STUDY QUESTION
Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features?
SUMMARY ANSWER
The mitochondrial DNA (mtDNA) content of ...culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤35 or >35 years old.
WHAT IS KNOWN ALREADY
Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis.
STUDY DESIGN, SIZE, DURATION
Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age.
MAIN RESULTS AND THE ROLE OF CHANCE
Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age.
LIMITATIONS, REASONS FOR CAUTION
It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate.
WIDER IMPLICATIONS OF THE FINDINGS
The approach we are proposing may provide a novel, non-invasive, objective tool for embryo quality grading. The correlation between a high mtDNA concentration and the fragmentation rate of embryos is suggestive that fragments are mainly anuclear cytoplasmatic debris arising during cleavage. Therefore, blastomere shaping as an early event during in vitro development may play a homeostatic role and be related to embryo competence.
STUDY FUNDING/COMPETING INTEREST
This project was funded by Merck Serono (Grant for Fertility Innovation 2011). The sponsor had no role in study design, data collection, data analysis, data interpretation and writing of the paper. Authors declare no conflicts of interest.
TRIAL REGISTRATION NUMBER
ClinicalTrials.gov Identifier: NCT01397136.
BACKGROUND The available data on effectiveness of aromatase inhibitors in treating pain symptoms related to endometriosis is limited. We compared the efficacy and tolerability of the aromatase ...inhibitor letrozole combined with norethisterone acetate versus norethisterone acetate alone in treating pain symptoms. METHODS This prospective, open-label, non-randomized trial included 82 women with pain symptoms caused by rectovaginal endometriosis. Patients received either a combination of letrozole and norethisterone acetate (group L) or norethisterone acetate alone (group N) for 6 months. Changes in pain symptoms during treatment and in the 12 months of follow-up were evaluated. Side effects of each treatment protocol were recorded. RESULTS Intensity of chronic pelvic pain and deep dyspareunia significantly decreased during treatment (P < 0.001 versus baseline by 3 months) in both study groups. At both 3- and 6-month assessment, the intensity of chronic pelvic pain (P < 0.001, P = 0.002, respectively) and deep dyspareunia (P < 0.001, P = 0.005, respectively) was significantly lower in group L than group N. At completion of treatment, 63.4% of women in group N were satisfied with treatment compared with 56.1% in group L (P = 0.49). Pain symptoms recurred after the completion of treatment; at 6-month follow-up no difference was observed in the intensity of pain symptoms between the groups. Adverse effects were more frequent in group L than in group N (P = 0.02). CONCLUSIONS The combination drug regimen was more effective in reducing pain and deep dyspareunia than norethisterone acetate; however, letrozole caused a higher incidence of adverse effects, cost more and did not improve patients' satisfaction or influence recurrence of pain.
In assisted reproduction technology, embryo competence is routinely evaluated on morphological criteria. Over the last decade, efforts in improving non-invasive embryo assessment have looked into the ...secretome of embryos. Human embryos release genomic DNA (gDNA) and mitochondrial DNA (mtDNA) into the culture medium, and the mtDNA/gDNA ratio is significantly correlated with embryo fragmentation. Here, we investigate whether mtDNA/gDNA ratio in embryo spent medium is correlated with blastulation potential and implantation. The mtDNA/gDNA ratio was assessed in 699 Day 3 culture media by quantitative polymerase chain reaction (qPCR) to investigate its correlation with embryo morphology, blastocyst development and implantation. A logistic regression model evaluated whether mtDNA/gDNA ratio in the secretome may improve the prediction of blastulation. We found that embryos that successfully developed into blastocysts exhibited a significantly higher mtDNA/gDNA ratio in the culture medium compared with those that arrest (P = 0.0251), and mtDNA/gDNA, combined with morphological grading, has the potential to predict blastulation better than morphology alone (P = 0.02). Moreover, mtDNA/gDNA ratio was higher in the media from good-quality embryos that reached the full blastocyst stage on Day 5 compared with those that developed more slowly (P < 0.0001). With respect to blastocyst morphology, higher trophectoderm quality was associated with a higher mtDNA/gDNA ratio in the culture medium. Finally, a high mtDNA/gDNA ratio in spent medium was associated with successful implantation outcome (P = 0.0452) of good-quality embryos. In summary, the mtDNA/gDNA ratio in the Day 3 embryo secretome, in combination with morphological grading, may be a novel, non-invasive, early biomarker to improve identification of viable embryos with high developmental potential.
Innate lymphoid cells (ILCs) are developmentally related cells that play an important role in innate defenses and tissue remodeling. So far, only natural killer (NK) cells have been identified and ...functionally characterized in human decidua where they contribute to induction of immune suppression, neo-angiogenesis, and tissue building/remodeling. The presence of other ILC subsets in human decidua has not been yet characterized. Here we identify in human decidua, during early pregnancy, two subsets of decidual group 3 ILC (ILC3), including lymphoid tissue inducer (LTi)-like cells and natural cytotoxicity receptors (NCRs)(+)ILC3 and interferon-(IFN)γ-producing ILC1, different from NK cells. Decidual LTi-like cells produced interleukin -17 (IL-17) and tumor necrosis factor (TNF), while NCR(+)ILC3 released IL-22 and IL-8. Importantly, NCR(+)ILC3 and LTi-like cells established functional interactions with stromal cells. Decidual LTi-like cells differentiated into NCR(+)ILC3, whereas they marginally contributed to NK cell generation. Our data suggest that decidual ILC3 may play a role in innate defenses and in vessel and tissue building, thus contributing to maintenance of pregnancy.
STUDY QUESTION
Do endometriotic ovarian cysts influence the rate of spontaneous ovulation?
SUMMARY ANSWER
Endometriotic cysts, no matter what their volume, do not influence the rate of spontaneous ...ovulation in the affected ovary.
WHAT IS KNOWN ALREADY
Endometriotic ovarian cysts may negatively affect spontaneous ovulation in the affected ovary.
STUDY DESIGN, SIZE, DURATION
This was a prospective observational study performed between September 2009 and June 2013.
PARTICIPANTS/MATERIALS, SETTING, METHODS
This study included women of reproductive age with regular menstrual cycles and unilateral ovarian endometriomas (diameter ≥20 mm) desiring to conceive. Exclusion criteria were: hormonal therapies in the 3 months prior to study entry and previous adnexal surgery. Patients underwent serial transvaginal ultrasound to assess the side of ovulation (for up to six cycles).
MAIN RESULTS AND THE ROLE OF CHANCE
Ovulation was monitored in 1199 cycles in 244 women (age, mean ± SD, 34.3 ± 4.9 years). 55.3% of the patients had left endometriomas and 44.7% had right endometriomas (P = 0.024). The mean (±SD) diameter of the endometriomas was 5.3 cm (±1.7 cm). Ultrasonographically documented ovulation occurred in 596 cycles in the healthy ovary (49.7%; 95% CI, 46.8–52.6%) and in 603 cycles in the affected ovary (50.3%; 95% CI, 47.1–53.2%; P = 0.919). This observation was confirmed in patients with diameter of the cyst ≥4 cm (n = 166) and in those with diameter of the cyst ≥6 cm (n = 45). One hundred and five patients spontaneously conceived (43.0%; 95% CI, 36.7–49.5%).
LIMITATIONS, REASON FOR CAUTION
The high pregnancy rate reported in this study was observed in a selected population of women with endometriomas and cannot be extrapolated to all patients with endometriosis.
WIDER IMPLICATIONS OF THE FINDINGS
Since ovarian endometriomas do not impair spontaneous ovulation, the impact on fertility of surgical excision of ovarian endometriomas should be further investigated.
STUDY FUNDING/COMPETING INTEREST(S)
None.
TRIAL REGISTRATION NUMBER
None.
BACKGROUND This pilot study evaluates the efficacy of norethisterone acetate in treating pain and gastrointestinal symptoms of women with colorectal endometriosis. METHODS This prospective study ...included 40 women with colorectal endometriosis, who had pain and gastrointestinal symptoms. Patients received norethisterone acetate (2.5 mg/day) for 12 months; in case of breakthrough bleeding, the dose of norethisterone acetate was increased by 2.5 mg/day. The degree of patient satisfaction with treatment (primary end-point) and the changes in symptoms (secondary end-point) were evaluated. Side effects of treatment were recorded. RESULTS Norethisterone acetate determined a significant improvement in the intensity of chronic pelvic pain, deep dyspareunia, dyschezia. Treatment determined the disappearance of symptoms related to the menstrual cycle (dysmenorrhea, constipation during the menstrual cycle, diarrhoea during the menstrual cycle and cyclical rectal bleeding). The severity of diarrhoea, intestinal cramping and passage of mucus significantly improved during treatment. On the contrary, the administration of norethisterone acetate did not determine a significant effect on constipation, abdominal bloating and feeling of incomplete evacuation after bowel movements. At the completion of treatment, 57% of the patients with diarrhoea or diarrhoea during the menstrual cycle continued the treatment with norethisterone acetate compared with 17% of the patients with constipation or constipation during the menstrual cycle. CONCLUSIONS In some patients with bowel endometriosis, the administration of norethisterone acetate may determine a relief of pain and gastrointestinal symptoms. This therapy has greater benefits in patients with gastrointestinal symptoms related to the menstrual cycle, diarrhoea and intestinal cramping.
Innate lymphoid cells (ILCs) have a central role in innate defenses against pathogens, lymphoid organogenesis, and tissue remodeling. They have been detected in human decidua, however, their role in ...this tissue remains unclear. Successful pregnancy requires an early inflammatory phase favoring implantation and tissue remodeling as well as a subsequent regulatory phase to prevent fetal rejection and supporting neoangiogenesis. Here, we show that, during the first trimester of pregnancy, neutrophils infiltrate decidua basalis and are more abundant in normal pregnancy than in spontaneous miscarriages. Decidual neutrophils localize in proximity of NCR
ILC3, which may influence neutrophil migration and survival given their production of CXCL8 and granulocyte macrophage colony-stimulating factor (GM-CSF). Moreover, NCR
ILC3-derived GM-CSF was found to induce the expression of heparin-binding EGF-like growth factor and IL1ra in neutrophils, two proteins/cytokines involved in tissue remodeling and maintenance of pregnancy. Our data suggest that the simultaneous presence of NCR
ILC3 and neutrophils in decidual tissues and their possible cross talk, may have a role in the early phases of pregnancy.
Peripheral T-cell lymphoma unspecified (PTCLU) and mycosis fungoides (MF) often show resistance to conventional chemotherapy. Gemcitabine should be considered a suitable option. We report the ...long-term update of 39 pretreated T-cell lymphoma patients treated with gemcitabine.
From May 1997 to September 2007, 39 pretreated MF and PTCLU patients received gemcitabine. Inclusion criteria were as follows: histologic diagnosis of MF or PTCLU; relapsed/refractory disease; age ≥18 years; and World Health Organization performance status of two or less. Nineteen patients had MF and 20 PTCLU. All patients with MF had a T3–T4, N0, and M0 disease and patients with PTCLU had stage III–IV disease. Gemcitabine was given on days 1, 8, and 15 on a 28-day schedule (1200 mg/m2/day) for a total of three to six cycles.
Overall response rate was 51% (20 of 39 patients); complete response (CR) and partial response (PR) rates were 23% (9 of 39 patients) and 28% (11 of 39 patients), respectively. Patients with MF had a CR rate of 16% and a PR rate of 32% compared with a CR rate of 30% and a PR rate of 25% of PTCLU patients. Among the CR patients, 7 of 9 are in continuous complete response with a variable disease-free interval (15–120 months).
In our experience, gemcitabine proved to be effective in pretreated MF and PTCLU patients, even in the long term.
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