Single oral dose of vitamin D3 supplementation prior to in vitro fertilization and embryo transfer in normal weight women: the SUNDRO randomized controlled trial.

BACKGROUND: Improving in vitro fertilization success is an unmet need. Observational studies have suggested that women with deficient or insufficient vitamin D have lower chances of in vitro fertilization success, but whether supplementation improves clinical pregnancy rate remains unclear. OBJECTIVE: This study aimed to determine whether oral vitamin D3 supplementation improves clinical pregnancy in women undergoing an in vitro fertilization cycle. STUDY DESIGN: The "supplementation of vitamin D and reproductive outcome" trial is a 2-center randomized superiority double-blind placebo-controlled trial. Subjects were recruited between October 2016 and January 2019. Participants were women aged 18 to 39 years with low vitamin D (peripheral 25-hydroxyvitamin D of <30 ng/mL), serum calcium of ≥10.6 mg/dL, body mass index of 18 to 25 kg/m2, and antimüllerian hormone levels of >0.5 ng/mL and starting their first, second, or third treatment cycle of conventional in vitro fertilization or intracytoplasmic sperm injection. The primary outcome was the cumulative clinical pregnancy rate per cycle. Pregnancies obtained with both fresh or frozen embryo transfers were included. Clinical pregnancy was defined as an intrauterine gestational sac with a viable fetus. The primary analysis was performed according to the intention-to-treat principle and could also include natural conceptions. Secondary outcomes included total dose of gonadotropins used, embryologic variables (number of oocytes retrieved, number of suitable oocytes retrieved, fertilization rate, and rate of top-quality embryos), and clinical outcomes (miscarriage rate and live birth rate). RESULTS: Overall, 630 women were randomized 2 to 12 weeks before the initiation of the in vitro fertilization cycle to receive either a single dose of 600,000 IU of vitamin D3 (n=308) or placebo (n=322). Interestingly, 113 (37%) and 130 (40%) women achieved a clinical pregnancy in the treatment and placebo groups, respectively (P=.37). The risk ratio of clinical pregnancy in women receiving vitamin D3 was 0.91 (95% confidence interval, 0.75-1.11). Compared with the placebo, vitamin D3 supplementation did not improve the rate of clinical pregnancy. Exploratory subgroup analyses for body mass index, age, indication to in vitro fertilization, ovarian reserve, interval between drug administration and initiation of the cycle, and basal levels of 25-hydroxyvitamin D failed to highlight any clinical situation that could benefit from the supplementation. CONCLUSION: In women with normal weight with preserved ovarian reserve and low vitamin D levels undergoing in vitro fertilization cycles, a single oral dose of 600,000 IU of vitamin D3 did not improve the rate of clinical pregnancy. Although the findings do not support the use of vitamin D3 supplementation to improve in vitro fertilization success rates, further studies are required to rule out milder but potentially interesting benefits and explore the effectiveness of alternative modalities of supplementation.

Oral Vitamin D supplementation impacts gene expression in granulosa cells in women undergoing IVF.

STUDY QUESTION: Does oral Vitamin D supplementation alter the hormonal milieu of follicular fluid (FF) and the transcriptomic profile of luteinised granulosa cells (GCs) in women with Vitamin D deficiency undergoing IVF? SUMMARY ANSWER: A transcriptomic signature relevant to oral Vitamin D supplementation in luteinised GCs was demonstrated, although Vitamin D supplementation did not alter hormone levels in FF. WHAT IS KNOWN ALREADY: Vitamin D deficiency is linked to lower live birth rates among women undergoing IVF. It is unclear whether Vitamin D elicits a targeted action in reproductive physiology or is a surrogate marker of overall well-being. Several in-vitro studies, but none in vivo, have examined the impact of Vitamin D on the periovulatory follicle, focusing on GCs as a proxy marker of oocyte competence. STUDY DESIGN, SIZE, DURATION: We present a report of secondary outcomes from the SUNDRO clinical trial, which was launched in 2016 to determine whether Vitamin D supplementation can improve the IVF outcomes of women who are deficient in Vitamin D (<30 ng/ml). FF samples of 145 women who were randomised to receive Vitamin D or placebo from March 2017 to January 2019 were collected. All follicles that were aspirated in our study measured ≥11 mm on the day of hCG trigger. The first cohort of samples was collected from the dominant follicle of each participant and utilised for hormone profiling (n = 50 Vitamin D, n = 45 Placebo). For the second cohort, the follicle aspirates of each participant were pooled to create a single FF sample, which was used for the isolation of GCs for gene expression studies (n = 20 Vitamin D, n = 30 placebo). Six of the samples from the second cohort were used for RNA-sequencing analysis (n = 3 Vitamin D, n = 3 placebo). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two academic infertility units were involved in the recruitment of the participants, who received a single dose of oral 25-hydroxyvitamin D (600 000 IU) or placebo, 2-12 weeks before oocyte retrieval. Women in both groups were deficient in Vitamin D, aged 18-39 years with a normal BMI (18-25 kg/m2) and <3 previous IVF cycles. The FF was aspirated at the time of oocyte retrieval and stored. Liquid chromatography tandem mass spectrometry was used to measure FF abundance of 25-hydroxyvitamin D, aldosterone, androstenedione, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, oestradiol (E2), 17-OH-hydroxyprogesterone, progesterone (P4) and testosterone. GCs were isolated from pooled FFs and the transcriptome was evaluated by RNA-sequencing and RT-PCR. Ingenuity pathway analysis (IPA) was used to assess the top canonical pathways and upstream regulators mediating the action of Vitamin D. MAIN RESULTS AND THE ROLE OF CHANCE: At oocyte retrieval, FF concentration of 25-hydroxyvitamin D was 2.8-fold higher (P < 0.001) in the Vitamin D group (39.5 ng/ml; n = 50) compared to placebo (13.8 ng/ml; n = 45) but no other hormonal differences were detected. In the placebo group, but not the Vitamin D group, weak correlations of 25-hydroxyvitamin D concentration with P4 (r = 0.31, P = 0.03) and E2 (r = 0.45, P = 0.002) were observed. RNA-sequencing identified 44 differentially expressed genes in the GCs of patients who received Vitamin D (n = 3) compared to placebo (n = 3). RT-PCR demonstrated upregulation of VDR (vitamin D receptor), GSTA3 (glutathione S-transferase A3) and IL21R (interleukin 21 receptor), and downregulation of P T GS2 (prostaglandin-endoperoxide synthase 2), KLF4 (kruppel-like factor 4), T RP C4 (transient receptor potential cation channel subfamily C member 4), VEGF (vascular endothelial growth factor), RXRB (retinoid X receptor beta) and AGER (advanced glycosylation end-product specific receptor) genes in the Vitamin D (n = 17) versus placebo (n = 27) group. IPA suggested roles of Vitamin D in antioxidant defence. LIMITATIONS, REASONS FOR CAUTION: Interpretation of the data is influenced by our intervention strategy (2-12 weeks prior to retrieval). As folliculogenesis may last 5-6 months, our protocol can only examine with confidence the impact of Vitamin D on the final stages of follicular growth. Furthermore, we examined the hormonal profile of the dominant follicle only, while the GC data reflect the transcriptome of all (pooled) follicles large enough to be used for IVF. Luteinised GCs from controlled ovarian stimulation were used in this study, which may be functionally distinct from the GCs of developing follicles. Moreover, the sample size for RNA-sequencing analysis was low (n = 3 per group), regardless of validation by RT-PCR that was performed on a larger cohort, introducing complexity to the IPA analysis, which required an input of data with P-adjusted <0.08 instead of <0.05 to be informative. WIDER IMPLICATIONS OF THE FINDINGS: This is the first in-vivo study to show that Vitamin D supplementation alters gene expression in luteinised GCs. In contrast to some in-vitro evidence, no effect of the intervention on expression of genes encoding steroidogenic enzymes was observed. Unlike other studies, our results suggest that supplementation with Vitamin D is unlikely to directly influence hormone availability in FF. Our findings instead reinforce the hypothesis that Vitamin D could be considered one of the gatekeepers in protecting against an exaggerated response to ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The study has been funded by the Italian Ministry of Health (RF-2013-02358757) following peer review in the competitive 'Bando di Ricerca Finalizzata e Giovani Ricercatori 2013' for the clinical trial SUNDRO (EudraCT registration number 2015-004233-27). There are no competing interests. TRIAL REGISTRATION NUMBER: EudraCT registration number 2015-004233-27.

Effect of vitamin D supplementation on assisted reproduction technology (ART) outcomes and underlying biological mechanisms: protocol of a randomized clinical controlled trial. The "supplementation of vitamin D and reproductive outcome" (SUNDRO) study.

BACKGROUND: Vitamin D plays an important role in human physiology and pathology. The receptor for vitamin D regulates 0.5-5% of the human genome. Accordingly, vitamin D insufficiency has been shown to increase the risk of several diseases. In recent years, based on growing evidence, on a role of vitamin D has been also postulated in reproductive health both in animals and humans, especially in female fertility female fertility. In vitro fertilization success was shown to be higher in women with appropriate reserves of vitamin D. However a causal relation has not been demonstrated and randomized controlled trials testing the effectiveness of vitamin D supplementation in IVF are warranted. METHODS: This is a multicenter randomized double blinded placebo controlled study aimed at determining the benefits of vitamin D [25(OH)D] supplementation in improving clinical pregnancy rate in women undergoing IVF. Eligible women with a serum level of 25-hydroxyvitamin D [25(OH)D] < 30 ng/ml will be randomized. Recruited women will be given the drug (either 600,000 IU of 25(OH) D or placebo in a single oral administration) at the time of randomization. Two centres will participate and the sample size (700 women) is foreseen to be equally distributed between the two. Patients will be treated according to standard IVF protocols. DISCUSSION: The primary aim of the study is the cumulative clinical pregnancy rate per oocyte retrieval. Clinical pregnancy is defined as the presence of at least one intrauterine gestational sac with viable foetus at first ultrasound assessment (3 weeks after a positive human chorionic gonadotropin [hCG] assessment). Secondary outcomes include: 1) clinical and embryological variables; 2) oocyte and endometrium quality at a molecular level. To investigate this latter aspect, samples of cumulus cells, follicular and endometrial fluids will be obtained from a subgroup of 50 age-matched good-prognosis cases and controls. TRIAL REGISTRATION: The protocol was included in EudraCT on 22nd September 2015 with the registration number assigned ' 2015-004233-27 '; it was submitted through the database of the Italian "Osservatorio Nazionale della Sperimentazione Clinica (OsSC)" - (National Monitoring Centre of Clinical Trials) to the National Competent Authority on 8th March 2016 and approved on 23rd June 2016.

Discovery of a benzothiophene-flavonol halting miltefosine and antimonial drug resistance in Leishmania parasites through the application of medicinal chemistry, screening and genomics.

Leishmaniasis, a major health problem worldwide, has a limited arsenal of drugs for its control. The appearance of resistance to first- and second-line anti-leishmanial drugs confirms the need to develop new and less toxic drugs that overcome spontaneous resistance. In the present study, we report the design and synthesis of a novel library of 38 flavonol-like compounds and their evaluation in a panel of assays encompassing parasite killing, pharmacokinetics, genomics and ADME-Toxicity resulting in the progression of a compound in the drug discovery value chain. Compound 19, 2-(benzo[b]thiophen-3-yl)-3-hydroxy-6-methoxy-4H-chromen-4-one, exhibited a broad-spectrum activity against Leishmania spp. (EC50 1.9 µM for Leishmania infantum, 3.4 µM for L. donovani, 6.7 µM for L. major), Trypanosoma cruzi (EC50 7.5 µM) and T. brucei (EC50 0.8 µM). Focusing on anti-Leishmania activity, compound 19 challenge in vitro did not select for resistance markers in L. donovani, while a Cos-Seq screening for dominant resistance genes identified a gene locus on chromosome 36 that became ineffective at concentrations beyond EC50. Thus, compound 19 is a promising scaffold to tackle drug resistance in Leishmania infection. In vivo pharmacokinetic studies indicated that compound 19 has a long half-life (intravenous (IV): 63.2 h; per os (PO): 46.9 h) with an acceptable ADME-Toxicity profile. When tested in Leishmania infected hamsters, no toxicity and limited efficacy were observed. Low solubility and degradation were investigated spectroscopically as possible causes for the sub-optimal pharmacokinetic properties. Compound 19 resulted a specific compound based on the screening against a protein set, following the intrinsic fluorescence changes.

Inside the biochemical pathways of thymidylate synthase perturbed by anticancer drugs: Novel strategies to overcome cancer chemoresistance.

Our current understanding of the mechanisms of action of antitumor agents and the precise mechanisms underlying drug resistance is that these two processes are directly linked. Moreover, it is often possible to delineate chemoresistance mechanisms based on the specific mechanism of action of a given anticancer drug. A more holistic approach to the chemoresistance problem suggests that entire metabolic pathways, rather than single enzyme targets may better explain and educate us about the complexity of the cellular responses upon cytotoxic drug administration. Drugs, which target thymidylate synthase and folate-dependent enzymes, represent an important therapeutic arm in the treatment of various human malignancies. However, prolonged patient treatment often provokes drug resistance phenomena that render the chemotherapeutic treatment highly ineffective. Hence, strategies to overcome drug resistance are primarily designed to achieve either enhanced intracellular drug accumulation, to avoid the upregulation of folate-dependent enzymes, and to circumvent the impairment of DNA repair enzymes which are also responsible for cross-resistance to various anticancer drugs. The current clinical practice based on drug combination therapeutic regimens represents the most effective approach to counteract drug resistance. In the current paper, we review the molecular aspects of the activity of TS-targeting drugs and describe how such mechanisms are related to the emergence of clinical drug resistance. We also discuss the current possibilities to overcome drug resistance by using a molecular mechanistic approach based on medicinal chemistry methods focusing on rational structural modifications of novel antitumor agents. This paper also focuses on the importance of the modulation of metabolic pathways upon drug administration, their analysis and the assessment of their putative roles in the networks involved using a meta-analysis approach. The present review describes the main pathways that are modulated by TS-targeting anticancer drugs starting from the description of the normal functioning of the folate metabolic pathway, through the protein modulation occurring upon drug delivery to cultured tumor cells as well as cancer patients, finally describing how the pathways are modulated by drug resistance development. The data collected are then analyzed using network/netwire connecting methods in order to provide a wider view of the pathways involved and of the importance of such information in identifying additional proteins that could serve as novel druggable targets for efficacious cancer therapy.

The Hippo Pathway and YAP/TAZ-TEAD Protein-Protein Interaction as Targets for Regenerative Medicine and Cancer Treatment.

The Hippo pathway is an important organ size control signaling network and the major regulatory mechanism of cell-contact inhibition. Yes associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are its targets and terminal effectors: inhibition of the pathway promotes YAP/TAZ translocation to the nucleus, where they interact with transcriptional enhancer associate domain (TEAD) transcription factors and coactivate the expression of target genes, promoting cell proliferation. Defects in the pathway can result in overgrowth phenotypes due to deregulation of stem-cell proliferation and apoptosis; members of the pathway are directly involved in cancer development. The pharmacological regulation of the pathway might be useful in cancer prevention, treatment, and regenerative medicine applications; currently, a few compounds can selectively modulate the pathway. In this review, we present an overview of the Hippo pathway, the sequence and structural analysis of YAP/TAZ, the known pharmacological modulators of the pathway, especially those targeting YAP/TAZ-TEAD interaction.

Vitamin D deficiency and infertility: insights from in vitro fertilization cycles.

CONTEXT: Vitamin D deficiency has been proven to affect fertility in mammals, but data in human is less convincing. In particular, data on in vitro fertilization (IVF), an attractive model to draw information on this topic, are sparse and conflicting. OBJECTIVE: Our objective was to investigate IVF outcome in women with deficient 25-hydroxy-vitamin D [25(OH)D] serum levels (<20 ng/mL). DESIGN AND SETTING: This prospective cross-sectional study was conducted at the infertility unit of an academic setting. PATIENTS: The main inclusion criteria were as follows: (1) indication to IVF, (2) age 18-42 years, (3) BMI 18-25 kg/m(2), (4) adequate ovarian reserve according to Bologna criteria. Eligible women provided a serum sample for 25(OH)D measurement at the time of cycle preparation. Subjects were subsequently excluded if the cycle was cancelled or if the attempt was excessively delayed. INTERVENTION: Quantitative detection of serum 25(OH)D. MAIN OUTCOME MEASURE: Clinical pregnancy rate. RESULTS: The number of recruited women with serum 25(OH)D <20 ng/mL and ≥ 20 ng/mL was 154 and 181, respectively. The clinical pregnancy rates were 20% (30/154) and 31% (56/181), respectively (P = .02); the adjusted odds ratio for clinical pregnancy in women with vitamin D ≥ 20 ng/mL was 2.15 (95% CI: 1.23-3.77). Subgroup analyses showed that the group of women with the highest serum levels (>30 ng/mL) had the highest chances of pregnancy. CONCLUSIONS: Vitamin D is an emerging factor influencing female fertility and IVF outcome. Additional studies are pressingly needed to confirm a causal relationship and to investigate the potential therapeutic benefits of vitamin D supplementation.

Modulation of the expression of folate cycle enzymes and polyamine metabolism by berberine in cisplatin-sensitive and -resistant human ovarian cancer cells.

Berberine is a natural isoquinoline alkaloid with significant antitumor activity against many types of cancer cells, including ovarian tumors. This study investigated the molecular mechanisms by which berberine differently affects cell growth of cisplatin (cDDP)-sensitive and -resistant and polyamine analogue cross-resistant human ovarian cancer cells. The results show that berberine suppresses the growth of cDDP-resistant cells more than the sensitive counterparts, by interfering with the expression of folate cycle enzymes, dihydrofolate reductase (DHFR) and thymidylate synthase (TS). In addition, the impairment of the folate cycle also seems partly ascribable to a reduced accumulation of folate, a vitamin which plays an essential role in the biosynthesis of nucleic acids and amino acids. This effect was observed in both lines, but especially in the resistant cells, correlating again with the reduced tolerance to this isoquinoline alkaloid. The data also indicate that berberine inhibits cellular growth by affecting polyamine metabolism, in particular through the upregulation of the key catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT). In this regard, berberine is shown to stimulate the SSAT induction by the spermine analogue N1, N12 bisethylspermine (BESpm), which alone was also able to downregulate DHFR mRNA more than TS mRNA. We report that the sensitivity of resistant cells to cisplatin or to BESpm is reverted to the levels of sensitive cells by the co-treatment with berberine. These data confirm the intimate inter-relationships between folate cycle and polyamine pathways and suggest that this isoquinoline plant alkaloid could be a useful adjuvant therapeutic agent in the treatment of ovarian carcinoma.

Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development.

Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, we combined a rapid-screening strategy using a folate-based library with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determined selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivatives, and nine compounds showed greater activity against parasite enzymes compared with human enzymes. Compound 6a displayed a K(i) of 100 nM toward LmPTR1, and the crystal structure of the LmPTR1:NADPH:6a ternary complex revealed a substrate-like binding mode distinct from that previously observed for similar compounds. A second round of design, synthesis, and assay produced a compound (6b) with a significantly improved K(i) (37 nM) against LmPTR1, and the structure of this complex was also determined. Biological evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T. cruzi. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs.

Synthesis of N-(5,7-diamino-3-phenyl-quinoxalin-2-yl)-3,4,5-substituted anilines and N-[4[(5,7-diamino-3-phenylquinoxalin-2-yl)amino]benzoyl]-l-glutamic acid diethyl ester: evaluation of in vitro anti-cancer and anti-folate activities.

Several diamino quinoxalines were designed, synthesized and evaluated as anti-tumor agents. Two compounds showed the most potent cytotoxic activities against the leukemia CCRF-CEM cell line (GI(50)<0.01microM) and the ovarian cancer cell line OVCAR-4 (GI(50)=0.03microM), respectively, with comparable/better activities than Methotrexate (MTX). Docking calculations of the complexes of hDHFR with the most active compounds identified the binding mode of the described molecules with respect to MTX.