were obtained from the following two groups of women who had undergone IVF: (a) 24 patients with RIF and (b) 18 patients with infertility due to tubal obstruction, who had achieved a successful clinical pregnancy after the first embryo transfer in IVF (control group). Identification of differentially expressed CD44 isoforms in endometrial tissues was assessed using immunohistochemistry, qPCR, and western blotting. Effects of overexpression and knockdown of CD44v3 on proliferation and decidualization of immortalized human endometrial stromal cells (T-HESCs) and primary HESCs were investigated by qPCR and western blot analysis. A heterologous coculture system of embryo implantation was constructed to mimic the process of trophoblast invasion during implantation. Results The expression of CD44v3 was significantly higher in the mid-secretory phase of endometrial stromal cells than in the proliferation phase, but was notably lower in RIF patients. Knockdown of CD44v3 significantly downregulated cell proliferation both in T-HESCs and HESCs. The expression of decidualization markers, prolactin (PRL) and insulin like growth factor binding protein-1 (IGFBP1), was notably decreased following the knockdown of CD44v3, whereas the expression of both PRL and IGFBP1 increased after its overexpression in HESCs. Furthermore, the CD44v3-knockdown HESCs displayed significant deficiency in supporting trophoblast outgrowth in a coculture system of embryo implantation; however, overexpression of CD44v3 in HESCs promoted trophoblast outgrowth. Conclusion The reduced expression of CD44v3 suppresses the proliferation and decidualization of HESCs, which might play a pivotal role in poor endometrial receptivity in women with RIF.
The PRISMA flowchart for the process of literature search and study selection
The relationship between ADAMTS family members and oocyte maturity rate
The relationship between ADAMTS family members and oocyte recovery rate
The relationship between ADAMTS family members and fertilization rate
The relationship between ADAMTS-1 levels and cleavage, good-quality embryo, and blastocyst formation rates
Abstract Background A disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS) is involved in inflammation and fertility in women with polycystic ovary syndrome (PCOS). This study aims to assess the role of ADAMTS level in the outcomes of in vitro fertilization and embryo transfer (IVF-ET) in women with PCOS, using a meta-analytic approach. Methods We systematically searched Web of Science, PubMed, EmBase, and the Cochrane library to identify potentially eligible studies from inception until December 2021. Study assess the role of ADAMTS levels in patients with PCOS was eligible in this study. The pooled effect estimates for the association between ADAMTS level and IVF-ET outcomes were calculated using the random-effects model. Results Five studies involving a total of 181 patients, were selected for final analysis. We noted that ADAMTS-1 levels were positively correlated to oocyte maturity (r = 0.67; P = 0.004), oocyte recovery (r = 0.74; P = 0.006), and fertilization (r = 0.46; P = 0.041) rates. Moreover, ADAMTS-4 levels were positively correlated to oocyte recovery (r = 0.91; P = 0.001), and fertilization (r = 0.85; P = 0.017) rates. Furthermore, downregulation of ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMTS-9 was associated with elevated follicle puncture (ADAMTS-1: weighted mean difference [WMD], 7.24, P
Background Preimplantation embryonic lethality is a driver of female infertility. Certain microRNAs (miRNAs) have previously been demonstrated to play important roles in the regulation of embryogenesis. Methods Normally developing blastocysts and arrested embryos were collected from patients undergoing intracytoplasmic sperm injection (ICSI), and the expression of specific miRNAs therein was evaluated by qPCR. The overexpression of target molecule miR-145 in early mice embryos was achieved via oocyte microinjection, enabling the subsequent monitoring of how such overexpression impacted embryonic development. Bioinformatics approaches were utilized to identify putative miR-145 target mRNAs, and luciferase reporter assessments were implemented to confirm the ability of miR-145 to regulate Abca1 in HEK293T cells. The functional relationship between miR-145 and Abca1 in the mice’s embryonic development was then confirmed through rescue assays. Results Abnormally increased miR-145 expression was observed in patients’ arrested embryos, and the exogenous overexpression of this miRNA significantly suppressed mural blastocyst formation. Mechanistically, miR-145 was found to bind to the 3′-untranslated area of the Abca1 mRNA in HK293T cells, thus suppressing its expression and increasing embryonic cholesterol levels. In line with the importance of these cholesterol levels to embryogenesis, the upregulation of Abca1 was sufficient to rescue the observed change in cholesterol levels and the associated retardation of mice embryonic development that occurred in response to the overexpression of miR-145. Conclusion The regulatory dynamics of the miR-145/Abca1 axis play an important role in shaping normal embryonic development.
Abstract Purpose To investigate the effects of coenzyme Q10 (CoQ10) and transcutaneous electrical acupoint stimulation (TEAS) pretreatment on pregnancy in patients with poor ovarian response (POR). Methods A total of 330 POR patients who were pretreated with CoQ10 or CoQ10 combined with TEAS before their in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET) cycles and who were not pretreated were selected and divided into CoQ10 group (group A, n = 110), CoQ10 + TEAS group (group B, n = 110) and control group (group C, n = 110). For patients with 2 or more transfer cycles, only the information of the first cycle was included. Ovarian function, response to gonadotropin (Gn) stimulation, and pregnancy outcomes of the three groups were compared in the IVF/ICSI-ET cycles. Results After pretreatment, basal FSH, total Gn dosage and duration were comparable among the three groups (all p-value > 0.05), basal E2 in group B decreased significantly compared with the control group (p = 0.022). Endometrial thickness on the human chorionic gonadotropin (hCG) day, antral follicle counts (AFC), the numbers of oocytes, metaphase II (MII) eggs and excellent embryos in the two pretreatment groups were significantly increased compared with group C (all p-value
PRISMA flowchart detailing the selection of publications for this literature review of identified ZP mutations
Mutations in ZP genes and ZP proteins
Different phenotypes of patients with ZP mutations. Different mutations of ZP can cause different clinical phenotypes, including empty follicle syndrome, ZP-thin oocytes, ZP-free oocytes, degenerated oocytes, and immature oocytes. COC, cumulus-oocyte complexes; EFS, empty follicle syndrome
Prediction model of pregnancy outcome. A. The receiver operating characteristic (ROC) curves of a mixture of four features, including the mutated ZP gene, the protein domain where the mutation is located, the type of mutation, and the clinical phenotype of the patient. The area under the curve (AUC) is 0.898. B. The normalized confusion matrix shows the true positive rate is 1.00 and the true negative rate is 0.38. The study used one-hot coding, coding non-pregnancy as 1 and pregnancy as 0
Three dimensional models of ZP proteins with missense mutations. The structures of ZP proteins are predicted by AlphaFold from AlphaFold Protein Structure Database ( Alpha helixes are shown in red, beta folds are shown in yellow, loops are shown in green. Amino acid residues with missense mutations are shown in blue
Background In mammals, normal fertilization depends on the structural and functional integrity of the zona pellucida (ZP), which is an extracellular matrix surrounding oocytes. Mutations in ZP may affect oogenesis, fertilization and early embryonic development, which may cause female infertility. Methods A PubMed literature search using the keywords ‘zona pellucida’, ‘mutation’ and ‘variant’ limited to humans was performed, with the last research on June 30, 2022. The mutation types, clinical phenotypes and pregnancy outcomes were summarized and analyzed. The naive Bayes classifier was used to predict clinical pregnancy outcomes for patients with ZP mutations. Results A total of 29 publications were included in the final analysis. Sixty-nine mutations of the ZP genes were reported in 87 patients with different clinical phenotypes, including empty follicle syndrome (EFS), ZP-free oocytes (ZFO), ZP-thin oocytes (ZTO), degenerated and immature oocytes. The phenotypes of patients were influenced by the types and location of the mutations. The most common effects of ZP mutations are protein truncation and dysfunction. Three patients with ZP1 mutations, two with ZP2 mutations, and three with ZP4 mutations had successful pregnancies through Intracytoplasmic sperm injection (ICSI) from ZFO or ZTO. A prediction model of pregnancy outcome in patients with ZP mutation was constructed to assess the chance of pregnancy with the area under the curve (AUC) of 0.898. The normalized confusion matrix showed the true positive rate was 1.00 and the true negative rate was 0.38. Conclusion Phenotypes in patients with ZP mutations might be associated with mutation sites or the degree of protein dysfunction. Successful pregnancy outcomes could be achieved in some patients with identified ZP mutations. Clinical pregnancy prediction model based on ZP mutations and clinical characteristics will be helpful to precisely evaluate pregnancy chance and provide references and guidance for the clinical treatment of relevant patients.
Background Polycystic ovary syndrome is associated with an increased rate of spontaneous abortion/early pregnancy loss and pups delivered to PCOS animals were abnormal. Currently, assisted reproductive technology has been used to help numerous infertile couples to have their babies. However, there is a low implantation rate after the transfer of embryos. Till now, it could not be concluded whether the reduced pregnancy rates observed were due to abnormal embryos or endometrial modification. Further, transgenic mouse models have been used to find out the molecular deficits behind early pregnancy complications. But, the deletion of crucial genes could lead to systemic deficiencies/embryonic lethality. Also, pregnancy is a complex process with overlapping expression patterns making it challenging to mimic their stage-specific role. Therefore, the motive of the current study was to investigate the probable molecular cascade to decipher the early pregnancy loss in the letrozole-induced PCOS mouse model. Methods PCOS was induced in mice by oral administration of letrozole daily for 21 days. Following, the pregnancy was established and animals were sacrificed on the day 6th of pregnancy. Animals were assessed for early pregnancy loss, hormonal profile, mRNA expression of steroid receptors ( Ar, Pr, Esr1/2 ), decidualization markers ( Hox10/11a ), adhesion markers ( Itgavb3, Itga4b1 ), matrix metalloproteinases and their endogenous inhibitor ( Mmp2/9, Timp1/2) and key mediators of LIF/STAT pathway ( Lif, Lifr, gp130 , stat3 ) were analyzed in the embryo implanted region of the uterus. Morphological changes in ovaries and implanted regions of the uterus were assessed. Results Mice treated with letrozole demonstrated significant increases in testosterone levels along with a decline in progesterone levels as compared to control animals. PCOS animals also exhibited decreased fertility index and disrupted ovarian and embryo-containing uterus histopathology. Altered gene expression of the steroid receptors and reduced expression of Hox10a , integrins, Mmp9, Timp1/3 , Gp130 & Stat3 was observed in the implanted region of the uterus of PCOS animals. Conclusion Our results reveal that majority of the molecular markers alteration in the establishment of early pregnancy could be due to the aberrant progesterone signaling in the embryonic-uterine tissue of PCOS animals, which further translates into poor fetal outcomes as observed in the current study and in several IVF patients.
Globozoospermia (OMIM: 102530) is a rare type of teratozoospermia (< 0.1%). The etiology of globozoospermia is complicated and has not been fully revealed. Here, we report an infertile patient with globozoospermia. Variational analysis revealed a homozygous missense variant in the SSFA2 gene (NM_001130445.3: c.3671G > A; p.R1224Q) in the patient. This variant significantly reduced the protein expression of SSFA2. Immunofluorescence staining showed positive SSFA2 expression in the acrosome of human sperm. Liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) and Coimmunoprecipitation (Co-IP) analyses identified that GSTM3 and Actin interact with SSFA2. Further investigation revealed that for the patient, regular intracytoplasmic sperm injection (ICSI) treatment had a poor prognosis. However, Artificial oocyte activation (AOA) by a calcium ionophore (A23187) after ICSI successfully rescued the oocyte activation failure for the patient with the SSFA2 variant, and the couple achieved a live birth. This study revealed that SSFA2 plays an important role in acrosome formation, and the homozygous c.3671G > A loss-of-function variant in SSFA2 caused globozoospermia. SSFA2 may represent a new gene in the genetic diagnosis of globozoospermia, especially the successful outcome of AOA-ICSI treatment for couples, which has potential value for clinicians in their treatment regimen selections.
Background Ovarian tissue cryopreservation and transplantation (OTCTP) is currently the main option available to preserve fertility in prepubertal patients undergoing aggressive cancer therapy treatments. However, a major limitation of OTCTP is follicle loss after transplantation. The mouse is a model of choice for studying ovarian function and follicle development after ovarian tissue grafting in vivo. In these mouse models, ovarian tissue or ovaries can be transplanted to different sites. Our aim was to evaluate a new alternative to heterotopic transplantation models that could be useful to test pharmaceutical improvement for ovarian grafts after OTCTP. Methods Slow frozen murine whole ovaries were transplanted into the mouse ears (between the external ear skin layer and the cartilage). Ovarian transplants were recovered after 3, 14 or 21 days. Grafts were analyzed by immunohistochemistry and follicle density analyses were performed. Results An increase of ovarian vascularization (CD31 and Dextran-FITC positive staining), as well as cellular proliferation (Ki67 staining) were observed 3 weeks after transplantation in comparison to 3 days. Fibrosis density, evaluated after Van Gieson staining, decreased 3 weeks after transplantation. Furthermore, transplantation of cryopreserved ovaries into ovariectomized mice favored follicle activation compared to transplantation into non-ovariectomized mice. Conclusion The present study indicates that surgical tissue insertion in the highly vascularized murine ear is an effective model for ovarian grafting. This model could be helpful in research to test pharmaceutical strategies to improve the function and survival of cryopreserved and transplanted ovarian tissue.
Background Sperm, during epididymal transit, acquires microRNAs(miRNAs), which are crucial for embryonic development. However, whether sperm miRNAs influenced by an obstructive epididymal environment affect embryonic development remains unknown. Method The sham operation and vasectomy were performed in C57BL/6 J mice to create the control group (CON) and the obstructive epididymal environment group(OEE) group, respectively. The morphology of the testis and epididymis was observed using hematoxylin and eosin staining (HE staining) to establish the OEE mice model. The sperm quality test, intracytoplasmic sperm injection (ICSI), and epididymosomes fusion were employed to observe the effect of the obstructive epididymal environment on sperm and resultant embryonic development. The alteration of the sperm small RNA (sRNA) profile was analyzed by sRNA sequencing. RT-qPCR and DNA methylation were applied to observe the effect of obstructive epididymis on the expression of sperm miRNAs. The miRNAs microinjection was used to explore the impacts of sperm miRNAs on embryonic development. Results We confirmed postoperative 8-week mice as the OEE mice model by examining the morphology of the testis and epididymis. In the OEE group, we observed that sperm quality degraded and the development potential of embryos was reduced, which can be saved by the normal epididymal environment. The sperm sRNA sequencing revealed that the expression of the developmental miR-17–92 cluster and the Sfmbt2 miRNA cluster was downregulated in the OEE group. The expression of these two miRNA clusters in epididymis was also downregulated and regulated by DNA methylation. However, the downregulation of either the miR-17–92 cluster or the Sfmbt2 miRNA cluster in normal zygotes did not impair embryonic development. Conclusion The obstructive epididymal environment influences sperm quality and resultant embryonic development, as well as the abundance of the developmental miR-17–92 cluster and the Sfmbt2 miRNA cluster in sperm, but these miRNA clusters are not the cause of abnormal embryonic development. It implies that epididymis is important in early embryonic development and may play a potential role in sperm epigenome.
Obesity impacts fertility and is positively correlated with endometrial hyperplasia and endometrial cancer occurrence. Endometrial epithelia often harbor disease driver-mutations, while endometrial stroma are highly regulative of neighboring epithelia. Here, we sought to determine distinct transcriptome changes occurring in individual cell types in the obese mouse uterus. Outbred CD-1 mice were fed high-fat or control diets for 18 weeks, estrous cycle staged, and endometrial epithelia, macrophages, and stroma isolated for transcriptomic analysis. High-fat diet mice displayed increased body mass and developed glucose intolerance, hyperinsulinemia, and fatty liver. Obese mouse epithelia displayed differential gene expression for genes related to innate immunity and leukocyte chemotaxis. The obese mouse stroma differentially expressed factors related to circadian rhythm, and expression of these genes correlated with glucose tolerance or body mass. We observed correlations between F4/80 + macrophage numbers, Cleaved Caspase 3 (CC3) apoptosis marker staining and glucose intolerance among obese mice, including a subgroup of obese mice with high CC3 + luminal epithelia. This subgroup displayed differential gene expression among all cell types, with pathways related to immune escape in epithelia and macrophages, while the stroma dysregulated pathways related to regulation of epithelia. These results suggest an important role for differential response of both the epithelia and stroma in their response to obesity, while macrophages are dysregulated in the context of apoptotic epithelia. The obesity-related gene expression programs in cells within the uterine microenvironment may influence the ability of the endometrium to function during pregnancy and influence disease pathogenesis.
The relationship between E2 concentration (pg/ml) on hCG trigger day and adjust mean birthweight(g)
The relationship between E2 concentration (pg/ml) on hCG trigger day and adjust of SGA
*Analyses of both in Fig. 1 and Fig. 2 were adjusted for couple’s age, maternal BMI, basal FSH, AMH, prior gonadotropin cycle, COS protocols, total Gn dose, stimulation duration, fertilization method, number of oocytes retrieved, endometrial type, number and stage of embryos transferred, newborn sex
Patient clinical characteristics, cycle parameters and neonatal outcomes by different E2 level on hCG trigger day
Background Previous studies have demonstrated that the supraphysiological E2 level is negatively correlated with birthweight. However, the cut-off value of E2 level that significantly affects birthweight is unknown, and there is no definite conclusion regarding this level. Our study aimed to explore the threshold of the effect of E2 levels on birthweight. Design A retrospective cohort study of 1846 samples was performed. All patients ≤42-years-old underwent autologous IVF cycles between August 1st, 2016 and April 30th, 2020. We categorized our data into four groups according to the E2 level: Group 1: ≤2000 pg/mL; Group 2: 2001–3000 pg/mL; Group 3: 3001–4000 pg/mL; and Group 4: > 4000 pg/mL. Results The results of the multivariate regression analyses showed that when the E2 level was 3001–4000 pg/mL (adjusted β: − 89.64, 95% [CI]: − 180.29 to − 6.01; P = 0.0336) and greater than 4000 pg/mL (adjusted β: − 138.10, 95% [CI]: − 272.87 to − 10.33; P = 0.0181), weight loss was significant. Furthermore, the odds of full-term SGA were 1.40 times higher with E2 levels of 3001–4000 pg/mL (adjusted OR: 1.40, 95% [CI]: 1.090 to 3.18; P = 0.0256) and 2.55 times higher with E2 > 4000 pg/mL (adjusted OR: 2.55, 95% [CI]: 1.84 to 3.86; P = 0.0063) compared to the reference group. It can also be seen from the adjusted curves and the threshold effects that when the E2 level > 2950 pg/mL and > 3121 pg/mL, the incidence of SGA increased and the birthweight decreased, respectively. Conclusions Our data suggest that E2 levels > 2950 pg/mL is an independent predictor for greater odds of full-term SGA singletons born after fresh embryo transfer.
A representative image of immunofluorescence staining in human ovarian granulosa cells (n = 5). The Red (× 400) expressed FSHR, the blue (× 400) expressed nuclear staining using 4 ‘, 6-diamino-2-phenylindole (DAPI). Non-specific staining can be observed with PBS instead of primary or secondary antibodies
Abstract Background MicroRNAs (miRNAs) are considered potential biomarkers for various diseases. This study investigated whether hsa-miR-320a-3p and hsa-miR-483-5p levels in human ovarian granulosa cells derived from follicular fluids are associated with embryo developmental competence. Methods We collected 195 granulosa cells samples and analyzed the treatment outcomes in patients undergoing in vitro fertilization (n = 147) or intracytoplasmic sperm injection (n = 48) cycles. The hsa-miR-320a-3p and hsa-miR-483-5p levels in granulosa cells were measured using quantitative reverse transcription-polymerase chain reaction. Results Patients were subdivided into four groups according to the granulosa cells hsa-miR-320a-3p and hsa-miR-483-5p levels quartiles (Q1–Q4). Embryo developmental competence was compared using the chi-square test. Patients in Q3 were less likely to achieve a normal fertilization rate for in vitro fertilization and blastocyst formation than those in Q1 as they expressed high levels of hsa-miR-320a-3p and hsa-miR-483-5p (P
Background Mammalian sperm maturation in the epididymis is mainly modulated by exosomes that are secreted into the epididymal lumen from epididymal epithelial cells (EECs). Exposure to oxidative stress (OS) resulting from being fed a high fat diet (HFD) reduces sperm fertility, which is one of the cause inducing male infertility. Thus, we hypothesize that stress-induced changes in exosome content play a critical role in mediating this detrimental process. Methods An obese mouse model was established by feeding a HFD. Then oxidative stress status was measured in the mouse caput epididymis, epididymal fluid and spermatozoa. Meanwhile, epididymis-derived purified exosomes were isolated and validated. Subsequently, liquid chromatography tandem mass spectrometry (LC-MS) was used to perform proteomic analysis of purified exosomes. Gene Ontology (GO) analysis was performed along with pathway enrichment to identify differentially expressed proteins (DEPs). Results Two hundred and two DEPs mostly related to endoplasmic reticulum (ER) function were identified in the exosomes separated from the epididymis of control mice and obese mice. The ER stress and CD63 (an exosome marker), both increased in the caput epididymis of obese mice. Furthermore, an in vitro study showed that palmitic acid (PA), an-oxidative stress inducer, increased exosome biogenesis and secretion in the EECs. Conclusion Oxidative stress in the epididymal microenvironment induces ER stress in the EECs. This effect alters the epididymis-derived exosome content, profile and amounts of their differentially expressed ER proteins. Such changes may affect exosome biogenesis and cargo packaging, finally leading to abnormalities in sperm maturation and fertility.
Top-cited authors
Ashok Agarwal
  • Cleveland Clinic
Rakesh K Sharma
  • Cleveland Clinic
Sajal Gupta
  • Cleveland Clinic
Norbert Gleicher
  • The Center for Human Reproduction. New York, N.Y.
David Barad
  • Center for Human Reproduction