Paola Anserini

Cancer and fertility preservation: International recommendations from an expert meeting

In the last years, thanks to the improvement in the prognosis of cancer patients, a growing attention has been given to the fertility issues. International guidelines on fertility preservation in cancer patients recommend that physicians discuss, as early as possible, with all patients of reproductive age their risk of infertility from the disease and/or treatment and their interest in having children after cancer, and help with informed fertility preservation decisions. As recommended by the American Society of Clinical Oncology and the European Society for Medical Oncology, sperm cryopreservation and embryo/oocyte cryopreservation are standard strategies for fertility preservations in male and female patients, respectively; other strategies (e.g. pharmacological protection of the gonads and gonadal tissue cryopreservation) are considered experimental techniques. However, since then, new data have become available, and several issues in this field are still controversial and should be addressed by both patients and their treating physicians.In April 2015, physicians with expertise in the field of fertility preservation in cancer patients from several European countries were invited in Genova (Italy) to participate in a workshop on the topic of “cancer and fertility preservation”. A total of ten controversial issues were discussed at the conference. Experts were asked to present an up-to-date review of the literature published on these topics and the presentation of own unpublished data was encouraged. On the basis of the data presented, as well as the expertise of the invited speakers, a total of ten recommendations were discussed and prepared with the aim to help physicians in counseling their young patients interested in fertility preservation.Although there is a great interest in this field, due to the lack of large prospective cohort studies and randomized trials on these topics, the level of evidence is not higher than 3 for most of the recommendations highlighting the need of further research efforts in many areas of this field. The participation to the ongoing registries and prospective studies is crucial to acquire more robust information in order to provide evidence-based recommendations.

Fertility after bowel resection for endometriosis.

OBJECTIVE To determine the pregnancy rate after bowel resection for rectosigmoid endometriosis. DESIGN Prospective cohort study. SETTING University hospital. PATIENT(S) Forty-six symptomatic women with bowel endometriosis requiring colorectal resection. INTERVENTION(S) Bowel resection by either laparoscopy or laparotomy. MAIN OUTCOME MEASURE(S) Pregnancy rate after surgery. RESULT(S) The pregnancy rate was higher in women who underwent bowel resection by laparoscopy (57.6%) than in those who underwent laparotomy (23.1%). No significant difference was observed in pregnancy rate and mode of conception between women with different fertility status before bowel resection. Women who conceived were significantly younger than those who did not conceive; only 26.7% of women aged > or =35 years conceived after bowel resection. Uterine adenomyosis was more frequently present in women who did not conceive than in those who conceived. Infertile women who conceived had a shorter length of infertility before surgery than those who did not conceive. CONCLUSION(S) Laparoscopic colorectal resection is less likely to impact negatively on fertility than the laparotomy approach. Previous laparotomies, age > or =35 years, uterine adenomyosis, and longer duration of infertility before surgery are associated with decreased pregnancy rate.

Multicenter observational study on slow-cooling oocyte cryopreservation: clinical outcome

OBJECTIVE To evaluate the efficacy of oocyte cryopreservation by a single slow-cooling protocol involving sucrose (0.2 mol/L) in the freezing solution. DESIGN Observational comparison of the clinical outcome in fresh and frozen thawed cycles. SETTING Public and private IVF centers. PATIENT(S) Infertile couples undergoing IVF treatment. INTERVENTION(S) Use of a maximum three oocytes in fresh cycles, as established by local law, and cryopreservation and later use of surplus oocytes. Likewise fresh cycles, maximum three thawed oocytes were used per cycle. All thawed oocytes were microinjected. MAIN OUTCOME MEASURE(S) Embryologic and clinical parameters of fresh and thawed cycles. RESULT(S) Two thousand forty-six patients underwent 2,209 oocyte retrievals involving oocyte cryopreservation. Overall, the survival rate of thawed oocytes was 55.8%. In 940 thaw cycles, the mean numbers of inseminated oocytes and fertilization rates were significantly decreased vs. fresh cycles outcomes (2.6 ± 0.7 vs. 2.9 ± 0.2 and 72.5% vs. 78.3%, respectively), as were the rates of implantation (10.1% vs. 15.4%), pregnancy rates per transfer (17.0% vs. 27.9%), and pregnancy rates per cycle (13.7% vs. 26.2%). Differences in clinical outcome were found among centers. A pregnancy rate per thawing cycle above 14% was achieved by most clinics. Fifty-seven retrievals involving oocyte cryopreservation achieved a pregnancy after fresh embryo replacement. Implantation and pregnancy rates per embryo transfer and per thawing cycles were 17.5%, 28.6%, and 24.6%, respectively. CONCLUSION(S) Under the conditions tested, the clinical outcome of oocyte slow-cooling cryopreservation is reduced compared with fresh cycles. Nevertheless, in cases of inapplicability of embryo cryopreservation, oocyte cryopreservation should be offered to patients with surplus oocytes.

Transcervical selective salpingography: a diagnostic and therapeutic approach to cases of proximal tubal injection failure *

OBJECTIVE Evaluation of selective salpingography for diagnosis and treatment of tubal injection failure during hysterosalpingography (HSG). DESIGN Prospective study. SETTING Obstetrics and Gynecology Department, University of Genoa (Italy)--tertiary care. PATIENTS One hundred eighty infertile women with unilateral or bilateral proximal tubal injection failure during HSG were submitted to the procedure. INTERVENTION Under fluoroscopy, a 4.5-F nylon catheter (3-F tip) was inserted into the ostium with or without the aid of a J-shaped, coaxial, angiographic guide wire, and 2 to 3 mL of contrast medium were injected. The procedure lasts 20 to 30 sec/tube. MAIN OUTCOME MEASURES Of 155 tubal ostia, 145 (94.2%) were catheterized. RESULTS Of the 146 catheterized tubes, 110 (75%) were rendered patent. Of the others, 21 (14.3%) presented hydrosalpinx or distal obstructions, and isthmic obstruction was present in 5 (3.4%). Patency of at least one tube was achieved in 82 (81.2%) of the 101 catheterized women; 8 conceived spontaneously and 11 after gamete intrafallopian transfer to the recanalized tube. CONCLUSIONS During HSG, selective salpinography can be performed when proximal injection failure is observed to determine its cause or to restore patency.

Future perspectives in the medical treatment of endometriosis.

In the last few years, our understanding of the pathogenesis of endometriosis at the cellular and molecular levels has improved significantly. This may give us the opportunity to use new, specific agents for the treatment of this disorder. Despite the effectiveness of the available treatments, novel therapeutic strategies may improve our ability to eliminate endometriotic lesions when present and to prevent the recurrence of endometriosis after surgical treatment. This review focuses on the new, experimental approaches to the medical treatment of endometriosis and its symptoms. The blockage of aromatase activity in endometriotic lesions with an aromatase inhibitor may represent a new step in the medical treatment of endometriosis. Preliminary clinical studies have demonstrated the efficacy of third-generation nonsteroidal aromatase inhibitors (ie, anastrozole and letrozole) in reducing the intensity of pain symptoms associated with the presence of endometriosis. The new selective progesterone receptor modulators may represent a valid hormonal treatment option. Therapeutic manipulation of the immune system through TNFα inhibitors may be beneficial in women with endometriosis. New pharmaceutical agents affecting inflammation, angiogenesis, and matrix metalloproteinase activity may prevent or inhibit the development of endometriosis. Further clinical trials may determine if these new therapies are superior to current medical treatment strategies for endometriosis. Target Audience: Obstetricians & Gynecologists, Family Physicians Learning Objectives: After completion of this article, the reader should be able to describe the new experimental medical treatments of endometriosis, state that the clinical use of nonsteroidal aromatase inhibitors for endometriosis appears to be efficacious but is based on preliminary clinical data, and recall that the drugs used for endometriosis in the future may include manipulation of the immune system.

Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation

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.

Vitamin D binding protein in endometriosis

Objective: Two-dimensional gel electrophoresis is a powerful method for identifying post-translationally modified molecules in biological fluids. We examined the presence and expression of vitamin D binding protein (DBP) in the peritoneal fluid (PF) and plasma (PL) of women with endometriosis. Methods: PL and PF samples were obtained from 36 women with untreated mild endometriosis (revised classification of the American Fertility Society [rAFS] stage I-II), 52 women with untreated severe endometriosis (rAFS stage III-IV), 17 women with endometriosis treated with the oral contraceptive (OC), and 40 controls (infertility, n = 23; tubal sterilization, n = 12; pelvic pain, n = 5). PF and PL samples were analyzed by quantitative, high-resolution 2-dimensional gel electrophoresis. Results: The expression of one DBP isoform (DBPE) in the PF of patients with untreated endometriosis was signficantly lower than in the control group (P <.05). The levels of PF DBPE in patients with endometriosis using OC were signficantly higher than in women with untreated endometriosis (P < .05). No significant difference was observed in PL DBPE expression between women with and without endometriosis, while it was significantly increased in patients with endometriosis using OC (P<. 05). DBP expression was not correlated with the stage of endometriosis (rAFS classification) or the phase of the menstrual cycle. Conclusion: The decreased level of DBPE in the PF but not in PL of women with untreated endometriosis suggests that this molecule may be relevant in the pathogenesis of this disease.

Treatment of endometriosis with goserelin depot, a long-acting gonadotropin-releasing hormone agonist analog: endocrine and clinical results

Thirty-two patients with laparoscopic diagnosis of endometriosis were treated for 6 months with the gonadotropin-releasing hormone agonist goserelin depot. Hormonal and clinical evaluations were conducted during treatment and for a 6-month follow-up period. Serum 17 beta-estradiol levels were sharply suppressed. Luteinizing hormone was also decreased, whereas follicle-stimulating hormone, after an initial fall, gradually rose to pretreatment levels. Ovarian androgenic production was less inhibited, total testosterone being the only significantly suppressed hormone. There was a marked improvement of signs and symptoms of endometriosis and a 47.3% reduction of laparoscopic score. The results of this study suggest that goserelin depot provides a very good suppression of ovarian estrogen production and is highly effective in decreasing the symptoms of endometriosis with an acceptable compliance by the patient.

Four year experience in laparoscopic dissection of intact ovarian dermoid cysts.

BACKGROUND Intraperitoneal spillage of dermoid cyst content, if not followed immediately by abundant peritoneal lavage, can cause a chemical peritonitis with subsequent adhesion formation. STUDY DESIGN We performed an open clinical study in a university hospital. Forty-four consecutive ovarian dermoid cysts were removed intact from 40 premenopausal women operated on between October 1993 and December 1997. The laparoscopic technique included: 1) creation of a cleavage plane between the cyst and the ovary; 2) dissection of the cyst by a combination of water, scissors, and gravity without direct traction on the cyst; and 3) extraction of the cyst after its placement inside a laparoscopic bag. RESULTS The mean cyst diameter was 6.5 cm (range 3 to 12 cm). Mean operating time was 125 minutes (range 50 to 180 minutes). All patients were discharged within 48 hours. The cysts were dissected completely intact and were extracted without spillage in the abdominal cavity in all cases. Operative followup was available in 15 of the 40 patients; mild adhesions were found on the treated ovary in 3 (20%). CONCLUSIONS It is always possible to prevent rupture and spillage of dermoid cysts during laparoscopic operations, but this approach is time consuming and needs expert surgical technique.

Mitochondrial DNA in day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome

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.