Oocyte competence is comparable between progestin primed ovarian stimulation with Norethisterone acetate (NETA-PPOS) and GnRH-antagonist protocols: A matched case-control study in PGT-A cycles.

OBJECTIVE: To outline oocyte competence after progestin primed ovarian stimulation with Norethisterone acetate (NETA-PPOS) compared to conventional GnRH-antagonist protocol. STUDY DESIGN: Retrospective matched case-control study involving advanced-maternal-age women undergoing ICSI with PGT-A. 89 NETA-PPOS were matched with 178 control patients based on maternal age and ovarian reserve biomarkers. Both groups underwent recombinant-FSH OS with GnRH-agonist ovulation trigger and collected ≥1 MII. In the study group, NETA (10 mg/day) was administered orally starting from day2 of the menstrual cycle. Euploid blastocyst rate per cohort of metaphase-II oocytes (EBR per MII) was the primary outcome. All other embryological and clinical outcomes were reported. Gestational age, birthweight and length were also assessed. RESULTS: The EBR per MII was comparable among PPOS and control (13.9 % ± 19.3 % versus 13.3 % ± 17.9 %; the sample size allowed to exclude up to a 10 % difference). Blastocysts morphology and developmental rate were similar. No difference was reported for all clinical outcomes among the 61 and 107 vitrified-warmed euploid single blastocyst transfers respectively conducted. The cumulative live birth delivery rate per concluded cycles was also comparable (24.7 % versus 21.9 %). Neonatal outcomes were analogous. CONCLUSIONS: Oocyte competence after NETA-PPOS and standard OS is comparable. This evidence is reassuring and, because of its lower cost and possibly higher patients' compliance, supports PPOS administration whenever the patients are indicated to freeze-all (e.g., fertility preservation, PGT-A, oocyte donation). More data are required about follicle recruitment, oocyte yield, gestational and perinatal outcomes. Randomized-controlled-trials are advisable to confirm our evidence.

The euploid blastocysts obtained after luteal phase stimulation show the same clinical, obstetric and perinatal outcomes as follicular phase stimulation-derived ones: a multicenter study.

STUDY QUESTION: Are the reproductive outcomes (clinical, obstetric and perinatal) different between follicular phase stimulation (FPS)- and luteal phase stimulation (LPS)-derived euploid blastocysts? SUMMARY ANSWER: No difference was observed between FPS- and LPS-derived euploid blastocysts after vitrified-warmed single embryo transfer (SET). WHAT IS KNOWN ALREADY: Technical improvements in IVF allow the implementation non-conventional controlled ovarian stimulation (COS) protocols for oncologic and poor prognosis patients. One of these protocols begins LPS 5 days after FPS is ended (DuoStim). Although, several studies have reported similar embryological outcomes (e.g. fertilization, blastulation, euploidy) between FPS- and LPS-derived cohort of oocytes, information on the reproductive (clinical, obstetric and perinatal) outcomes of LPS-derived blastocysts is limited to small and retrospective studies. STUDY DESIGN, SIZE, DURATION: Multicenter study conducted between October 2015 and March 2019 including all vitrified-warmed euploid single blastocyst transfers after DuoStim. Only first transfers of good quality blastocysts (≥BB according to Gardner and Schoolcraft's classification) were included. If euploid blastocysts obtained after both FPS and LPS were available the embryo to transfer was chosen blindly. The primary outcome was the live birth rate (LBR) per vitrified-warmed single euploid blastocyst transfer in the two groups. To achieve 80% power (α = 0.05) to rule-out a 15% difference in the LBR, a total of 366 first transfers were required. Every other clinical, as well as obstetric and perinatal outcomes, were recorded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Throughout the study period, 827 patients concluded a DuoStim cycle and among them, 339 did not identify any transferable blastocyst, 145 had an euploid blastocyst after FPS, 186 after LPS and 157 after both FPS and LPS. Fifty transfers of poor quality euploid blastocysts were excluded and 49 patients did not undergo an embryo transfer during the study period. Thus, 389 patients had a vitrified-warmed SET of a good quality euploid blastocyst (182 after FPS and 207 after LPS). For 126 cases (32%) where both FPS- and LPS-derived good quality blastocysts were available, the embryo transferred was chosen blindly with a 'True Random Number Generator' function where '0' stood for FPS-derived euploid blastocysts and '1' for LPS-derived ones (n = 70 and 56, respectively) on the website random.org. All embryos were obtained with the same ovarian stimulation protocol in FPS and LPS (GnRH antagonist protocol with fixed dose of rec-FSH plus rec-LH and GnRH-agonist trigger), culture conditions (continuous culture in a humidified atmosphere with 37°C, 6% CO2 and 5% O2) and laboratory protocols (ICSI, trophectoderm biopsy in Day 5-7 without assisted hatching in Day 3, vitrification and comprehensive chromosome testing). The women whose embryos were included had similar age (FPS: 38.5 ± 3.1 and LPS: 38.5 ± 3.2 years), prevalence of male factor, antral follicle count, basal hormonal characteristics, main cause of infertility and previous reproductive history (i.e. previous live births, miscarriages and implantation failures) whether the embryo came from FPS or LPS. All transfers were conducted after warming in an artificial cycle. The blastocysts transferred after FPS and LPS were similar in terms of day of full-development and morphological quality. MAIN RESULTS AND THE ROLE OF CHANCE: The positive pregnancy test rates for FPS- and LPS-derived euploid blastocysts were 57% and 62%, biochemical pregnancy loss rates were 10% and 8%, miscarriage rates were 15% and 14% and LBRs were 44% (n = 80/182, 95% CI 37-51%) and 49% (n = 102/207, 95% CI 42-56%; P = 0.3), respectively. The overall odds ratio for live birth (LPS vs FPS (reference)) adjusted for day of blastocyst development and quality, was 1.3, 95% CI 0.8-2.0, P = 0.2. Among patients with euploid blastocysts obtained following both FPS and LPS, the LBRs were also similar (53% (n = 37/70, 95% CI 41-65%) and 48% (n = 27/56, 95% CI 35-62%) respectively; P = 0.7). Gestational issues were experienced by 7.5% of pregnant women after FPS- and 10% of women following LPS-derived euploid single blastocyst transfer. Perinatal issues were reported in 5% and 0% of the FPS- and LPS-derived newborns, respectively. The gestational weeks and birthweight were similar in the two groups. A 5% pre-term delivery rate was reported in both groups. A low birthweight was registered in 2.5% and 5% of the newborns, while 4% and 7% showed high birthweight, in FPS- and LPS-derived euploid blastocyst, respectively. Encompassing the 81 FPS-derived newborns, a total of 9% were small and 11% large for gestational age. Among the 102 LPS-derived newborns, 8% were small and 6% large for gestational age. No significant difference was reported for all these comparisons. LIMITATIONS, REASONS FOR CAUTION: The LPS-derived blastocysts were all obtained after FPS in a DuoStim protocol. Therefore, studies are required with LPS-only, late-FPS and random start approaches. The study is powered to assess differences in the LBR per embryo transfer, therefore obstetric and perinatal outcomes should be considered observational. Although prospective, the study was not registered. WIDER IMPLICATIONS OF THE FINDINGS: This study represents a further backing of the safety of non-conventional COS protocols. Therefore, LPS after FPS (DuoStim protocol) is confirmed a feasible and efficient approach also from clinical, obstetric and perinatal perspectives, targeted at patients who need to reach the transfer of an euploid blastocyst in the shortest timeframe possible due to reasons such as cancer, advanced maternal age and/or reduced ovarian reserve and poor ovarian response. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Double stimulation in the same ovarian cycle (DuoStim) is an intriguing strategy to improve oocyte yield and the number of competent embryos in a short timeframe.

Proper ovarian stimulation regimens are crucial for any patient undergoing in-vitro fertilization (IVF). However, maximizing the oocyte yield in advanced maternal age patients with poor or suboptimal response is still a challenge. In fact, no standard treatment has been outlined yet to manage these women. Across the last years, an improved efficiency of the IVF units via blastocyst culture, vitrification and reliable embryo selection approaches paved the way to the investigation of novel unconventional stimulation protocols, like double stimulation in a single ovarian cycle (DuoStim). DuoStim, by conjugating follicular phase stimulation (FPS) and luteal phase stimulation (LPS) in the same ovarian cycle, allows to maximize the number of oocytes obtained in a short timeframe, a precious outcome when we aim at shortening time to pregnancy. In this regard, LPS seems to contribute to conventional stimulation with more oocytes with a comparable competence as FPS, retrieved per ovarian cycle. Although any stimulation protocol which exploits anovulatory waves of follicular growth needs a thorough investigation, no evidence has been produced to question the safety of DuoStim, which to date represents the most intriguing strategy to treat poor prognosis in IVF.

Double Stimulation in the Same Ovarian Cycle (DuoStim) to Maximize the Number of Oocytes Retrieved From Poor Prognosis Patients: A Multicenter Experience and SWOT Analysis.

A panel of experts known as the POSEIDON group has recently redefined the spectrum of poor responder patients and introduced the concept of suboptimal response. Since an ideal management for these patients is still missing, they highlighted the importance of tailoring the ovarian stimulation based on the chance of each woman to obtain an euploid blastocyst. Interestingly, a novel pattern of follicle recruitment has been defined: multiple waves may arise during a single ovarian cycle. This evidence opened important clinical implications for the treatment of poor responders. For instance, double stimulation in the follicular (FPS) and luteal phase (LPS) of the same ovarian cycle (DuoStim) is an intriguing option to perform two oocyte retrievals in the shortest possible time. Here, we reported our 2-year experience of DuoStim application in four private IVF centers. To date, 310 poor prognosis patients completed a DuoStim protocol and underwent IVF with blastocyst-stage preimplantation-genetic-testing. LPS resulted into a higher mean number of oocytes collected than FPS; however, their competence (i.e., fertilization, blastocyst, euploidy rates, and clinical outcomes after euploid single-embryo-transfer) was comparable. Importantly, the rate of patients obtaining at least one euploid blastocyst increased from 42.3% (n = 131/310) after FPS to 65.5% (n = 203/310) with the contribution of LPS. A summary of the putative advantages and disadvantages of DuoStim was reported here through a Strengths-Weaknesses-Opportunities-Threats analysis. The strengths of this approach make it very promising. However, more studies are needed in the future to limit its weaknesses, shed light on its putative threats, and realize its opportunities.

Luteal phase anovulatory follicles result in the production of competent oocytes: intra-patient paired case-control study comparing follicular versus luteal phase stimulations in the same ovarian cycle.

STUDY QUESTION: Are the mean numbers of blastocysts obtained from sibling cohorts of oocytes recruited after follicular phase and luteal phase stimulations (FPS and LPS) in the same ovarian cycle similar? SUMMARY ANSWER: The cohorts of oocytes obtained after LPS are larger than their paired-FPS-derived cohorts and show a comparable competence, thus resulting in a larger mean number of blastocysts. WHAT IS KNOWN ALREADY: Three theories of follicle recruitment have been postulated to date: (i) the 'continuous recruitment' theory, (ii) the 'single recruitment episode' theory and (iii) the 'wave' theory. Yet, a clear characterization of this crucial biological process for human reproduction is missing. Recent advances implemented in in vitro fertilization (IVF), such as blastocyst culture, aneuploidy testing and vitrification, have encouraged clinicians to maximize the exploitation of the ovarian reserve through tailored stimulation protocols, which is crucial especially for poor prognosis patients aiming to conceive after IVF. LPS has been already successfully adopted to treat poor prognosis or oncological patients through Duostim, LPS-only or random-start ovarian stimulation approaches. Nevertheless, little, and mainly retrospective, evidence has been produced to support the safety of LPS in general. Feasibility of the LPS approach would severely question the classic 'single recruitment episode' theory of follicular development. STUDY DESIGN, SIZE, DURATION: This case-control study was conducted with paired follicular phase- and luteal phase-derived cohorts of oocytes collected after stimulations in the same ovarian cycle (DuoStim) at two private IVF clinics between October 2015 and December 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included 188 poor prognosis patients undergoing DuoStim with preimplantation genetic testing for aneuploidies (PGT-A). FPS and LPS were performed with the same daily dose of recombinant-gonadotrophins in an antagonist protocol. Blastocyst culture, trophectoderm biopsy, vitrification and frozen-warmed euploid single blastocyst transfers were performed. The primary outcome was the mean number of blastocysts obtained per oocyte retrieval from paired-FPS- and LPS-derived cohorts (required sample size = 165 patients; power = 90%). Mean blastulation and euploidy rates were monitored, along with the number of oocytes, euploid blastocysts and clinical outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: Significantly fewer blastocysts were obtained after FPS than LPS (1.2 ± 1.1 vs. 1.6 ± 1.6, P < 0.01), due to fewer oocytes collected (3.6 ± 2.1 vs. 4.3 ± 2.8, P < 0.01) and a similar mean blastocyst rates per retrieval (33.1% ± 30.3% vs. 37.4% ± 30.8%, P = NS). The number of oocytes collected were correlated (R = 0.5, P < 0.01), while the blastocyst rates were uncorrelated among paired-FPS- and LPS-derived cohorts. Overall, a significantly lower chance of producing blastocyst(s) was reported after FPS than after LPS: 67.6% (n = 127/188, 95%CI: 60.3-74.1) vs. 77.1% (n = 145/188, 95%CI: 70.3-82.8; P = 0.05). The mean euploidy rates per retrieval were similar between FPS- and LPS-derived cohorts of oocytes (13.6% ± 22.8% vs. 16.3% ± 23.4%, P = NS). Therefore, on average fewer euploid blastocysts (0.5 ± 0.8 vs. 0.7 ± 1.0, P = 0.02) resulted from FPS. Similar ongoing-pregnancy/delivery rates were reported, to date, after FPS- and LPS-derived euploid single blastocyst transfers: 42.4% (n = 28/66, 95%CI: 30.5-55.2) vs. 53.8% (n = 35/65, 95%CI: 41.1-66.1; P = NS). LIMITATIONS, REASONS FOR CAUTION: More studies need to be conducted in the future to confirm the safety of LPS, especially in terms of ovarian and follicular environment, as well as the clinical, peri-natal and post-natal outcomes. Here, we showed preliminary data suggesting a similar ongoing implantation/delivery rate (>22 weeks) between FPS- and LPS-derived euploid blastocysts, that need to be extended in the future, to populations other than poor prognosis patients and using approaches other than DuoStim together with a constant monitoring of the related peri-natal and post-natal outcomes. WIDER IMPLICATIONS OF THE FINDINGS: These data, from a paired study design, highlight that LPS-derived oocytes are as competent as FPS-derived oocytes, thereby adding some evidence to support the use of LPS for poor prognosis and oncological patients and to question the 'single recruitment episode' theory of follicle recruitment. These findings also encourage additional studies of the basics of folliculogenesis, with direct clinical implications for the management of ovarian stimulation in IVF. TRIAL REGISTRATION: None. STUDY FUNDING/COMPETING INTEREST(S): No external funds were used for this study and there are no conflicts of interest.

Preimplantation genetic diagnosis for aneuploidy testing in women older than 44 years: a multicenter experience.

OBJECTIVE: To report laboratory and clinical outcomes in preimplantation genetic diagnosis for aneuploidies (PGD-A) cycles for women 44 to 47 years old. DESIGN: Multicenter, longitudinal, observational study. SETTING: In vitro fertilization (IVF) centers. PATIENT(S): One hundred and thirty-seven women aged 44.7 ± 0.7 years (range: 44.0-46.7) undergoing 150 PGD-A cycles during April 2013 to January 2016. INTERVENTION(S): Quantitative polymerase chain reaction-based PGD-A on trophectoderm biopsies and cryopreserved euploid single-embryo transfer (SET). MAIN OUTCOMES MEASURE(S): Primary outcome measure: delivery rate per cycle; secondary outcome measures: miscarriage rate, and the rate and reasons for cycle cancelation with subanalyses for female age and number of metaphase 2 oocytes retrieved. RESULT(S): In 102 (68.0%) of 150 cycles blastocyst development was obtained, but only 21 (14.0%) were euploid blastocysts. The overall euploidy rate was 11.8% (22 of 187). Twenty-one SET procedures were performed, resulting in 13 clinical pregnancies, of which 1 miscarried and 12 delivered. The delivery rate was 57.1% per transfer, 8.0% per cycle, and 8.8% per patient. The logistic regression analysis found that only female age (odds ratio 0.78) and number of metaphase 2 oocytes retrieved (odds ratio 1.25) statistically significantly correlated with the likelihood of delivery. The delivery rate per cycle was 10.6% (11 of 104) in patients aged 44.0 to 44.9 years and 2.6% in patients aged 45.0 to 45.9 years (n = 1 of 38). No euploid blastocysts were found for patients older than 45.0 years. CONCLUSION(S): Extensive counseling based on biological and clinical data should be provided to women older than 43 years who are requesting IVF because of their very low odds of success and high risk for embryonic aneuploidies. Nevertheless, the low miscarriage and good delivery rates reported in this study in women with good ovarian reserve aged 44 should encourage the use of PGD-A in this population.

Follicular versus luteal phase ovarian stimulation during the same menstrual cycle (DuoStim) in a reduced ovarian reserve population results in a similar euploid blastocyst formation rate: new insight in ovarian reserve exploitation.

OBJECTIVE: To compare the euploid blastocyst formation rates obtained after follicular phase (FP) versus luteal phase (LP) stimulation performed in the same menstrual cycle in a preimplantation genetic diagnosis for aneuploidy testing (PGD-A) program in patients with reduced ovarian reserve. DESIGN: Prospective paired noninferiority observational study. SETTING: Private infertility program. PATIENT(S): Forty-three reduced ovarian reserve patients undergoing a PGD-A. INTERVENTION(S): Both FP and LP stimulations using follicle-stimulating hormone and luteinizing hormone in combination with gonadotropin-releasing hormone (GnRH) antagonist starting on day 2 of the cycle and 5 days after the first oocyte retrieval, respectively, where GnRH agonist was used for both FP and LP ovulation triggering; a trophectoderm biopsy quantitative polymerase chain reaction-based PGD-A strategy; and single euploid blastocyst transfers during a subsequent natural cycle. PRIMARY OUTCOME MEASURE: euploid blastocyst rate per injected metaphase 2 (MII) oocyte; secondary outcome measures: number of cumulus-oocyte complexes (COCs), MII oocytes, and blastocysts. RESULT(S): Patients with an antimüllerian hormone level of <1.5 ng/mL, antral follicle count of <6 follicles, and/or <5 oocytes retrieved in a previous cycle were included. No statistically significant differences were found in the number of retrieved COCs (5.1 ± 3.4 vs. 5.7 ± 3.3), MII oocytes (3.4 ± 1.9 vs. 4.1 ± 2.5), or biopsied blastocysts per stimulated cycle (1.2 ± 1.2 vs. 1.4 ± 1.7) from FP versus LP stimulation, respectively. No differences were observed in the euploid blastocyst rate calculated either per biopsied blastocyst (46.9% vs. 44.8%) or injected MII oocyte (16.2% vs. 15.0%). CONCLUSION(S): Stimulation with an identical protocol in the FP and LP of the same menstrual cycle resulted in a similar number of blastocysts in patients with reduced ovarian response. The LP stimulation statistically significantly contributed to the final transferable blastocyst yield, thus increasing the number of patients undergoing transfer per menstrual cycle.

Small leucine rich proteoglycans are differently distributed in normal and pathological endometrium.

BACKGROUND: During the woman's fertile period, the non-pregnant uterus is subject to constant cyclic changes. The complex mechanisms that control the balance among proliferation, differentiation, cell death and the structural remodeling of the extracellular matrix can contribute to the benign or malignant endometrial pathological state. The small leucine-rich proteoglycans (SLRPs) are important components of cell surface and extracellular matrices. MATERIALS AND METHODS: Using immunohistochemistry, we showed that the distribution patterns of SLRPs were completely modified in the pathological compared to normal endometrium. RESULTS: The expression of SLRPs was low/absent in all endometrial pathologies examined compared to normal endometrium. We observed an increase of lumican from proliferative to secretory phase of the endometrium and a decrease of fibromodulin, biglycan and decorin. In menopause endometrial tissue, the level of expression of fibromodulin, biglycan, decorin and lumican dramatically decreased. CONCLUSION: The results revealed the prominence and importance of proteoglycans in the tissue architecture and extracellular matrix organization.

Minilaparoscopic myomectomy: a mini-invasive technical variant.

OBJECTIVE: We propose a mini-invasive technical variant for laparoscopic myomectomy, which is currently less invasive and more feasible. STUDY DESIGN: This was a prospective, controlled, randomized trial, involving 170 patients, who underwent laparoscopic myomectomy. Patients were randomized into two groups: Group A (n=98) underwent standard laparoscopic myomectomy, and Group B (n=72) underwent the mini-invasive technique. The current mini-invasive variant is performed with a 10-mm umbilical trocar and only two 5-mm ancillary trocars. Morcellation is transumbilical: a 0° 5-mm optical system is used and is inserted either in the left or in the right iliac trocar according to the surgeon's preference. RESULTS: The degree of surgical difficulty, evaluated using a visual analog scale (VAS), was similar in the two groups (P=nonsignificant). Postoperative pain measured on a VAS scale showed there was less pain experienced in patients in Group B than in Group A (P<.01). Esthetic results measured on a VAS scale showed a higher compliance for patients in Group B than Group A (P<.01). CONCLUSIONS: The mini-invasive laparoscopic myomectomy described is, in our opinion, currently less invasive and more feasible than techniques usually used. Finally, this technique is a valid approach for the surgeon, and it gives women very acceptable aesthetic results.

Localization and modulation of NEDD8 protein in the human placenta.

BACKGROUND: Neural precursor cell-expressed, developmentally down-regulated-8 (NEDD8) is a 76-amino-acid ubiquitin-like polypeptide. NEDD8 affects the signaling of various molecules but the major cellular target proteins are cullins. The neddylation process is correlated closely with apoptosis, cell-cycle regulation, embryogenesis and development. AIM: The purpose of the present work was to investigate NEDD8 distribution and expression in the human placenta during gestation. MATERIALS AND METHODS: A total of 30 samples, 15 chorionic villous samples from first trimester and 15 from full-term placentae, were used for the immunohistochemical analysis of NEDD8 expression. The gestation period ranged from 5 to 40 weeks. RESULTS: NEDD8 was highly expressed in the cytotrophoblast of the first trimester of gestation, whereas in the third trimester, it was localized in the endothelial cells and stroma of placental villi. CONCLUSION: Our results suggest that NEDD8 may play an important role in the control of proliferation and differentiation of human placenta throughout pregnancy.

Effectiveness of the association micronized N-Palmitoylethanolamine (PEA)-transpolydatin in the treatment of chronic pelvic pain related to endometriosis after laparoscopic assessment: a pilot study.

OBJECTIVE: Aim of our study was to evaluate the effectiveness of the association between N-Palmitoylethanolamine and transpolydatin in the management of chronic pelvic pain related to EMS. STUDY DESIGN: This was a randomized, double-blind, parallel-group, placebo-controlled clinical trial involving 61 subjects, submitted to a first line laparoscopic conservative surgery, who were randomized into 3 groups receiving: group A (n=21) the association N-Palmitoylethanolamine-transpolydatin 400 mg + 40 mg twice a day for 3 months; group B (n=20) the placebo for 3 months; group C (n=20) a single course of Celecoxib 200mg twice a day for 7 consecutive days. Assessments of the severity of pelvic endometriosis (pelvic pain, dysmenorrhoea and dyspareunia) were recorded before and after treatment on a questionnaire and a 10-point VAS. Differences between groups were verified with Kruskal-Wallis ANOVA for non-parametric multiple comparisons. RESULTS: A marked decrease in dysmenorrhoea, dyspareunia and pelvic pain was observed in all groups, and the association between N-Palmitoylethanolamine and transpolydatin resulted to be more effective than placebo (P<.001). Additionally, the treatment with Celecoxib resulted in a decrease in pelvic pain more effective either than the association N-Palmitoylethanolamine and transpolydatin or placebo. CONCLUSION: These preliminary results show that the association between micronized N-Palmitoylethanolamine and transpolydatin is effective in the management of pelvic pain related to endometriosis after laparoscopy. Additionally, this association seems to be safe, shows an optimal control of pain and can be used in patients who are unable to receive other therapies.

Measurement of bisphenol A and bisphenol B levels in human blood sera from healthy and endometriotic women.

A sensitive HPLC method with fluorescence detection was developed for the determination of bisphenol A (BPA) and bisphenol B (BPB) in human blood serum. The detection limits of the method were 0.18 and 0.20 ng/mL for BPA and BPB, respectively. A single-step liquid-liquid extraction was used for the pre-treatment of serum samples. The recoveries of BPA and BPB spiked to sera were 85.6 and 87.7%, respectively. The analyses of sera from both healthy and endometriotic women emphasized the absence of bisphenols in all the control cases (11 women), whereas BPA was found in 30 sera (51.7%) and BPB was found in 16 sera (27.6%) in the group of 58 patients with endometriosis; in nine of such sera BPA and BPB were present simultaneously. Only relatively to the sera quantitated, BPA concentrations ranged from 0.79 to 7.12 ng/mL (mean concentration 2.91 +/- 1.74 ng/mL), whereas BPB concentrations ranged from 0.88 to 11.94 ng/mL (mean concentration 5.15 +/- 4.16 ng/mL). Therefore, the presence of at least one of the two bisphenols was verified in a percentage as high as 63.8% in the sera from endometriotic women, suggesting the existence of a relationship between endometriosis and BPA and/or BPB exposure. Indeed, it is well known that bisphenols can work as xenoestrogens, owing to their structural similarity to natural and synthetic estrogens (e.g. estradiol and dietilstilbestrol). However, further studies are necessary to confirm this hypothesis and to assess the actual dose at which exposures to bisphenols are able to increase the sensitivity of the endometriotic cells to estradiol.

CYP17 and CYP19 gene polymorphisms in women affected with endometriosis.

OBJECTIVE: To investigate whether CYP17 T>C polymorphism and polymorphisms C1558T and Val80 of CYP19 are related to endometriosis. DESIGN: Clinical study. PATIENT(S): Women affected with endometriosis (n = 104) and control group (n = 86). The diagnosis of endometriosis was confirmed by the histologic examination of the endometriotic lesions. RESULT(S): In patients affected with endometriosis, we observed that AA and CC genotypes were significantly represented in Val80 and C1558T polymorphisms of CYP19. CONCLUSION(S): The molecular mechanisms that underlie the development of endometriosis are unclear. Both environmental and genetic factors are involved in the pathogenesis of the disease. The inheritable susceptibility to endometriosis justifies the growing interest in identifying genes and/or genetic polymorphisms that predispose women to an increased risk of developing endometriosis. The identification of single-nucleotide polymorphism (SNP), probably linked to endometriosis, could help to explain its pathogenesis.

The pattern of expression of Notch protein members in normal and pathological endometrium.

The objective of this study was to investigate the pattern of expression and the localization of Notch-1, Notch-4 and Jagged-1 in physiological and pathological human endometrium and to evaluate the expression levels of two major regulators of the G1 checkpoint, namely cyclin D1 and p21. Sixty samples of physiological endometrium and 60 samples of pathological endometrium were used for the study. Evaluation of the expression level and the distribution of Notch pathway members and cell-cycle proteins was performed by immunohistochemistry. In the physiological endometrium we observed an increase of Notch-1 and Jagged-1 from proliferative to secretory phase and an opposite trend for Notch-4. In menopause, the level of expression of all three members of the Notch pathway decreased. We also observed a cyclin D1 increase from proliferative to secretory phase. By contrast, p21 showed a slight increase from proliferative to secretory phase. In the pathological endometrium, we observed an increase of Notch-1 expression from polyps to carcinoma and decrease for Notch-4 and Jagged-1. Moreover, we observed a higher expression of cyclin D1 in all the endometrial pathologies. By contrast, the expression level of p21 slightly increased from polyps to carcinoma. We concluded that in human endometrium Notch-4 seems to be more involved in controlling proliferation, whereas Notch-1 seems to be more involved in differentiation programming. Deregulation of these functions may induce the onset of several endometrial pathologies from polyps to cancer.

Role of proteoglycans in the organization of periurethral connective tissue in women with stress urinary incontinence.

OBJECTIVES: Connective tissue consists of fibroblasts and extracellular matrix (ECM) with collagen and elastic fibers, glycoproteins and proteoglycans (PGs) and it is considered an important factor of the supportive structures of the genitourinary region. Since PGs are essential for the organization of the collagen fibrils in the ECM, we investigated the presence of two PGs, fibromodulin and lumican, and of collagen type I in the periurethral connective tissue from women with stress urinary incontinence (SUI), compared to asymptomatic controls. METHODS: Thirty-two patients participated in the study and they were divided into four groups: premenopausal incontinents, premenopausal controls, postmenopausal incontinents and postmenopausal controls. All patients underwent gynaecologic surgical procedures and punch biopsies from the periurethral tissue were obtained. Immunohistochemistry for collagen type I, fibromodulin and lumican was performed on the histological slides. RESULTS: In premenopausal incontinents the immunoreactivity for collagen type I was weaker with an irregular distribution compared to premenopausal controls; while for fibromodulin, the staining was stronger in premenopausal incontinents than in premenopausal controls. Between the two postmenopausal groups there was not a significant difference in the intensity of collagen type I and fibromodulin staining that instead were less strong than in premenopausal groups. Lumican staining had the same distribution in the four groups. CONCLUSIONS: Our results suggest an altered remodelling of connective tissue in the periurethral region of premenopausal patients with SUI, with a significant decrease of collagen content and an irregular organization and distribution of the collagen fibrils, compared to premenopausal controls. In the SUI patients this abnormal ECM remodelling, mainly related to the observed change in PGs expression, might affect significantly the tensile strength of the connective tissue and consequently the support that is provided by the urogenital suspensory apparatus to urethra and bladder base. Moreover, the significant decrease in collagen type I content in postmenopausal patients respect to premenopausal patients, suggests that age and hormonal factors could contribute to the pathological modifications of the supportive genitourinary connective tissues in the SUI patients.

Modulation of Bax expression in physiological and pathological human placentas throughout pregnancy.

Apoptosis is intimately involved in placental homeostasis, growth and remodelling, and apoptotic rates increase progressively during normal pregnancy as part of normal placental development. Moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction and diabetes. In the present study, we describe differences in the expression of proapoptotic protein Bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. We first observed a strong increase of Bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate Bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. Secondly, we showed a more intense immunopositivity for Bax in the third trimester spontaneous birth with respect to the third trimester caesarean birth. Thirdly, we observed an increase of Bax expression in preeclamptic placentas compared to the normal full-term placentas. In contrast, we observed a moderate Bax expression in diabetic placentas only slightly lower than the normal full-term placentas. Our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies.