Impact of different typologies of smoking on ovarian reserve and oocyte quality in women performing ICSI cycles: an observational prospective study.

OBJECTIVE: Within the last few years smoking activities, as well as infertility, have increased in Italy, and so has the consumption of alternative cigarette devices among women of childbearing age. The aim of this observational study was to evaluate the impact of the consumption of cigarettes and alternatives devices, such as electronic cigarettes and heat-not-burn (HnB) products, on infertile women performing in vitro fertilization (IVF), in specific on the quality of oocytes retrieved in women performing intracytoplasmic sperm injection (ICSI) cycles. PATIENTS AND METHODS: Prospective observational longitudinal study involving 410 women referring to the Reproductive Physiopathology and Andrology Unit, Sandro Pertini Hospital, Rome, from 2019-2022. All the women enrolled filled out an elaborate questionnaire investigating smoking consumption, before the beginning of ovarian stimulation by antagonist protocol, ovarian pick-up, and subsequent ICSI technique. The outcomes of the study were the evaluation of clinical and ICSI features between the groups of smokers and non-smokers: the number of retrieved oocytes, immature oocytes, and fertilization rate were confronted between the two groups and between cigarette smokers vs. e-cigarette and heat-not-burn (HnB) products smokers. RESULTS: Clinical parameters were comparable between the group of smokers compared to one of the non-smokers, except for anti-Müllerian hormone (AMH), which was statistically lower in smokers (p<0.05). Regarding IVF hormonal stimulations it appears that the total dose of gonadotropin was statistically lower in the non-smoker's group, compared to smokers (1850±860 UI vs. 1,730±780 p<0.05). Regarding ICSI techniques interestingly the number of oocytes retrieved was lower in the smokers' group compared to non-smokers (5.21±0.9 vs. 6.55±3.5, p<0.001), and the number of empty zona pellucida oocytes was statistically higher in the smokers' group (0.51±0.1 vs. 0.2±0.1, p<0.05). On the other hand, the fertilization rate (FR) was statistically higher in non-smokers compared to the smokers' group (72.16±3.05 vs. 68.12±2.21, p=0.03). Out of the 203 smokers, overall, any statistically significant difference, regarding ICSI results, has been found between the group of cigarette smokers, compared to the group of e-cigarettes plus HnB products smokers. CONCLUSIONS: Smoking negatively impacts human fertility, leading to a reduction of ovarian reserve and ovarian quality, which can negatively impact results in women performing ICSI cycles. Despite the limitation of the study, our results underline that consumption of cigarette alternative devices seems to have a similar negative impact on the quantity and quality of oocytes retrieved in ICSI cycles. Clinicians should emphasize the reduction of exposure to harmful substances derived from the combustion of tobacco smoking, as well as alternative devices, in women of childbearing age.

Characterization of epidermal growth factor-like domain 7 (EGFL7) expression in normal endometrium and in the endometrium of women with poor reproductive outcomes.

STUDY QUESTION: Could epidermal growth factor-like domain 7 (EGFL7) be a factor involved in the preparation of the endometrium for implantation and could its dysregulation be implicated in poor reproductive outcomes? SUMMARY ANSWER: EGFL7 is highly expressed in the endothelium and glandular epithelium throughout the menstrual cycle; it is upregulated by stromal cells in secretory phase and appears strongly reduced in endometrial biopsies and isolated stromal cells of women with unexplained recurrent pregnancy loss (uRPL) and recurrent implantation failure (RIF). WHAT IS KNOWN ALREADY: The secreted factor EGFL7, originally identified as a gene primarily expressed in endothelial cells, is also expressed by the mouse blastocyst and by mouse and human trophoblast cells. It regulates trophoblast migration and invasion by activating NOTCH1 signaling. NOTCH1 has been demonstrated to play a fundamental role in endometrial receptivity and its dysregulation may be involved in selected pregnancy complications characterized by altered endometrial receptivity, such as uRPL. STUDY DESIGN, SIZE, DURATION: This is an exploratory study for which 84 endometrial biopsies were collected from normally fertile women, as well as from women with uRPL and RIF. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were collected from women in both the proliferative and secretory phases of the menstrual cycle and stratified into three sub-groups according to the patient clinical history: 20 fertile women (8 in proliferative and 12 in secretory phase), 41 women with uRPL (6 in proliferative and 35 in secretory phase), and 27 women with RIF (8 in proliferative and 19 in secretory phase). Immunohistochemistry, real-time PCR, and western blot analyses were performed to study the expression of EGFL7 and NOTCH1, as well as the NOTCH target genes. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis of spatial and temporal distribution of EGFL7 in endometrial biopsies from fertile women revealed higher levels of EGFL7 in samples from the secretory phase compared to proliferative phase. The expected expression of EGFL7 in endothelial cells was shown as well as the novel, not previously reported, expression in endometrial glands and stromal cells. EGFL7 was significantly reduced in the endometrium of women with uRPL and RIF in the secretory phases and this was associated with a downregulation of the NOTCH1 signaling pathway. Human recombinant EGFL7 was able to activate the NOTCH1 signaling pathway in endometrial stromal cells (EndSCs) obtained from fertile women but not in cells from uRPL or RIF patients. EndSCs from fertile women and decidualized in vitro for three days showed an upregulation of EGFL7 expression, whereas cells obtained from women with uRPL and RIF and decidualized in vitro did not. LIMITATIONS, REASONS FOR CAUTION: This study was conducted with a relatively small number of patient samples. Although results are highly reproducible and consistent, additional observations from multicentric cohorts would strengthen the relevance of the data. Moreover, this is an in vitro study, which might only partially represent the in vivo conditions. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate for the first time that EGFL7 is new player involved in decidualization and provide new insights into the pathophysiology of selected implantation defects and early pregnancy complications. Our studies have revealed that alterations in EGFL7 expression and the consequent dysregulation of NOTCH signaling are potential underlying causes of RIF and uRPL. Our results might have therapeutic relevance, as the EGFL7/NOTCH pathway may represent a potential target for medical intervention. STUDY FUNDING/COMPETING INTEREST(S): This study has been supported by the Grant for Fertility Innovation 2017 (Merck KGaA). There are no competing interests to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Uterine fibroids treatment: do we have new valid alternative? Experiencing the combination of vitamin D plus epigallocatechin gallate in childbearing age affected women.

OBJECTIVE: Uterine myomas are the most common benign tumors in females, and at least 25% of affected patients experience symptoms severe enough to need treatment, like heavy hemorrhage, pelvic pain, and infertility. Currently, a non-invasive approach is preferred in women of childbearing age who desire pregnancy. The aim of our study was to determine the effect of oral supplementation with a combination of vitamin D plus epigallocatechin gallate (EGCG) and vitamin B6 in women with myomas. PATIENTS AND METHODS: Between April and December 2020, we enrolled 95 women of childbearing age, afferent to our hospital, displaying at least one myoma with a diameter <4 cm. Patients were divided in two groups: 41 women were treated daily with two tablets of 25 µg vitamin D + 150 mg EGCG + 5 mg vitamin B6 for 4 months; 54 women, representing the control group, received no treatment. Total volume and vascularization of myomas were analyzed ultrasonographically. Bleeding and pelvic pain was also evaluated, as well as patients' quality of life and health through questionnaire Short Form Health Survey (SF-36) and Patient Global Impression of improvement (PGI-I). RESULTS: After treatment myomas' total volume and peripherical vascularization significantly decreased respectively by 37.9% (p<0.001) and 7.7%. On the other hand, we observed an increase in myomas' volume by 5.5 % and of peripherical vascularization by 5% in the control group. The treated group reported an improvement in SF-36 (p<0.001) and PGI-I (85.4%) questionnaire scores. CONCLUSIONS: We demonstrated, in young women who want to preserve fertility, that the combined supplementation of vitamin D, EGCG, and vitamin B6 reduced myomas' volume and improved patients' quality of life, without side effects.

Controlled, prospective, observational study on the efficiency and tolerability of a combination of potential Nrf2-inducing antioxidants and micronutrients as pre-treatment for ICSI in dyspermic patients with previous failure.

OBJECTIVE: To evaluate the efficiency of pre-treatment in dyspermic males in IVF couples with a combination of micronutrients, for the purpose of improving the fertilization rate, the implantation rate and the outcome of the pregnancy. PATIENTS AND METHODS: This controlled prospective clinical study was performed in two medically assisted reproduction centers. 59 males with mild oligo-astheno-teratospermia (OAT) were admitted to the study. All of them had a history of previous in vitro fertilization (IVF) attempts with female partners aged < 40 diagnosed having tubal or idiopathic infertility. The subjects upon enrolment underwent a semen test and afterward were treated with alpha lipoic acid and glutathione (Fertiplus SOD®, Idi-Pharma, Catania, Italy) for 4 weeks (short-term). The primary endpoints that were evaluated are the following: fertilization rate (mean fertilization), implantation rate and pregnancy rate. RESULTS: At the end of this study all the males (mean age 39.5 ± 5.1) reported in not having any side effects during the administration of Fertiplus. Their female partners (mean age 34.9 ± 4.5) underwent IVF using the ICSI technique. The number of oocytes retrieved and inseminated was not statistically different in comparison to previous attempts, but with the same number of oocytes treated, the fertilization rate per couple demonstrated statistically significant increase (p<0.001). We did not observe a percentage increase in evolutionary embryos, but we noticed an improvement in embryo quality per individual couple (p<0.001), associated with a net increase in the implantation rate per couple (p<0.001) in terms of clinical pregnancy. The estimated miscarriage risk after treatment was five times lower (p<0.001). CONCLUSIONS: Short-term treatment with micronutrients in dyspermic subjects can improve the reproductive outcome of the IVF procedure.