Markers of Local and Systemic Estrogen Metabolism in Endometriosis.

Estrogen metabolites (EMs) can work independently from their parent hormones. We hypothesize that in endometriosis, estrogen is metabolized preferentially along hormonally active pathways. We recruited 62 women with endometriosis (proven laparoscopically and histologically) and 52 control women (normal findings with laparoscopy) among patients undergoing surgery for pelvic pain and/or infertility during the proliferative phase of the menstrual cycle. Urinary samples were collected preoperatively. Biopsies from eutopic endometrium of control women and women with endometriosis were collected during surgery. EMs in urine and endometrial tissues were extracted and determined using Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS). These included: 2-hydroxyestrone (2OHE1), 16-α hydroxyestrone (16α-OHE1), 2OHE1/16α-OHE1 ratio, 4-hydroxyestrone (4OHE1), 2-hydroxyestradiol (2OHE2), and 4-hydroxyestradiol (4OHE2). Eutopic endometrium of endometriosis patients, as compared to control endometrium, contained significantly higher level of 4OHE1 (0.03 (IQR: 0.03-0.265) versus 0.03 (IQR: 0.03-0.03) µg/g, respectively, P = 0.005), 2-OHE2 (0.241 (IQR: 0.1-0.960) versus 0.1 (IQR: 0.1-0.1) µg/g, respectively, P < 0.001), and 4-OHE2 (0.225 (IQR: 0.22-1.29) versus 0.0.2 (IQR: 0.2-0.2) µg/g, respectively, P < 0.001). Only 2OHE1 showed higher concentration in urine of women with endometriosis than controls (9.9 (IQR: 3.64-14.88) versus 4.5 (IQR: 1.37-17.00) µg/mg creatinine, respectively, P = 0.042). Eutopic endometrium of women with endometriosis metabolizes estrogen preferentially to the biologically active 2OHE2, and potentially genotoxic 4OHE1 and 4OHE2 metabolites. This contributes to further understanding of endometriosis etiology, its link to ovarian cancer, and could help identifying an endometrial biomarker of the disease.

A randomized controlled trial of laser-assisted ICSI.

STUDY QUESTION: Does the use of a laser to open the zona pellucida during ICSI (laser assisted or LA-ICSI) improve oocyte survival, embryo development and clinical outcomes? SUMMARY ANSWER: Compared to conventional ICSI, LA-ICSI increased rates of oocyte survival and some aspects of embryo development but it did not alter the ongoing pregnancy rate; after adjusting for oocyte survival, there was no beneficial effect of LA-ICSI on embryo development and utilization. WHAT IS KNOWN ALREADY: Oocyte degeneration occurs in a 10th of mature oocytes after ICSI. Pilot studies suggest that LA-ICSI may improve oocyte survivability. STUDY DESIGN, SIZE, DURATION: In a randomized controlled trial, 966 couples (16 122 metaphase II oocytes) were allocated to receive LA-ICSI (intervention) or conventional ICSI (control) between 17 September 2018 and 5 August 2019. Oocyte survival (primary endpoint), embryo development and ongoing pregnancy rates were compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: Couples included in this study were recommended for ICSI due to female or male factor, unexplained infertility or a combination of factors. Patients were ineligible to participate in the study if they had uterine abnormality including thin endometrium, recurrent pregnancy loss, endometriosis or a severe medical condition. Concealed randomization to LA-ICSI or conventional ICSI, allocated in a 1:1 ratio, took place on stimulation Day 1 with replacement of blastocysts on only Day 5. The primary endpoint was oocyte survival with membrane integrity 24 h after the ICSI procedure. The sample size was estimated to detect a 3% increase in oocyte survival after LA-ICSI with 99% power at a 1% significance level. This also permitted the detection of 10% increase in ongoing pregnancy rate after LA-ICSI with 85% power at 5% alpha level. We used Poisson regression with zero-inflation for count data to estimate relative risk (RR) with 95% CI and logistic regression for clinical outcomes to estimate odds ratio (OR) with 95% CI. Both models adjusted for age as a covariate. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with conventional ICSI, LA-ICSI resulted in a higher number of surviving oocytes (RR 1.08, 95% CI 1.05-1.12, P < 0.001), as well as a higher number of fertilized oocytes (RR 1.08, 95% CI 1.04-1.13, P < 0.001) and utilizable blastocysts (RR 1.09, 95% CI 1.04-1.15, P < 0.001). Sensitivity analyses adjusted for oocyte survival showed no between-group difference in utilizable blastocysts (OR 1.01, 95% CI 0.95-1.08, P = 0.73) and by calculating the mean rate, a reduction in utilizable blastocysts was shown (RR 0.95, 95% CI 0.94-0.97, P < 0.001). Ongoing pregnancy showed no between-group difference (LA-ICSI 179/489 (37%) vs ICSI 201/477 (42%), OR 0.79, 95% CI 0.61-1.03, P = 0.09). LIMITATIONS, REASONS FOR CAUTION: It was not possible to blind the embryologists involved in the ICSI procedure. However, there was concealment of randomization and blinding of outcome assessments reducing the risk of selection and measurement bias. WIDER IMPLICATIONS OF THE FINDINGS: A beneficial effect of LA-ICSI on oocyte survival should be shown to improve clinical outcomes, before its use in clinical practice is justified. STUDY FUNDING/COMPETING INTEREST(S): The study received no funding, and the authors declare that there are no conflicts of interest. TRIAL REGISTRATION NUMBER: NCT03665103. TRIAL REGISTRATION DATE: 11 September 2018. DATE OF FIRST PATIENT'S ENROLMENT: 17 September 2018.

Cytokines hold promise for human embryo culture in vitro: results of a randomized clinical trial.

OBJECTIVE: To evaluate the effects of cytokine enrichment of culture medium on embryological and clinical outcomes after intracytoplasmic sperm injection (ICSI). DESIGN: A randomized clinical trial. SETTING: In vitro fertilization centers. PATIENT(S): This trial included 443 ICSI cycles randomized into two groups. INTERVENTION(S): This study evaluated the influence of integration of granulocyte-macrophage colony-stimulating factor, heparin-binding epidermal growth factor-like growth factor, and leukemia inhibitory factor into culture media on human embryo development after ICSI. MAIN OUTCOME MEASURE(S): Ongoing pregnancy rate per a randomized participant. RESULT(S): Cytokine enrichment of culture medium showed improvement in ongoing pregnancy rate compared with no cytokines (106/224 [47%] vs. 78/219 [36%]; absolute rate difference [ARD] = 12; 95% confidence interval [CI], 2.5-21). This integration of cytokines also showed better rates of live birth (101/224 [45%] vs. 71/219 [33%]; ARD = 13; 95% CI, 4-21) and cumulative live birth (132/224 [60%] vs. 97/219 [44%]; ARD = 12; 95% CI, 4-20) and lower rate of pregnancy loss (27/124 [22%] vs. 37/103 [36%]; ARD = -14; 95% CI, -26 to -2) than conventional medium. Embryos developed in the cytokine-supplemented medium showed better blastocyst formation, quality, cryopreservation, and use than control medium. CONCLUSION(S): Integration of cytokines into human embryo culture media showed improvement in embryological and clinical outcomes after ICSI. However, the long-term effect of cytokine enrichment of a medium is still unclear and warrants further studies with longitudinal follow-up. CLINICAL TRIAL REGISTRATION NUMBER: NCT02420886 at ClinicalTrials.gov.

Human uterine leiomyoma contains low levels of 1, 25 dihdroxyvitamin D3, and shows dysregulated expression of vitamin D metabolizing enzymes.

OBJECTIVES: To evaluate tissue concentration of 1, 25 dihydroxyvitamin D3, and gene expression level of CYP27B1 that codes for 1-α hydroxylase (vitamin D activating enzyme), and CYP24A1 that codes for 24-hydroxylase (vitamin D catabolizing enzyme) in human uterine leiomyoma (ULM), its adjacent myometrium (Myo-F), and normal myometrium (Myo-N). STUDY DESIGN: Levels of 1, 25 dihydroxyvitamin D3 were measured using HPLC and Diode detectors whereas CYP27B1, and CYP24A1 expressions were assessed using Real-Time PCR in ULM, Myo-F, and Myo-N. Non-parametric statistics were used. RESULTS: ULMs contained significantly less 1, 25 dihydroxy vitamin D3 compared to Myo-F (3.0, IQR: 1.0-9.0 versus 6.0, IQR: 3.0-13.0 µg/ kg, P value is 0.03). No significant difference was detected between ULM and Myo-N, or Myo-F and Myo-N. Intratumoral level of the active form of vitamin D did not differ according to the type of ULM (submucous or interstitial/subserous), or to the ULM volume. CYP27B1 was expressed in ULM (2.17, IQR: 0.65-4.9), Myo-F (4.94, IQR: 1.04-22.59), and Myo-N (0.99, IQR: 0.49-1.71) to a comparable level. CYP24A1 expression was significantly higher in ULM compared to Myo-N (2.00, IQR: 0.69-10.77 versus 0.22, IQR: 00- 0.96, respectively, P value is 0.04). CONCLUSIONS: Human ULMs contain significantly lower 1, 25 dihydroxyvitamin D3 than its adjacent myometrium. ULM, Myo-F, and Myo-N express CYP27B1 and CYP24A1. ULMs express significantly higher level of CYP24A1 than normal myometrium indicating that over expression of 24-hydroxylase is a mechanism by which ULMs sustain a relative state of hypovitaminosis D.

Artificial oocyte activation with SrCl2 or calcimycin after ICSI improves clinical and embryological outcomes compared with ICSI alone: results of a randomized clinical trial.

STUDY QUESTION: Are pregnancy and birth rates affected by artificial oocyte activation (AOA) with SrCl2 or calcimycin after ICSI for couples with male-factor infertility linked to abnormal sperm morphology or for couples with previous ICSI cycles of unexplained low fertilization or inadequate fertilization associated with impaired oocyte morphology? SUMMARY ANSWER: AOA with either SrCl2 or calcimycin can improve the rates of clinical pregnancy, ongoing pregnancy and live birth compared with ICSI alone, and the two agents have diverse effects for different subgroups of patients. WHAT IS KNOWN ALREADY: ICSI is a successful treatment for infertility, but not in all individuals. AOA has potential to overcome inadequate fertilization in ICSI. Calcimycin and SrCl2 are candidate agents for AOA, but their effectiveness remains to be compared. STUDY DESIGN, SIZE, DURATION: This study was a randomized, open-label, three-arm, parallel-group, double-centre, superiority trial conducted between April 2015 and January 2016. The study evaluated the effects of AOA with calcimycin or SrCl2 for clinical pregnancy rates after ICSI and included 343 couples divided into three groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: Couples were included if they had two previous ICSI cycles of no or low fertilization (0-30%) with unknown causes or impaired oocyte morphology. Male-factor infertility cycles (frozen-thawed sperm, surgically retrieved sperm or ejaculates contained <10 millions spermatozoa/ml) undergoing their first ICSI attempt were also included if they had 100% abnormal sperm morphology (including globozoospermia and tapered-head). Couples were randomized to undergo ICSI with SrCl2 AOA, ICSI with calcimycin AOA or ICSI alone, with clinical pregnancy as the primary endpoint. Effect sizes were summarized as absolute rate differences (ARDs) and odds ratios (ORs), with precision evaluated by 95% CIs. MAIN RESULTS AND THE ROLE OF CHANCE: Both SrCl2 and calcimycin AOA improved clinical pregnancy rates compared to ICSI alone (49, 42 and 27%; ARD 22, 95% CI: 9-33; P = 0.0007 and ARD 16, 95% CI: 3-27; P = 0.014). SrCl2 and calcimycin AOA were also superior to ICSI alone on the rates of ongoing pregnancy (42, 36 and 23%; P = 0.0019 and P = 0.023) and live birth (40, 33 and 18%; P = 0.0002 and P = 0.012). Among couples with previous ICSI cycles of low fertilization, AOA with SrCl2 (but not with calcimycin) was superior to ICSI alone for rates of clinical pregnancy (ARD 35 percentage points (pp), P = 0.0007), ongoing pregnancy (ARD 27 pp, P = 0.009) and live birth (ARD 37 pp, P = 0.002). Among couples affected by male-factor infertility, AOA with calcimycin (but not with SrCl2) was superior to ICSI alone for rates of clinical pregnancy (ARD 22 pp, P = 0.006), ongoing pregnancy (ARD 19 pp, P = 0.013) and live birth (ARD 17 pp, P = 0.02). LIMITATIONS, REASONS FOR CAUTION: This study was an open-label trial, and this design might have introduced bias, although randomization methods were used. The study did not include a longitudinal follow-up, so further evidence is required to demonstrate the safety of AOA. WIDER IMPLICATIONS OF THE FINDINGS: The decision to use SrCl2 or calcimycin for AOA after ICSI may depend on whether the activation failure originates in the oocyte or the sperm. STUDY FUNDING/COMPETING INTEREST(S): The study received no funding and the authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: NCT02424214. TRIAL REGISTRATION DATE: 22 April 2015. DATE OF FIRST PATIENT'S ENROLMENT: 27 April 2015.

Soluble tumor necrosis factor-alpha receptors in the serum of endometriosis patients.

INTRODUCTION: We examine serum levels sTNFR-I and sTNFR-II in endometriosis patients, and their role as biomarkers of endometriosis. MATERIAL AND METHODS: Women were diagnosed with endometriosis during laparoscopy to investigate pelvic pain and/or infertility (N=62). Control group included women with pelvic pain and/or infertility, whose laparoscopy showed no abnormalities (N=55). Serum concentrations of sTNFR-I and sTNFR-II were measured using Bioplex Protein Array system. Non-parametric statistics were used. RESULTS: Endometriosis patients had significantly higher levels of sTNFR-I than controls (257.46pg/ml, IQR=2.37-1048.92 versus 130.39pg/ml, IQR=0.99-361.1 respectively, P value=0.01). For TNFR-II, difference between women with (232pg/ml, IQR=0.0-624.4), and women without (132.93pg/ml, IQR=0.0-312.81) endometriosis was not significant (P value=0.05). Early stage endometriosis patients had significantly higher level of sTNFR-I (559.13, IQR=1.82-1289.86) and sTNFR-II (248.8, IQR=0-644.65) than control women (P value is 0.01 for TNFR-I and 0.04 for TNFR-II). Levels of sTNFR-I and sTNFR-II were comparable for advanced endometriosis and controls, and between early and advanced endometriosis. As a biomarker for all- stage endometriosis, sTNFR-I produces AUC of 0.62, sensitivity of 61%, and specificity of 47.3%, at a cutoff of 81.87pg/ml. For early stage disease, sTNFR-I yields AUC of 0.68, sensitivity of 60.7%, specificity of 75%, at a cutoff of 351.22pg/ml. CONCLUSION: sTNFR-I is significantly higher in serum of endometriosis patients than controls. As an endometriosis biomarker, sTNFR-I achieves better performance for early stage disease.

Enhancing Adenoviral-Mediated Gene Transfer and Expression to Endometrial Cells.

OBJECTIVE: Our aim was to screen a panel of modified adenoviral gene transfer vectors to identify those which can sustain high gene expression in human endometrial cells. METHODS: Normal endometrial stromal cell cultures were established from endometrial lining of hysterectomy specimens performed for benign gynecologic indications. Human endometrial stromal cells were transfected by modified adenoviruses expressing luciferase reporter gene. Luciferase activity mediated by each virus was expressed as a percentage of adenovirus serotype 5 (Ad5-CMV-luc) activity. The 2-tailed Student t test was used to compare data. RESULTS: At a multiplicity of infection (MOI) of 10 pfu/cell, of the transductionally modified adenoviruses, adenovirus-RGD (Ad-RGD-luc) mediated highest level of endometrial cell transduction with transgene expression around 4 times higher when compared to Ad5 (P < .001). Of the transcriptionally targeted adenoviruses, adenovirus under secretory leukocyte protease inhibitor promoter (Ad-SLPI-luc) and adenovirus under heparanase promoter (Ad-heparanase-luc)-mediated luciferase activation were 5.8- and 4.3-folds higher than Ad5-CMV-luc, respectively (P = .02 and .03, respectively). At MOI of 50 pfu/cell, Ad-RGD-luc and AD-SLPI-luc mediated significantly higher gene transfer efficiency compared to Ad5-CMV-luc (P values < .001, for each virus). Ad-heparanase-luc achieved higher gene activity, but difference was not significant (P = .1). Ad-SLPI-luc, at low viral dose (10 pfu/ cell), mediated gene expression effect comparable to Ad5-CMV-luc at a high dose (50 pfu/cell), with no significant difference. CONCLUSIONS: We conclude that when compared to the wild-type adenovirus, Ad-RGD-luc, Ad-SLPI-luc, and Ad-heparanase-luc mediate higher reporter gene activity in endometrial cells and can work as effective gene transfer vectors in gene therapy applications to the endometrium.

Expulsion of Nova-T380, Multiload 375, and Copper-T380A contraceptive devices inserted during cesarean delivery.

OBJECTIVE: To compare the expulsion rate of Nova-T380, Multiload 375, and Copper-T380A intrauterine contraceptive devices (IUCDs) inserted during cesarean delivery. METHODS: A comparative randomized study was conducted between January 1, 2013, and June 30, 2014, in three maternity centers in Egypt and Saudi Arabia. All women scheduled for an elective cesarean and accepting intraoperative insertion of an IUCD were randomly allocated to receive the Nova-T380 (group 1), Multiload 375 (group 2), or Cu-T380A (group 3) using a computer-generated table. Researchers and participants were not masked to the type of IUCD. Follow-up was for 1 year. The primary outcome was IUCD expulsion (complete or partial [i.e. displacement]). RESULTS: Each group contained 40 participants. At 1 year, expulsion had been reported for 5 (13%) women in group 1, 2 (5%) in group 2, and 6 (15%) in group 3 (P>0.05 for all). The frequency of displacement was significantly lower in group 2 (5 [13%] participants) than in group 1 (15 [38%]; P=0.001) and group 3 (14 [35%]; P=0.008). CONCLUSION: Despite a comparable risk of expulsion following IUCD insertion during cesarean delivery, the Multiload 375 device showed the lowest risk of displacement.

Modified cervicopexy: a novel, less-invasive technique for Stages III and IV uterine prolapse.

OBJECTIVE: To evaluate modified anterior abdominal wall cervicopexy (AWC) as a less invasive (via 3-cm minilaparotomy) and more augmented (via securing posterior vaginal wall to uterosacral ligaments) technique. METHODS: Case series of 30 women with Stages III and IV apical uterine prolapse assessed by the pelvic organ prolapse quantification system. RESULTS: The modified AWC procedure was performed successfully for 17 cases with Stage III uterovaginal prolapse and 13 cases with Stage IV uterovaginal prolapse. The procedure was conducted safely with no operative or postoperative complications, apart from two cases with postoperative urinary retention. Operative time ranged from 45 to 70min. Follow-up was available for 1-3 years. Overall, 27 cases were satisfied with the procedure, and three cases developed recurrence after caesarean section due to cutting the supporting sutures. CONCLUSIONS: The modified AWC procedure is less invasive, simple and effective for Stages III and IV uterine prolapse.