Effect of endometrial biopsy method on ribonucleic acid quality and gene expression analysis in patients with leiomyoma.

Objective: To compare ribonucleic acid (RNA) quantity and purity in tissue collected with different endometrial sampling methods to establish the optimal tool for use in endometrial gene expression studies. Design: Observational study. Setting: University hospital. Patients: Fourteen patients with submucosal leiomyomas. Interventions: Unguided biopsies were obtained using a low-pressure suction device before hysteroscopy from 14 patients with submucosal leiomyomas followed by guided biopsy with a resectoscope loop. Fifty-seven samples were collected: 25 obtained using a suction device and 32 with a loop. Main Outcome Measures: Total biopsy weight, RNA purity, and RNA yield for each collection method. After complementary deoxyribonucleic acid synthesis, HOXA10 expression was measured by quantitative polymerase chain reaction in the endometrium overlying and remote from the leiomyoma, as similar expression throughout the cavity was a prerequisite for the use of unguided biopsy method. Results: The median weight of the samples was significantly larger when obtained with the low-pressure suction device than with the resectoscope loop (153 vs. 20 mg). The RNA yield was similar (suction curette, 1,625 ng/mg; resectoscope loop, 1,779 ng/mg). The A260-to-A280 ratio was satisfactory for 94.7 % of the samples, with no difference between the groups. The endometrial expression of HOXA10 was similar in areas overlying the leiomyoma compared with that in remote endometrial sites (2-ΔCt = 0.0224 vs. 0.0225). Conclusions: Low-pressure endometrial suction devices provide tissue samples with acceptable RNA purity and quantity for gene expression studies. The expression of HOXA10 did not differ between endometrial sampling sites even in the presence of leiomyomas.

Single-cell m6A mapping in vivo using picoMeRIP-seq.

Current N6-methyladenosine (m6A) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio, we developed picogram-scale m6A RNA immunoprecipitation and sequencing (picoMeRIP-seq) for studying m6A in vivo in single cells and scarce cell types using standard laboratory equipment. We benchmark m6A mapping on titrations of poly(A) RNA and embryonic stem cells and in single zebrafish zygotes, mouse oocytes and embryos.

Doppler ultrasonographic evaluation of tissue revascularization following connective tissue graft at implant sites.

AIM: To assess the Doppler ultrasonographic tissue perfusion at dental implant sites augmented with connective tissue graft (CTG) using coronally advanced flap (CAF) or tunnel technique (TUN). MATERIALS AND METHODS: Twenty-eight patients presenting with isolated healthy peri-implant soft-tissue dehiscence (PSTD) were included in this randomized clinical trial. PSTDs were treated with either CAF + CTG or TUN + CTG. Ultrasound scans were taken at baseline, 1 week, 1 month, 6 months and 12 months. Tissue perfusion at the mid-facial, mesial and distal aspects of the implant sites was assessed by colour Doppler velocity (CDV) and power Doppler imaging (PDI). Early vascularization of the graft and the flap at 1 week and at 1 month were evaluated via dynamic tissue perfusion measurements (DTPMs), including flow intensity (FI), mean perfusion relief intensity (pRI) and mean perfused area (pA). RESULTS: Regression analysis did not reveal significant differences in terms of mid-facial CDV and PDI changes between CAF and TUN over 12 months (p > .05), while significant differences between the two groups were observed at the interproximal areas (p < .001 for both CDV and PDI changes). Higher early DTPMs were observed at the TUN-treated sites in terms of mean FI of the graft (p = .027) and mean FI (p = .024) and pRI of the flap (p = .031) compared with CAF-treated sites at 1 week. Assessment of the FI direction showed that CTG perfusion at 1 week and at 1 month mainly occurred from the flap towards the implant/bone. Early tissue perfusion outcomes were found to be associated with the 12-month mean PSTD coverage and mucosal thickness gain. CONCLUSIONS: Doppler ultrasonography shows tissue perfusion changes occurring at implant sites augmented with CTG. The main differences in tissue perfusion between CAF and TUN were observed at the interproximal sites, with early perfusion associated with clinical and volumetric outcomes at 12 months.

Comprehension and recall of information about factors associated with peri-implantitis: A randomized controlled trial.

BACKGROUND: To evaluate the effect of different communication strategies on comprehension and recall of information about factors associated to peri-implantitis. METHODS: A prospective, randomized controlled trial was conducted in consecutive patients diagnosed with peri-implantitis. The sample was divided into three groups according to the communication strategy used: Test group 1-Written communication via leaflet with visual aids (L-VA); Test group 2-Written communication via leaflet with no visual aids (L-NVA); and control group-only verbal communication with no leaflet (NL). A questionnaire assessing comprehension at baseline (T0) and recall at 3 months (T1) and 6 months (T2) was administered following the fuzzy trace theory with a combination of 11 "gist" and seven "verbatim" items. The "health belief model" dimensions were further examined to test the influence of the communication strategy upon perceived severity, susceptibility, benefits, barriers, self-efficacy, and behavioral intentions. RESULTS: Ninety-nine patients that fulfilled the eligibility criteria were included. Gist and verbatim comprehension of the control, risk factors, and preventive measures for peri-implantitis overall was significantly greater in the test groups, in particular in L-VA at T0 (n = 99). Nevertheless, recall was not influenced by the communication strategy at T1 (n = 85) or T2 (n = 78). No significant differences were noted between groups or as a function of time for any of the "health belief model" constructs with the sole exception of perceived barriers (P = 0.045), which proved lower in the test groups. CONCLUSION: The comprehension of information about factors associated to peri-implantitis can be efficiently improved by using written communicative strategies, in particular when supplemented with visual aids. Nevertheless, this approach failed to show effectiveness in modulating recall or in changing behavioral intentions over follow-up (NCT04543604).

Early soft tissue changes following implant placement with or without soft tissue augmentation using a xenogeneic cross-link collagen scaffold: A volumetric comparative study.

OBJECTIVE: Soft tissue augmentation (STA) at implant sites has the potential of improving peri-implant health, esthetics, and marginal bone level stability. The present study aimed at evaluating the volumetric changes occurring following implant placement in sites that received STA compared to non-augmented sites. METHODS: A total of 26 subjects received a dental implant in a posterior edentulous site. Simultaneous STA with a xenogeneic cross-linked collagen scaffold was performed for the first 13 patients, while the remaining subjects served as the negative control. An intraoral optical scanner was used at baseline and at 12 weeks to generate digital models. RESULTS: The mean volume (Vol) gain of the test group was 38.43 mm3 , while a mean Vol of -16.82 mm3 was observed for the control group (p < 0.05). The mean thickness of the reconstructed volume (ΔD) was 0.61 and -0.24 mm, for the test and control group, respectively (p < 0.05). Higher linear dimensional changes were observed for the test group (p < 0.05), while no significant differences were observed in terms of keratinized mucosa width and pocket depth changes between the two groups. CONCLUSIONS: Simultaneous STA with xenogeneic collagen scaffold obtained statistically significant higher volumetric outcomes compared to the non-augmented group. CLINICAL SIGNIFICANCE: STA at the time of implant placement using a xenogeneic cross-linked collagen scaffold can prevent remodeling of the ridge during the first 12 weeks, as compared to non-grafted implant sites.

Spatial and temporal changes in follicle distribution in the human ovarian cortex.

RESEARCH QUESTION: How does follicle distribution evolve in the human ovarian cortex between the ages of 20 and 35 years? DESIGN: Fragments of ovarian cortex from women undergoing unilateral oophorectomy for fertility preservation were obtained for quantitative histological assessment, including recording the two-dimensional coordinates of the follicles. Data were analysed using spatial statistical methods. RESULTS: A total of 53 ovarian cortex tissue samples, containing 1-803 follicles each, were obtained from 14 women aged 20-35 years. Primordial and transitory follicles lay in a clustered manner in the human ovarian cortex, with an average cluster radius of around 270 µm (95% confidence interval 154-377 µm; n = 49). Follicle density declined with age (P = 0.006, n = 13), and the distance from the nearest neighbouring follicle increased (P = 0.004, n = 13). Cluster radius decreased with age (P = 0.02, n = 13), but the degree of clustering tended to increase (P = 0.11, n = 13). In the majority of the samples, follicles at different stages lay in different clusters (P < 0.05, n = 13). CONCLUSIONS: This study shows that primordial and transitory follicles lie in different clusters in the human ovarian cortex. Spatio-temporal computer simulation suggests that interfollicular signals may hinder follicle loss and may therefore drive clustered follicle distribution. In clinical practice, the woman's age should be taken into account when assessing follicle density, as follicle distribution is increasingly clustered with advancing age.

Assessing the true incidence of mosaicism in preimplantation embryos.

Modern technologies applied to the field of preimplantation genetic diagnosis for aneuploidy screening (PGD-A) have improved the ability to identify the presence of mosaicism. Consequently, new questions can now be addressed regarding the potential impact of embryo mosaicism on diagnosis accuracy and the feasibility of considering mosaic embryos for transfer. The frequency of chromosomal mosaicism in products of conception (POCs) of early miscarriages has been reported to be low. Mosaic embryos with an aneuploid inner cell mass are typically lost during the first trimester owing to spontaneous miscarriages. Most of the mosaics in established pregnancies would derive from placental mosaicism or placental aneuploidy, and mosaic embryos with aneuploid inner cell mass should be lost mainly due to first-trimester spontaneous miscarriages. The well described clinical outcomes of live births from mosaic embryos suggest a wide spectrum of phenotypes, from healthy to severely impaired. Therefore, there is a need to balance the risks of discarding a possibly viable embryo with that of transferring an embryo that may ultimately have a lower implantation potential.

Distribution patterns of segmental aneuploidies in human blastocysts identified by next-generation sequencing.

OBJECTIVE: To evaluate the ability of next-generation sequencing (NGS) to detect pure and mosaic segmental aneuploidies in trophectoderm biopsies and to identify distribution patterns in whole blastocysts. DESIGN: Validation study. SETTING: Reference laboratory. PATIENT(S): Seventy couples with known karyotypes who had undergone preimplantation genetic screening with diagnoses at the blastocyst stage using array comparative genomic hybridization (aCGH). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Concordance rates for segmental and whole-chromosome aneuploidies determined between aCGH and NGS, and estimates of mosaicism levels of segmental aneuploidies in fixed blastocysts. RESULT(S): We used NGS with amplified DNA from trophectoderm biopsies in which segmental aneuploidies had been previously detected by array comparative genomic hybridization (aCGH). Single-cell fluorescent in situ hybridization (FISH) was then used as an independent form of analysis. The concordance rate between NGS and aCGH was 124 (98.4%) of 126 for the detection of segmental aneuploidies, and 48 (96.0%) of 50 for whole-chromosome aneuploidies. The overall concordance rate was 99.8% (2,276 of 2,280 chromosomes assessed). After FISH analyses with 41.4 ± 24.3 cells per blastocyst, 26 (92.9%) of 28 segmentals detected by aCGH and NGS were confirmed. The FISH analysis did not detect the segmentals in two blastocysts, in which all cells analyzed were euploid. CONCLUSION(S): This is the first report analyzing distribution patterns of segmental aneuploidies in trophectoderm biopsy by NGS. We have demonstrated that NGS allows the detection of pure and mosaic segmental aneuploidies with the same efficiency as aCGH. The FISH analysis confirmed the existence of these events in the trophectoderm and the inner cell mass.

Prediction model for aneuploidy in early human embryo development revealed by single-cell analysis.

Aneuploidies are prevalent in the human embryo and impair proper development, leading to cell cycle arrest. Recent advances in imaging and molecular and genetic analyses are postulated as promising strategies to unveil the mechanisms involved in aneuploidy generation. Here we combine time-lapse, complete chromosomal assessment and single-cell RT-qPCR to simultaneously obtain information from all cells that compose a human embryo until the approximately eight-cell stage (n=85). Our data indicate that the chromosomal status of aneuploid embryos (n=26), including those that are mosaic (n=3), correlates with significant differences in the duration of the first mitotic phase when compared with euploid embryos (n=28). Moreover, gene expression profiling suggests that a subset of genes is differentially expressed in aneuploid embryos during the first 30 h of development. Thus, we propose that the chromosomal fate of an embryo is likely determined as early as the pronuclear stage and may be predicted by a 12-gene transcriptomic signature.