Effect of ejaculatory abstinence period on fertilization and clinical outcomes in ICSI cycles: a retrospective analysis.

RESEARCH QUESTION: Does ejaculatory abstinence impact fertilization outcomes in intracytoplasmic sperm injection (ICSI) cycles in infertile couples? DESIGN: This single-centre retrospective observational study included 6919 ICSI cycles from 2013 to 2022. The primary outcome was the assessment of oocyte fertilization, measured in terms of the rate of formation of two-pronuclear (2PN), 3PN and 1PN zygotes. Secondary outcomes were blastulation, cumulative positive ß-human chorionic gonadotrophin test and clinical pregnancy rates. Relationships between ejaculatory abstinence and fertilization outcomes, and ejaculatory abstinence and clinical outcomes were evaluated with multivariable analysis, including possible confounders. RESULTS: A positive association was observed between ejaculatory abstinence and semen sample volume (P < 0.001), sperm concentration (P < 0.001) and total motile sperm count (P < 0.001). No association was found between the 1PN zygote rate and ejaculatory abstinence (P = 0.97). Conversely, for each additional day of ejaculatory abstinence, the likelihood of obtaining 2PN zygotes from all inseminated oocytes decreased by 3% [adjusted odds ratio (aOR) 0.97, 95% CI 0.94-0.99], whilst the likelihood of obtaining 3PN zygotes from all inseminated oocytes increased significantly by 14% (aOR 1.14, 95% CI 1.07-1.22). No significant associations were found between ejaculatory abstinence and blastulation, cumulative pregnancy or miscarriage rates. CONCLUSIONS: A longer ejaculatory abstinence period significantly decreases the rate of 2PN zygotes, and increases the rate of 3PN zygotes without directly affect blastulation and pregnancy rates.

'Guess who'? An Italian multicentric study on pigmentation traits prevalence in endometriosis localizations.

OBJECTIVES: Previous evidence seems to support the more common presence of certain pigmentation types in women with endometriosis. The aim of this study was to assess the association of certain somatic phenotypes with specific localizations of the disease. The genetic makeup of those somatic traits may will help in better define the disease pathogenesis. STUDY DESIGN: Multicentric, retrospective study of women aged 18 to 45 with histologically confirmed endometriosis. 575 patients were recruited at eleven different Italian endometriosis clinics from March 2015 to January 2021. Data regarding clinical and surgical features were recorded following the self-administered endometriosis patient questionnaire and the surgical standard of reports approved by the World Endometriosis Research Foundation (WERF). Pigmentation types/somatic phenotypes frequencies among endometriosis localizations were reported. A logistic regression analysis was performed to determine somatic types independently associated with disease' localizations. RESULTS: Having green eyes increased by ∼4 folds (OR 3.7; 95% CI: 1.42-9.61; p = 0.007) the risk of having a ureteral nodule, whereas brown/black eyes decreased this risk (OR 0.34; 95% CI: 0.13-0.87; p = 0.025). Consistently, the combination of green eyes and blonde/light brown hairs increased the odds of ureteral endometriosis by more than 5 folds (OR 5.40; 95%CI: 2.02-14.49; p = 0.001), even after correction for anthropometric confounders (aOR 5.85; 95% CI: 2.13-16.09; p < 0.001). CONCLUSIONS: The association between endometriosis and pigmentary traits has been herein confirmed, with the novel finding of the possible predisposition of ureteral endometriosis in patients with green eyes and blonde/light brown hairs. Further investigation on the genetic makeup of somatic traits may provide new inroads also into the molecular aspects of endometriosis leading to a better understanding of this complex disease.

Endometriosis: seeking optimal management in women approaching menopause.

The incidence of endometriosis in middle-aged women is not minimal compared to that in the reproductive age group. The treatment of affected women after childbearing age to the natural transition toward menopause has received considerably poor attention. Disease management is problematic for these women due to increased contraindications regarding hormonal treatment and the possibility for malignant transformation, considering the increased cancer risk in patients with a long-standing history of the disease. This state-of-the-art review aims for the first time to assess the benefits of the available therapies to help guide treatment decisions for the care of endometriosis in women approaching menopause. Progestins are proven effective in reducing pain and should be preferred in these women. According to the international guidelines that lack precise recommendations, hysterectomy with bilateral salpingo-oophorectomy should be the definitive therapy in women who have completed their reproductive arc, if medical therapy has failed. Strict surveillance or surgery with removal of affected gonads should be considered in cases of long-standing or recurrent endometriomas, especially in the presence of modifications of ultrasonographic cyst patterns. Although rare, malignant transformation of various tissues in endometriosis patients has been described, and management is herein discussed.

Assessment of ovarian reserve after cystectomy versus 'one-step' laser vaporization in the treatment of ovarian endometrioma: a small randomized clinical trial.

STUDY QUESTION: Does CO2 laser vaporization offer better results in treating endometrioma in terms of ovarian reserve preservation compared to traditional cystectomy? SUMMARY ANSWER: Assessing both antral follicle count (AFC) and serum anti-Müllerian hormone (AMH) levels as measures of ovarian reserve, the results suggest that CO2 technology may be an alternative treatment for endometrioma, causing minimal damage to adjacent healthy ovarian tissue. WHAT IS KNOWN ALREADY: Excisional surgery has been questioned as an ideal surgical approach for endometriomas because it is associated with potential reduction of ovarian reserve. Recently, vaporization with CO2 laser in-line-of-sight, according to the 'three-step procedure', has been proposed as the best method to preserve ovarian function. However, no randomized controlled trials have been conducted to compare cystectomy and 'one-step' CO2 fiber laser vaporization (without GnRH agonist therapy) with respect to the ovarian reserve. STUDY DESIGN, SIZE, DURATION: A multicentre randomized clinical trial including 60 patients was performed between July 2017 and February 2018. Computerized randomization was conducted to allocate them in a proportion of 1:1 either to Group 1 (laparoscopic stripping: cystectomy) or Group 2 (CO2 laser vaporization). Patients in Group 1 underwent a standardized laparoscopic stripping technique; patients in Group 2 underwent drainage of the cyst content, biopsy and vaporization of the internal wall with a CO2 fiber laser. Patients underwent pelvic ultrasound examination to determine the AFC and blood sampling to determine AMH levels before surgery and at 1- and 3-month follow-up. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients undergoing surgery for symptomatic endometriomas (infertility and/or pelvic pain) larger than 3 cm were randomized in two groups according to the surgical technique. Patients aged ≥40 years, or with deep infiltrating endometriosis/adenomyosis, or previously submitted to surgical procedures on the ovaries or to hysterectomy were excluded from the study. The primary endpoint was the comparison of intra-group AFC changes before and after surgery (ΔAFC) between the two groups (ΔAFC Group 1 versus ΔAFC Group 2). The secondary endpoint was the modification of serum AMH before and after surgery (ΔAMH) between the two groups (ΔAMH Group 1 versus ΔAMH Group 2). MAIN RESULTS AND THE ROLE OF CHANCE: The AFC of the operated ovary was significantly increased in Group 2 (laser vaporization) compared with Group 1 (cystectomy) after surgery (Group 1: from 4.1 ± 2.2 [mean ± SD] at baseline to 6.3 ± 3.5 at 3-month follow-up; 95% CI: 0.9-4; Group 2: from 3.6 ± 1.9 at baseline to 8.6 ± 4.2 at 3-month follow-up; 95% CI: 2.8-7.1; P = 0.016); serum AMH levels were significantly reduced at 3 months in Group 1 (from 2.6 ± 1.4 ng/mL at baseline to 1.8 ± 0.8 ng/mL at 3-month follow-up; 95% CI: -1.3 to -0.2; P = 0.012) compared with no reduction in Group 2 (from 2.3 ± 1.1 ng/mL at baseline to 1.9 ± 0.9 ng/mL at 3-month follow-up; 95% CI: -1 to -0.2; P = 0.09). LIMITATIONS, REASON FOR CAUTION: The key limitations of the trial were the low accuracy of AFC in estimating the ovarian reserve in ovaries with endometriomas, the limited study size and the relatively short follow-up, which do not allow us to draw definitive conclusions. WIDER IMPLICATIONS OF THE FINDINGS: The present study suggests that CO2 technology may treat endometrioma with minimal damage to the adjacent healthy ovarian tissue; however, this study should be considered as a preliminary clinical trial, intended to stimulate future larger trials to address this clinically relevant issue. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT03227640. TRIAL REGISTRATION DATE: 9 July 2017. DATE OF FIRST PATIENT'S ENROLLMENT: 24 July 2017.

Multifactorial analysis of the stochastic epigenetic variability in cord blood confirmed an impact of common behavioral and environmental factors but not of in vitro conception.

Background: An increased incidence of imprint-associated disorders has been reported in babies born from assisted reproductive technology (ART). However, previous studies supporting an association between ART and an altered DNA methylation status of the conceived babies have been often conducted on a limited number of methylation sites and without correction for critical potential confounders. Moreover, all the previous studies focused on the identification of methylation changes shared among subjects while an evaluation of stochastic differences has never been conducted. This study aims to evaluate the effect of ART and other common behavioral or environmental factors associated with pregnancy on stochastic epigenetic variability using a multivariate approach. Results: DNA methylation levels of cord blood from 23 in vitro and 41 naturally conceived children were analyzed using the Infinium HumanMethylation450 BeadChips. After multiple testing correction, no statistically significant difference emerged in the number of cord blood stochastic epigenetic variations or in the methylation levels between in vitro- and in vivo-conceived babies. Conversely, four multiple factor analysis dimensions summarizing common phenotypic, behavioral, or environmental factors (cord blood cell composition, pre or post conception supplementation of folates, birth percentiles, gestational age, cesarean section, pre-gestational mother's weight, parents' BMI and obesity status, presence of adverse pregnancy outcomes, mother's smoking status, and season of birth) were significantly associated with stochastic epigenetic variability. The stochastic epigenetic variation analysis allowed the identification of a rare imprinting defect in the locus GNAS in one of the babies belonging to the control population, which would not have emerged using a classical case-control association analysis. Conclusions: We confirmed the effect of several common behavioral or environmental factors on the epigenome of newborns and described for the first time an epigenetic effect related to season of birth. Children born after ART did not appear to have an increased risk of genome-wide changes in DNA methylation either at specific loci or randomly scattered throughout the genome. The inability to identify differences between cases and controls suggests that the number of stochastic epigenetic variations potentially induced by ART was not greater than that naturally produced in response to maternal behavior or other common environmental factors.

Top quality blastocyst formation rates in relation to progesterone levels on the day of oocyte maturation in GnRH antagonist IVF/ICSI cycles.

Cycles with progesterone elevation during controlled ovarian stimulation (COS) for IVF/ICSI are commonly managed with a "freeze-all" strategy, due to a well-recognized detrimental effect of high progesterone levels on endometrial receptivity. However, also a detrimental effect of elevated progesterone on day-3 embryo quality has recently been found with regards to top quality embryo formation rate. Because blastocyst culture and cryopreservation are largely adopted, we deemed relevant to determine whether this detrimental effect is also seen on blastocyst quality on day 5-6. This issue was investigated through a large two-center retrospective study including 986 GnRH antagonist IVF/ICSI cycles and using top quality blastocyst formation rate as the main outcome. Results showed that on multivariate analysis sperm motility (p<0.01) and progesterone levels at ovulation triggering (p = 0.01) were the only two variables that significantly predicted top quality blastocyst formation rate after adjusting for relevant factors including female age, BMI, basal AMH and total dose of FSH used for COS. More specifically, progesterone levels at induction showed an inverse relation with top quality blastocyst formation (correlation coefficient B = -1.08, 95% CI -1.9 to -0.02) and ROC curve analysis identified P level >1.49 ng/ml as the best cut-off for identification of patients at risk for the absence of top quality blastocysts (AUC 0.55, p<0.01). Our study is the first to investigate the top quality blastocyst formation rate in relation to progesterone levels in IVF/ICSI cycles, showing that increasing progesterone is associated with lower rates of top quality blastocyst. Hence, the advantages of prolonging COS to maximize the number of collected oocytes might eventually be hindered by a decrease in top quality blastocysts available for transfer, if increasing progesterone levels are observed. This observation extends the results of two recent studies focused on day-3 embryos and deserves further research.

Are extremely high progesterone levels still an issue in IVF?

BACKGROUND: Premature luteinization of one or more developing follicles complicates 1-2 % of controlled ovarian stimulation cycles for assisted reproduction. The management of this complication is controversial, with cycle cancellation likely representing the most commonly used strategy. The aim of this study was to evaluate the efficacy of the "freeze-all" policy-where the entire cohort of blastocysts is cryopreserved for subsequent frozen-thawed embryo transfer-in treating cases of premature luteinization. METHODS: Patients experiencing premature luteinization during controlled ovarian stimulation-identified by extremely high progesterone levels at induction (P levels ≥3.0 ng/ml and/or P/estradiol ratio ≥1, n = 42)-were included in a "freeze-all" program and compared to controls undergoing a "freeze-all" program with normal progesterone levels at induction (P < 1.5 ng/ml, n = 67). RESULTS: Blastulation rate was comparable between patients with premature luteinization and controls (48.1 ± 20.5 % in Cases vs. 52.3 ± 24.9 % in Controls, p = 0.36). Ongoing pregnancy rates after the first frozen-thawed embryo transfer (38.1 % in Cases and 41.0 % in Controls, p = 0.83) and cumulative ongoing pregnancy rates after three frozen-thawed embryo transfer cycles (40.5 % in Cases vs. 47.8 % in Controls, p = 0.55) were also similar. CONCLUSIONS: These results show that extremely marked progesterone elevation throughout controlled ovarian stimulation does not impair blastocyst development and implantation potential in the context of a "freeze-all" strategy. Based on this, adoption of the "freeze-all" strategy represents a valuable tool in treating premature luteinization. In contrast, cycle cancellation-likely the most frequently used method for management of this complication-currently represents a misconduct.

Stimulatory effect of SOM230 on human and rat adrenal corticosteroid secretion in vitro.

SOM230 (pasireotide, Signifor), a recently developed somatostatin analog, has been tested in ACTH-secreting pituitary tumors with promising results. No study has yet evaluated whether this analog also directly affects adrenal steroid production. The aim of the current study was to evaluate whether SOM230 modulates corticosteroid secretion by normal adrenals in vitro. Primary cultures from normal human and rat adrenals were incubated with 10-100 nM SOM230 with and without 10nM ACTH. Dose-response studies with 1 nM-1 µM SOM230 were performed on rat adrenals. Cortisol/corticosterone levels in medium were measured after 4 and 24h. SOM230 (10nM) significantly increased corticosteroid levels after 24h incubation in both human (36.4 ± 0.43 ng/well vs 27.7 ± 3.17 ng/well, p<0.05) and rat (16.2 ± 1.16 ng/well vs 11.6 ± 0.92 ng/well p<0.05) adrenals; lesser effects were observed with 100 nM SOM (33.4 ± 2.59 ng/well vs 27.7 ± 3.17 ng/well p<0.05; 13.4 ± 0.82 ng/well vs 11.6 ± 0.92 ng/well, N.S. vs baseline secretion for human and rat adrenals, respectively). Dose-response curves confirmed maximal effect at 10nM SOM230. The corticosteroid secretory response to ACTH was unaffected by SOM230 co-incubation. In conclusion, SOM230 exerts a moderate stimulatory effect on adrenal corticosteroid secretion in vitro. This argues against a direct adrenal involvement in the clinical efficacy of SOM230 in patients with ACTH-secreting pituitary tumors and widens the known range of action of SOM230.

Responses to corticotrophin-releasing hormone and dexamethasone in a large series of human adrenocorticotrophic hormone-secreting pituitary adenomas in vitro reveal manifold corticotroph tumoural phenotypes.

Patients with Cushing's disease are known to present a variable secretory response to stimulatory and inhibitory challenges. Evaluation of the secretory behaviour of pituitary adrenocorticotrophic hormone (ACTH)-secreting adenomas in vitro aids in the comprehension of its behaviour in vivo; however, given the small size of these tumours and the consequent paucity of material available to in vitro studies, a comprehensive study on the secretory behaviour of human corticotroph tumours has not yet been performed. The present study aimed to assess the spectrum of responses to the two main corticotroph modulators, corticotrophin-releasing hormone (CRH) and dexamethasone, in a large series of human ACTH-secreting pituitary tumours. Seventy-two ACTH-secreting pituitary tumours were collected during surgery and established in culture. Specimens were incubated with 10 nm CRH and/or 10 nm dexamethasone for 4 h and 24 h. Secretion in unstimulated, control wells was set at 100% and changes in ACTH concentrations by at least 20% were considered as responses. Parallel experiments in 12 rat anterior pituitary primary cultures were evaluated. A marked ACTH increase was observed during incubation with CRH in 70% of tumoural specimens at 4 h (range 124-3500% of control wells) and in 57% at 24 h (range 122-3323%). Dexamethasone reduced ACTH secretion in almost 50% of tumours (range 78-2% of control at 4 h; 76-3% at 24 h), whereas it did not affect ACTH medium levels in 30% of specimens and induced a paradoxical ACTH increase in 20% of tumours (range 130-327% of control at 4 h; 156-348% at 24 h). By comparison, CRH uniformly increased ACTH levels in rat anterior pituitary primary cultures (mean 745 ± 84% at 4 h; 347 ± 25% at 24 h), whereas dexamethasone decreased ACTH levels by 40-50% in all experiments. In conclusion, the present study of a large series of human ACTH-secreting pituitary tumours in vitro revealed a considerable variability in the responses to CRH and dexamethasone. This finding indicates the existence of multiple corticotroph tumoural phenotypes and may account for the different responses to physiological and pharmacological modulators in vivo.

The human POMC gene promoter: where do we stand?

Proopiomelanocortin (POMC) is crucial for several life-essential functions and its regulation has been studied extensively in the past decades. The first studies provided the framework for POMC promoter activity, namely the identification for the major response elements contained in the promoter, e.g., the glucocorticoid response element, the Nur response element, while subsequent studies showed the importance of cooperation and interplay between transcription factors to achieve optimal promoter activity. The involvement of constitutive repressors of POMC transcription, such as Bmp4, provided the latest clues to our understanding of POMC promoter activity. This increased knowledge benefits the clinician as it allows genetic testing and early recognition of patients with congenital ACTH deficiency due to mutations in TPIT and paves the way to new medical treatments in Cushing's disease. The present review will illustrate the current standing on regulation of the human POMC promoter, focusing on its activity in corticotropes.

Inhibitory effect of prepro-thyrotrophin-releasing hormone (178-199) on adrenocorticotrophic hormone secretion by human corticotroph tumours.

Prepro-thyrotrophin-releasing hormone (TRH) (178-199), a 22-amino acid cleavage product of the TRH prohormone, has been postulated to act as an adrenocorticotrophin hormone (ACTH)-release inhibitor. Indeed, although in vitro evidence indicates that this peptide may inhibit basal and stimulated ACTH secretion in rodent anterior pituitary primary cultures and cell lines, not all studies concur and no study has as yet evaluated the effect of this peptide in Cushing's disease. The present study aimed to test the effect of preproTRH(178-199) in human tumoural corticotrophs. Twenty-four human ACTH-secreting pituitary tumours (13 macroadenomas, 11 microadenomas) were collected during surgery and incubated with 10 or 100 nm preproTRH(178-199). ACTH secretion was assessed after 4 and 24 h of incubation by immunometric assay and expressed relative to levels observed in control, unchallenged wells (= 100%). Parallel experiments were performed in rat anterior pituitary primary cultures. A clear inhibition of ACTH secretion at 4 and 24 h was observed in 12 specimens (for 10 nm ppTRH: 70 +/- 4% control at 4 h and 83 +/- 5% control at 24 h; for 100 nm ppTRH: 70 +/- 4% control at 4 h and 85 +/- 5% control at 24 h), whereas a mild and short-lasting stimulatory effect was observed in three tumours and no changes in ACTH secretion in the remaining nine tumoural specimens. The inhibitory effect of preproTRH(178-199) was more evident in macroadenomas and significantly correlated with sensitivity to dexamethasone inhibition. Significant inhibition of ACTH secretion by preproTRH(178-199) in rat pituitary cultures was observed after 24 h of incubation. The present study conducted in a large series of human corticotroph tumours shows that preproTRH(178-199) inhibits tumoural ACTH secretion in a sizable proportion of specimens, in close relation to the size of the tumour and its sensitivity to glucocorticoid negative feedback. This appears a promising avenue of research and further studies are warranted to explore the full scope of preproTRH(178-199) as a regulator of ACTH secretion.