Factors associated with embryo mosaicism: a systematic review and meta-analysis.

PURPOSE: Evaluate which factors are involved in the increased rate of mosaicism in embryos. METHODS: A systematic review and meta-analysis was performed. After an exhaustive search of the literature, a total of seven papers were included in the analysis. In addition, data collected from IVF cycles performed in our fertility clinic were also analysed. Day of biopsy, embryo quality, maternal and paternal age and seminal quality were the chosen factors to be studied. RESULTS: The results of the meta-analysis show that neither embryo quality nor seminal quality were related to mosaic embryo rate (OR: 1.09; 95% CI: 0.94-1.28 and OR: 1.10; 95% CI: 0.87-1.37, respectively). A positive association was observed for the variable "biopsy day" with embryos biopsied at day 6 or 7 having the highest rate of mosaicism (OR: 1.06; 95% CI: 1.01-1.11). In opposite to what happens with aneuploidy rate, which increases with maternal age, embryo mosaicism is higher in younger women (<34 years) rather than in older ones (≥34 years) (OR: 0.95; 95% CI: 0.92-0.98). However, for the "paternal age" factor, no association with mosaicism was found (OR: 1.04; 95% CI: 0.90-1.21). CONCLUSIONS: With the present study, we can conclude that the factors related to the presence of mosaicism in embryos are the embryo biopsy day and maternal age. The rest of the studied factors showed no significant relationship with mosaicism. These results are of great importance as knowing the possible causes leading to mosaicism helps to improve the clinical results of reproductive treatments.

Identification of novel candidate genes associated with meiotic aneuploidy in human embryos by whole-exome sequencing.

PURPOSE: To identify novel genetic variants responsible for meiotic embryonic aneuploidy. METHODS: A prospective observational cohort study that included 29 couples who underwent trophectoderm biopsies from 127 embryos and performed whole-exome sequencing (WES) between November 2019 and March 2022. Patients were divided into two groups according to the expected embryo aneuploidy rate based on maternal age. RESULTS: After variant filtering in the WES analysis of 58 patients/donors, five heterozygous variants were identified in female partners from the study group that had an impact on embryo aneuploidy. Additionally, a slowdown in embryo development and a decrease in the number of blastocysts available for biopsy were observed in the study group embryos. CONCLUSION: This study has identified new candidate genes and variants not previously associated with meiotic embryo aneuploidy, but which are involved in important biological processes related to cell division and chromosome segregation. WES may be an efficient tool to identify patients with a higher-than-expected risk of embryo aneuploidy based on maternal age and allow for individualized genetic counselling prior to treatment.

IL-6/IL-10 and IL-1ß/IL-4 ratios associated with poor ovarian response in women undergoing in-vitro fertilization.

The aim of this work was to evaluate whether serum cytokines levels are associated with ovarian response in IVF. 149 patients were included in a retrospective study. Cytokines IL-2, IL-4, IL- 6, IL-8, IL-10, VEGF, IFNγ, TNFα, IL-1α, IL-1ß, MCP-1 and EGF were measured by sandwich immunoassay previously to ovarian stimulation. Performing hierarchical cluster analysis, we observed that the antral follicle count, the total oocytes recovered and the MII recovered are grouped in the same cluster as the cytokines IL-2-4-6-10-1α-1ß, IFNγ y TNFα. Then, we found that the ratio between IL and 6 and IL-10 was higher in low responder women (2.15 versus 1.55; p = 0.035). If we establish 0.9 as a cut-off for the IL-6/IL-10, we observed that above this value the risk of having a low response to ovarian stimulation was more than 3 times greater than below this value (22.9 % versus 6.0 %; p = 0.007). Also, the ratio IL-1ß/IL-4 was higher in patients with normal or suboptimal response (0.62 versus 0.34; p = 0.034) and any patient with low response had a value greater than 0.7 (p = 0.003). As a conclusion, the IL-6/IL-10 and IL-1ß/IL-4 ratios showed differences between normoresponder women and patients with low ovarian response.

Identification of new variants and candidate genes in women with familial premature ovarian insufficiency using whole-exome sequencing.

PURPOSE: To identify candidate variants in genes possibly associated with premature ovarian insufficiency (POI). METHODS: Fourteen women, from 7 families, affected by idiopathic POI were included. Additionally, 98 oocyte donors of the same ethnicity were enrolled as a control group. Whole-exome sequencing (WES) was performed in 14 women with POI to identify possibly pathogenic variants in genes potentially associated with the ovarian function. The candidate genes selected in POI patients were analysed within the exome results of oocyte donors. RESULTS: After the variant filtering in the WES analysis of 7 POI families, 23 possibly damaging genetic variants were identified in 22 genes related to POI or linked to ovarian physiology. All variants were heterozygous and five of the seven families carried two or more variants in different genes. We have described genes that have never been associated to POI pathology; however, they are involved in important biological processes for ovarian function. In the 98 oocyte donors of the control group, we found no potentially pathogenic variants among the 22 candidate genes. CONCLUSION: WES has previously shown as an efficient tool to identify causative genes for ovarian failure. Although some studies have focused on it, and many genes are identified, this study proposes new candidate genes and variants, having potentially moderate/strong functional effects, associated with POI, and argues for a polygenic etiology of POI in some cases.

Identification of vaginal microbiome associated with IVF pregnancy.

The factors that cause a preterm birth (PTB) are not completely understood up to date. Moreover, PTB is more common in pregnancies achieved by in-vitro fertilization (IVF) than in spontaneous pregnancies. Our aim was to compare the composition of vaginal microbiome at 12 weeks of gestation between women who conceived naturally or through IVF in order to study whether IVF PTB-risk could be related to vaginal microbiome composition. We performed an observational, prospective and multicentre study among two public hospitals and a fertility private clinic in Spain. Vaginal swabs from 64 pregnant women at 12 weeks of gestation were collected to analyse the microbiome composition by sequencing the V3-V4 region of the 16S rRNA. Our results showed that the vaginal microbiome signature at 12 weeks of pregnancy was different from women who conceived naturally or through IVF. The beta diversity and the genus composition were different between both cohorts. Gardnerella, Neisseria, Prevotella, and Staphylococcus genus were enriched genus in the vaginal microbiome from the IVF group, allowing us to create a balance model to predict both cohorts. Moreover, at species level the L. iners abundance was higher and L. gasseri was lower in the IVF group. As a conclusion, our findings were consistent with a proposed framework in which IVF pregnancy are related to risk for preterm birth (PTB) suggesting vaginal microbiome could be the reason to the relation between IVF pregnancy and risk for PTB.

Characterization of the vaginal and endometrial microbiome in patients with chronic endometritis.

OBJECTIVE: To compare the endometrial and vaginal microbiome of women with and without chronic endometritis. STUDY DESIGN: A cohort study with 60 patients undergoing assisted reproductive treatment with their own or donated gametes was undertaken. Vaginal and endometrial samples were taken in the cycle prior to embryo transfer. The endometrial and vaginal microbiome was analysed by mass sequencing of the V3V4 region of 16S rRNA gene. Bioinformatics analysis was performed using QIIME2 and MicrobiomeAnalyst packages. Alpha diversity, beta diversity and taxonomic characterization were compared between samples that tested positive and negative for chronic endometritis on CD138 immunohistochemistry. RESULTS: Different bacterial communities were detected when vaginal and endometrial samples were analysed in patients with and without endometritis diagnosed using CD138 immunohistochemistry. In patients with endometritis, a higher alpha-diversity index was found in vaginal samples (p = 0.15 for the Shannon index) and significant differences were found in endometrial samples (p = 0.01 for the Shannon index). In the beta-diversity analysis, no significant differences were observed between the groups with and without endometritis. Vaginal and endometrial samples from women with endometritis showed a microbiome pattern that was not dominated by Lactobacillus spp. Relative abundance analysis identified Ralstonia and Gardnerella spp. in endometrial samples, and Streptoccoccus and Ureaplasma spp. in vaginal samples of patients diagnosed with chronic endometritis on CD138 immunohistochemistry. When comparing endometrial and vaginal samples diagnosed with endometritis on CD138 immunohistochemistry, both alpha diversity (p = 0.06 for the Shannon index and p = 0.08 for the Simpson index) and beta diversity (p < 0.001) showed significant differences. Lactobacillus spp. (p = 3.76E-4), Ralstonia spp. (p = 8.19E-4), Delftia spp. (p = 0.004) and Anaerobacillus spp. (p = 0.004) were identified in these sample groups. CONCLUSION: These results demonstrate the existence of a characteristic vaginal and endometrial microbiota in patients with chronic endometritis. Different genera and species were identified in patients with and without chronic endometritis depending on whether the sample was endometrial or vaginal. There is a clear relationship between changes in the vaginal microbiome and chronic endometritis. The microbiota is a continuum throughout the female reproductive tract, so study of the vaginal microbiota could be useful for the diagnosis of diseases of the upper reproductive tract, such as chronic endometritis.

Comprehensive mitochondrial DNA analysis and IVF outcome.

STUDY QUESTION: Do mitochondrial DNA (mtDNA) copy number and heteroplasmy in human embryos affect the ongoing pregnancy rate? SUMMARY ANSWER: Our study suggests that mtDNA copy number above a specific threshold is associated with the ongoing pregnancy rate. WHAT IS KNOWN ALREADY: Mitochondria play a vital role in cell function. Recently, there has been increasing research on mtDNA as a biomarker of embryo implantation. Although reports showed that high levels of mtDNA in the blastocyst are associated with low implantation potential, other publications were unable to confirm this. Confounding factors may influence the mtDNA copy number in euploid embryos. On the other hand it has been speculated that both mtDNA heteroplasmy and copy number contribute to mitochondrial function. Next generation sequencing (NGS) allows us to study in depth mtDNA heteroplasmy and copy number simultaneously. STUDY DESIGN SIZE DURATION: A prospective non-selection study was performed. We included 159 blastocyst biopsies from 142 couples who attended our clinic for preimplantation genetic testing for aneuploidies (PGT-A), from January 2017 to December 2017. All embryos were biopsied on Day 5 or Day 6. The aneuploid testing was performed by NGS. All blastocysts were diagnosed as euploid non-mosaic and were transferred. The mtDNA analysis was performed once the embryo diagnosis was known. PARTICIPANTS/MATERIALS SETTING METHODS: Sequencing reads mapping to the mtDNA genome were extracted from indexed bam files to identify copy number and heteroplasmy. The relative measure of mtDNA copy number was calculated by dividing the mtDNA reads by the nuclear DNA value to normalize for technical variants and the number of cells collected at the biopsy. All the results were subjected to a mathematical correction factor according to the embryo genome. Heteroplasmy was assigned by MitoSeek. MAIN RESULTS AND THE ROLE OF CHANCE: The mean average copy number and SD of mtDNA per genome was 0.0016 ± 0.0012. Regarding heteroplasmy, 40 embryos were heteroplasmy carriers (26.32%). MtDNA variants were detected in coding and non-coding regions and the highest number of variants in an embryo was eight. With respect to IVF outcome for mtDNA copy number analysis, we set a threshold of 0.003 for the following analysis. The vast majority of the embryos were below the threshold (142/159, 89.31%) and 17 embryos were classified as having higher mtDNA levels. We showed a reduction in ongoing pregnancy rate associated with elevated mtDNA copy number (42.96% versus 17.65%, P < 0.05). This result was independent of maternal age and day of the biopsy: these factors were included as confounding factors because mtDNA copy number was negatively correlated with female age (25 -30 y: 0.0017 ± 0.0011, 30 -35 y: 0.0012 ± 0.0007, 35 -40 y: 0.0016 ± 0.0009, over 40 y: 0.0024 + 0.0017, P < 0.05). Embryos biopsied on Day 5 were more likely to have higher quantities of mtDNA compared with those biopsied on Day 6 (0.0017 versus 0.0009, P < 0.001). According to IVF outcome and heteroplasmy, a lower ongoing pregnancy rate was reported for embryos that carried more than two variants. However, this did not reach statistical significance when we compared embryos with a number of variants lower or higher than two (39.15 versus 20.0, P = 0.188). Finally, a clear positive association between the mtDNA variants and copy number was reported when we compare embryos with or without heteroplasmy (0.0013 ± 0.0009 versus 0.0025 ± 0.0014, P < 0.001) and among different numbers of variants (0:0.0013 ± 0.0009, 1-2:0.0023 ± 0.0012, >2:0.0043 ± 0.0014, P < 0.05). LIMITATIONS REASONS FOR CAUTION: A limitation may be the size of the sample and the high-throughput sequencing technology that might not have detected heteroplasmy levels below 2% which requires high sequence depth A clinical randomized trial comparing the clinical outcome after the transfer of embryos selected according to mtDNA levels or only by morphological evaluation will be necessary. More research into the impact of mtDNA heteroplasmy and copy number on IVF outcome is needed. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that embryos with elevated mtDNA copy number have a lower chance of producing an ongoing pregnancy. MtDNA copy number is higher in older women and is dependent upon the number of cell divisions that preceded biopsy. Moreover, our data suggest that mitochondrial activity could be a balance between functional capacity and relative mtDNA copy number. STUDY FUNDING/COMPETING INTERESTS: There are no conflicts of interest or sources of funding to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Intermediate and normal sized CGG repeat on the FMR1 gene does not negatively affect donor ovarian response.

BACKGROUND: Fragile X syndrome is associated with low ovarian reserve and poor ovarian response. The aim of this study was to investigate whether CGG repeats on the fragile X mental retardation 1 (FMR1) gene have predictive value for ovarian response to stimulation with gonadotrophins and for clinical outcome in our oocyte donation program. METHODS: Oocyte donor candidates were selected according to Instituto Bernabeu oocyte donation program requirements. Fragile X genetic screening was performed in 204 oocyte donors, defining 141 controls and 63 cases: 35-39 repeats (n = 34), 40-45 (n = 12) and >45 (n = 17). All the patients underwent ovarian stimulation using a GnRH antagonist protocol and received a GnRH agonist trigger. The main factors used to measure outcome were oocyte yields, days of stimulation, gonadotrophin dosages, biochemical pregnancy, ongoing pregnancy and miscarriage rates. RESULTS: No differences between the study group and controls were reported in oocyte yields (17.5 versus 18.9) or days of stimulation (11.40 versus 9.82). The control group used significantly more gonadotrophin (2212 versus 1850 IU) than the study group. Clinical outcome was not affected by the CGG repeats on the FMR1 gene in oocyte donors. CONCLUSIONS: No negative effect was observed for intermediate-sized CGG repeats on ovarian stimulation and clinical outcome using a non-confounding model of oocyte donation. These results disagree with previous studies performed on infertility patients. Owing to the present study, fragile X genetic screening should not be considered for prediction of response to ovarian stimulation.

Respiratory nitrate reductase from haloarchaeon Haloferax mediterranei: biochemical and genetic analysis.

The Haloferax mediterranei nar operon has been sequenced and its regulation has been characterized at transcriptional level. The nar operon encodes seven open reading frames(ORFs) (ORF1 narB, narC, ORF4, narG, narH, ORF7 and narJ). ORF1, ORF4 and ORF7 are open reading frames with no assigned function, however the rest of them encoded different proteins. narB codes for a 219-amino-acid-residue iron Rieske protein. narC encodes a protein of 486 amino acid residues identified by databases searches as cytochrome-b (narC). The narG gene encodes a protein with 983 amino acid residues and is identified as a respiratory nitrate reductase catalytic subunit (narG). NarH protein has been identified as an electron transfer respiratory nitrate reductase subunit (narH). The last ORF encodes a chaperonin-like protein (narJ) of 242 amino acid residues. The respiratory nitrate reductase was purified 21-fold from H. mediterranei membranes. Based on SDS-PAGE and gel-filtration chromatography under native conditions, the enzyme complex consists of two subunits of 112 and 61 kDa. The optimum temperature for activity was 70 degrees C at 3.4 M NaCl and the stability did not show a direct dependence on salt concentration. Respiratory nitrate reductase showed maximum activity at pH 7.9 and pH 8.2 when assays were carried out at 40 and 60 degrees C, respectively. The absorption spectrum indicated that Nar contains Fe-S clusters. Reverse transcriptase (RT-PCR) shows that regulation of nar genes occurs at transcriptional level induced by oxygen-limiting conditions and the presence of nitrate.