Deregulation of oxidative phosphorylation pathways in embryos derived in vitro from prepubertal and pubertal heifers based on whole-transcriptome sequencing.

BACKGROUND: Although, oocytes from prepubertal donors are known to be less developmentally competent than those from adult donors it does not restrain their ability to produce full-term pregnancies. The transcriptomic profile of embryos could be used as a predictor for embryo's individual developmental competence. The aim of the study was to compare transcriptomic profile of blastocysts derived from prepubertal and pubertal heifers oocytes. Bovine cumulus-oocyte complexes (COCs) were obtained by ovum pick- up method from prepubertal and pubertal heifers. After in vitro maturation COCs were fertilized and cultured to the blastocyst stage. Total RNA was isolated from both groups of blastocysts and RNA-seq was performed. Gene ontology analysis was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery). RESULTS: A higher average blastocyst rate was obtained in the pubertal than in the pre-pubertal group. There were no differences in the quality of blastocysts between the examined groups. We identified 436 differentially expressed genes (DEGs) between blastocysts derived from researched groups, of which 247 DEGs were downregulated in blastocysts derived from pubertal compared to prepubertal heifers oocytes, and 189 DEGs were upregulated. The genes involved in mitochondrial function, including oxidative phosphorylation (OXPHOS) were found to be different in studied groups using Kyoto Encyclopedia of Genes (KEGG) pathway analysis and 8 of those DEGs were upregulated and 1 was downregulated in blastocysts derived from pubertal compared to prepubertal heifers oocytes. DEGs associated with mitochondrial function were found: ATP synthases (ATP5MF-ATP synthase membrane subunit f, ATP5PD- ATP synthase peripheral stalk subunit d, ATP12A- ATPase H+/K + transporting non-gastric alpha2 subunit), NADH dehydrogenases (NDUFS3- NADH: ubiquinone oxidoreductase subunit core subunit S3, NDUFA13- NADH: ubiquinone oxidoreductase subunit A13, NDUFA3- NADH: ubiquinone oxidoreductase subunit A3), cytochrome c oxidase (COX17), cytochrome c somatic (CYCS) and ubiquinol cytochrome c reductase core protein 1 (UQCRC1). We found lower number of apoptotic cells in blastocysts derived from oocytes collected from prepubertal than those obtained from pubertal donors. CONCLUSIONS: Despite decreased expression of genes associated with OXPHOS pathway in blastocysts from prepubertal heifers oocytes, the increased level of ATP12A together with the lower number of apoptotic cells in these blastocysts might support their survival after transfer.

Routine double-ovarian-stimulation (DuoStim) in poor responders lacks rationale, evidence, and follow-up.

Double ovarian stimulation (DuoStim), initially only suggested for fertility preservation in cancer patients, is now increasingly also used in routine clinical IVF, especially in poor responders. The claimed rational for this is the alleged existence of multiple follicular waves in a single intermenstrual interval, allowing for retrieval of more oocytes in a single IVF cycle. This commentary argues that this expansion of purpose lacks rationale, evidence, and follow-up. Consequently, we suggest that, unless valid clinical indications have been established, DuoStim be only subject of controlled clinical trials with appropriate experimental consents.

Comment on 'Importance of real-time measurement of sperm head morphology in intracytoplasmic sperm injection' by Fumiaki Itoi et al.

We present a commentary on the article published in the Zygote FirstView: 'Importance of real-time measurement of sperm head morphology in intracytoplasmic sperm injection' by Fumiaki Itoi and colleagues. We comment on the importance of providing the microscope setup details whenever sperm morphology visualization is discussed. The claim of ×6000-10,000 magnification is misleading as such levels of magnification are impossible to achieve.

Inflammation-Related Molecules at the Maternal-Fetal Interface during Pregnancy and in Pathologically Altered Endometrium.

The blastocyst expresses paternally derived alloantigens and induces inflammation during implantation. However, it is necessary for the onset of pregnancy. An abnormal response might result in a pathological course of pregnancy or pregnancy failure. On the other hand, a state of maternal immune tolerance is necessary to ensure the normal development of pregnancy by suppressing inflammatory processes. This article discusses recognized mechanisms and the significance of inflammatory processes for embryo implantation and pregnancy establishment. We would also like to present disorders involving excessive inflammatory response and their influence on events occurring during embryo implantation. The chain of correlation between the processes responsible for embryo implantation and the subsequent physiological course of pregnancy is complicated. Many of those interrelationships are still yet to be discovered. Undoubtedly, their recognition will give hope to infertile couples for the emergence of new treatments that will increase the chance of giving birth to a healthy child.

Mitochondrial DNA Copy Number in Cleavage Stage Human Embryos-Impact on Infertility Outcome.

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between mitochondrial DNA (mtDNA) content of cleavage-stage preimplantation embryos and their developmental potential. A total of 69 couples underwent IVF treatment (averaged women age: 36.5, SD 4.9) and produced a total of 314 embryos. A single blastomere was biopsied from each embryo at the cleavage stage (day-3 post-fertilization) subjected to low-pass next generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number amount was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 6.3 ± 7.5 versus 7.1 ± 5.8, p < 0.004; U Mann−Whitney test), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (6.6 ± 4.8 vs. 8.5 ± 13.6, p 0.09), sex (6.6 ± 4.1 vs. 6.2 ± 6.8, p 0.16), maternal age (6.9 ± 7.8 vs. 6.7 ± 4.5, p 0.14) or its ability to implant (7.4 ± 6.6 vs. 5.1 ± 4.6, p 0.18). The mtDNA content cannot serve as a useful biomarker at this point in development. However, further studies investigating both quantitative and qualitative aspects of mtDNA are still required to fully evaluate the relationship between mitochondrial DNA and human reproduction.

Vitamin D and anti-Müllerian hormone concentration in human follicular fluid individually aspirated from all patient follicles.

OBJECTIVE: The main aim of this prospective study was to investigate the relationship between intrafollicular vitamin D and anti-Müllerian hormone (AMH) concentration and its impact on oocyte quality and developmental competence. METHODS: The analysis was performed on 208 follicular fluid (FF) samples obtained from 33 patients undergoing ovarian stimulation as part of in vitro fertilization (IVF) treatment that included intracytoplasmic sperm injection. RESULTS: Our study shows that vitamin D concentration in FF varies according to the developmental stage of the oocyte and corelates with embryo development status on day 3, while AMH concentration in FF is not correlated with the developmental potential of an oocyte. We demonstrated that the levels of vitamin D and AMH were higher in FF than in serum. Moreover we showed that AMH and vitamin D levels were positively correlated in FF but not in serum. CONCLUSION: FF-AMH levels do not appear to be a suitable as noninvasive test of the developmental potential of an oocyte, while FF-vitamin D level can be used to evaluate whether embryos obtained from particular oocytes have potential of reaching the third day of culture. However, our results encourage further research to be carried out on a larger number of patients and testing additional components found in FF such as androgens.

Does Trophectoderm Mitochondrial DNA Content Affect Embryo Developmental and Implantation Potential?

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between the mitochondrial DNA (mtDNA) content of trophectoderm and embryo developmental potential. A total of 275 couples underwent IVF treatment, producing a total of 716 embryos. The trophectoderm was biopsied from each embryo at the blastocyst stage (day 5 or day 6 post-fertilization) subjected to low-pass next-generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 1.13 ± 1.37 versus 1.45 ± 1.78, p = 0.02) and in day 5 biopsies compared to day 6 biopsies (1.41 ± 1.66 vs. 1.19 ± 1.27, p = 0.001), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (1.58 ± 2.44 vs. 2.19 ± 2.89, p = 0.12), genetic sex (1.27 ± 1.29 vs. 1.27 ± 1.18, p = 0.99), maternal age (1.31 ± 1.41 vs. 1.33 ± 1.29, p = 0.43), or its ability to implant (1.14 ± 0.88 vs. 1.21 ± 1.16, p = 0.39). mtDNA has small potential to serve as an additional, independent biomarker for embryo selection.

Compatibility of Distinct Label-Free Proteomic Workflows in Absolute Quantification of Proteins Linked to the Oocyte Quality in Human Follicular Fluid.

We present two separate label-free quantitative workflows based on different high-resolution mass spectrometers and LC setups, which are termed after the utilized instrument: Quad-Orbitrap (nano-LC) and Triple Quad-TOF (micro-LC) and their directed adaptation toward the analysis of human follicular fluid proteome. We identified about 1000 proteins in each distinct workflow using various sample preparation methods. With assistance of the Total Protein Approach, we were able to obtain absolute protein concentrations for each workflow. In a pilot study of twenty samples linked to diverse oocyte quality status from four donors, 455 and 215 proteins were quantified by the Quad-Orbitrap and Triple Quad-TOF workflows, respectively. The concentration values obtained from both workflows correlated to a significant degree. We found reasonable agreement of both workflows in protein fold changes between tested groups, resulting in unified lists of 20 and 22 proteins linked to oocyte maturity and blastocyst development, respectively. The Quad-Orbitrap workflow was best suited for an in-depth analysis without the need of extensive fractionation, especially of low abundant proteome, whereas the Triple Quad-TOF workflow allowed a more robust approach with a greater potential to increase in effectiveness with the growing number of analyzed samples after the initial effort of building a comprehensive spectral library.

Prostaglandin E2 affects in vitro maturation of bovine oocytes.

The role of prostaglandin E2 (PGE2) in the successful resumption of oocyte meiosis and cumulus expansion has been well-documented. However, there remains very little information available on the influence of PGE2 on other processes that occur during oocyte maturation. In this study, we supplemented a maturation medium with PGE2 and monitored oocyte quality markers, glucose metabolism, mitochondrial status, oxidative stress, and apoptosis in the cumulus-oocyte complexes (COCs), using a well-established in vitro model of embryo production in cattle. We found that this increased availability of PGE2 during maturation led to an increase in the expression of genes associated with oocyte competence and improved the quality of blastocysts produced. Prostaglandin E2 also appeared to stimulate glucose uptake and lactate production in the COCs, both influencing the expression of enzymes involved in glycolysis and the hexosamine biosynthetic pathway. We found that PGE2 reduced intracellular reactive oxygen species levels, and simultaneously increased glutathione concentration and stimulated antioxidant gene expression in the oocyte. These results indicate that PGE2 has an important role in the protection of oocytes against oxidative stress. Mitochondrial membrane potential was also improved in PGE2-treated oocytes, and there was a reduction in the occurrence of apoptosis in the COCs. Promotion of an anti-apoptotic balance in transcription of genes involved in apoptosis was present in both oocytes and the cumulus cells. In summary, PGE2 could represent a novel autocrine/paracrine player in the mechanisms that can facilitate successful oocyte maturation and oocyte survival in the cow.

Potential synergism between ulipristal acetate and vitamin D3 in uterine fibroid pharmacotherapy - 2 case studies.

This is a preliminary report of the first cases of successful simultaneous use of ulipristal acetate (UPA) and vitamin D3 in uterine fibroid (UF) oral treatment in humans. We present two cases of 37- and 49-year-old females with clinically symptomatic UFs and vitamin D deficiency. Both patients were treated with a standard 3 months of UPA scheme (5 mg daily) with the additional use of vitamin D3 (7000 IU daily orally). In the 37-year-old female all the symptoms (pain, pressure, frequent urination) decreased, total tumor volume after the treatment changed by 47.8%. In the 49-year-old female most symptoms perished, total tumor volume was reduced by 63.3%. UPA and vitamin D share synergistic anti-fibroid properties. Further studies are necessary to show the exact effect of UPA and vitamin D as co-drugs.

Effect of next-generation sequencing in preimplantation genetic testing on live birth ratio.

The present study analysed live birth ratios in frozen embryo transfer (FET) cycles where embryo ploidy status was determined with preimplantation genetic testing (PGT) using next-generation sequencing (NGS). PGT was performed on trophectoderm cells biopsied at the blastocyst stage. The present prospective cohort study included 112 women undergoing frozen embryo transfer, with NGS PGT. The control group consisted of 85 patients who underwent the IVF procedure with FET planned for a subsequent cycle. The live birth rate per cycle was higher by ~18.5 percentage points in the investigated compared with control group (42.0% vs 23.5% respectively; P=0.012). The differences between the study and control groups were also significant for clinical pregnancy (42.0% vs 23.5% respectively; P=0.012), implantation (41.2% vs 22.2% respectively; P=0.001) and pregnancy loss rates (9.6% vs 28.6% respectively; P=0.027). The results show that PGT NGS is a useful method for embryo selection and it may be implemented in routine clinical practice with propitious results.

Age-related decline in AMH is assay dependent limiting clinical interpretation of repeat AMH measures across the reproductive lifespan.

PURPOSE: The aim of the study was to determine whether the assays exhibit an interaction with age and exhibit heterogeneous age related declines in AMH. Apart of chronological age, AMH variation was investigated with relation to menstrual cycle day (MCD). The goal implicates two questions: Are distributions of AMH concentrations homogenous after adjustment for the specific AMH assay? Does age-assay product has an effect on AMH depletion? METHODS: The study was conducted by examining results of AMH tests performed for 12,917 women with four types of AMH assays: Immunotech I generation kit (IMI, 4016 samples), Beckman Coulter II generation kit RUO (BCII RUO, 3430 samples), Beckman Coulter II generation kit with IVD certificate (BCII IVD, 830 samples), and Ansh Labs I generation kit (AnshLabs, 4641 samples). Statistical analysis included ACNOVA and least square regression technique. RESULTS: Menstrual cycle day has no effect on AMH measurements. On the other hand, AMH values differed substantially between the four assays, with a marked discordance in the rate of age-related AMH decline for the four assays (ranging from -8.16% (95% CI: -8.79, -7.54) to -11.53% (95% CI -12.20, -10.87), with a significant interaction between age and assay. CONCLUSIONS: (1) The distribution of AMH concentration is heterogeneous after controlling the age across assays; (2) the rate of AMH decline as a function of age is different for the four manual AMH ELISA assays.

Vitamin D and Uterine Fibroids-Review of the Literature and Novel Concepts.

This article provides a detailed review of current knowledge on the role of vitamin D and its receptor in the biology and management of uterine fibroids (UFs). Authors present ideas for future steps in this area. A literature search was conducted in PubMed using the following key words: "uterine fibroid" and "vitamin D". The results of the available studies, published in English from January 2002 up to April 2018, have been discussed. Vitamin D is a group of steroid compounds with a powerful impact on many parts of the human body. This vitamin is believed to regulate cell proliferation and differentiation, inhibit angiogenesis, and stimulate apoptosis. Nowadays, hypovitaminosis D is believed to be a major risk factor in the development of UFs. In many studies vitamin D appears to be a powerful factor against UFs, resulting in inhibition of tumor cell division and a significant reduction in its size, however, the exact role of this compound and its receptor in the pathophysiology of UFs is not fully understood. According to available studies, vitamin D and its analogs seem to be promising, effective, and low-cost compounds in the management of UFs and their clinical symptoms, and the anti-tumor activities of vitamin D play an important role in UF biology. The synergy between vitamin D and selected anti-UF drugs is a very interesting issue which requires further research. Further studies about the biological effect of vitamin D on UF biology are essential. Vitamin D preparations (alone or as a co-drugs) could become new tools in the fight with UFs, with the additional beneficial pleiotropic effect.

The Role of Tumor Necrosis Factor α in the Biology of Uterine Fibroids and the Related Symptoms.

Uterine fibroids (UFs) are the most common benign tumors of the female genital tract. The incidence of UFs has been estimated at 25⁻80% depending on selected population. The pathophysiology of UFs remains poorly understood. The transformation of smooth muscle cells of the uterus into abnormal, immortal cells, capable of clonal division, is the main component of all pathways leading to UF tumor formation and tumor necrosis factor α (TNF-α) is believed to be one of the key factors in this field. TNF-α is a cell signaling protein involved in systemic inflammation and is one of the cytokines responsible for the acute phase reaction. This publication presents current data about the role of tumor necrosis factor α in the biology of UFs and the related symptoms. TNF-α is an extremely important cytokine associated with the biology of UFs, UF-related symptoms and complaints. Its concentration has been proven to be elevated in women with clinically symptomatic UFs. The presented data suggest the presence of an "inflammation-like" state in women with UFs where TNF-α is a potent inflammation inducer. The origin of numerous symptoms reported by women with UFs can be traced back to the TNF-α influence. Nevertheless, our knowledge on this subject remains limited and TNF-α dependent pathways in UF pathophysiology should be investigated further.

Qualitative and Quantitative Analysis of Proteome and Peptidome of Human Follicular Fluid Using Multiple Samples from Single Donor with LC-MS and SWATH Methodology.

Human follicular fluid (hFF) is a natural environment of oocyte maturation, and some components of hFF could be used to judge oocyte capability for fertilization and further development. In our pilot small-scale study three samples from four donors (12 samples in total) were analyzed to determine which hFF proteins/peptides could be used to differentiate individual oocytes and which are patient-specific. Ultrafiltration was used to fractionate hFF to high-molecular-weight (HMW) proteome (>10 kDa) and low-molecular-weight (LMW) peptidome (<10 kDa) fractions. HMW and LMW compositions were analyzed using LC-MS in SWATH data acquisition and processing methodology. In total we were able to identify 158 proteins, from which 59 were never reported before as hFF components. 55 (45 not reported before) proteins were found by analyzing LMW fraction, 67 (14 not reported before) were found by analyzing HMW fraction, and 36 were identified in both fractions of hFF. We were able to perform quantitative analysis for 72 proteins from HMW fraction of hFF. We found that concentrations of 11 proteins varied substantially among hFF samples from single donors, and those proteins are promising targets to identify biomarkers useful in oocyte quality assessment.

Non-equivalence of anti-Müllerian hormone automated assays-clinical implications for use as a companion diagnostic for individualised gonadotrophin dosing.

STUDY QUESTION: Can anti-Müllerian hormone (AMH) automated immunoassays (Elecsys® and Access) be used interchangeably as a companion diagnostic for individualisation of follitropin delta dosing? SUMMARY ANSWER: The Access assay gives systematically higher AMH values than the Elecsys® assay which results in over 29% of women being misclassified to a different follitropin delta dose. WHAT IS KNOWN ALREADY: Follitropin delta is the first gonadotrophin to be licenced with a companion diagnostic, the Roche Elecsys® AMH Plus assay. Alternative automated AMH assays including the Beckman Coulter Access immunoassay are considered to provide similar results, but clarification of their suitability as an off-licence companion diagnostic for follitropin delta is required. STUDY DESIGN, SIZE, DURATION: We systematically searched the existing literature for studies that had measured AMH using both automated assays in the same cohort of women. Individual paired patient data were acquired from each author and combined with unpublished data. PARTICIPANTS/MATERIALS, SETTING, METHODS: We identified five eligible prospective published studies and one additional unpublished study. A 100% response from the authors was achieved. We collected paired AMH data on samples from 848 women. Passing-Bablok regression and Bland-Altman plots were used to compare the analytical performance of the two assays. The degree of misclassification to different treatment categories was estimated should the Access AMH be used as a companion diagnostic instead of the Elecsys AMH in determining the dosing of follitropin delta. MAIN RESULTS AND THE ROLE OF CHANCE: The Passing-Bablok regression shows a linear relationship (Access = -0.05 + 1.10 × Elecsys). The Access assay systematically gave higher values by an average of 10% compared with the Elecsys assay (slope = 1.10, 95% CI: 1.09 to 1.12). The average of the difference between the two assays was 2.7 pmol/l. The 95% limits of agreement were -11.7 to 6.3. Overall 253 (29.3%) women would have received an inappropriate follitropin delta dose if the Beckman Coulter Access assay was used. Specifically, a substantial proportion of women (ranging from 49% to 90% depending on the AMH category) would receive a lower dose of follitropin delta based on the Access AMH assay. Up to 10% (ranging from 2.5% to 10%) of women with high ovarian reserve would have been misclassified to a greater dose of follitropin delta based on the Access AMH assay. LIMITATIONS REASONS FOR CAUTION: We compared the values of the two principal automated assays, extrapolation of our findings to other automated AMH assays would require similar comprehensive examination. WIDER IMPLICATIONS OF THE FINDINGS: An international standard for the calibration of the automated AMH assays is warranted to facilitate efficient use of AMH as a companion diagnostic. The variable calibration of alternative automated AMH assays may adversely impact on the performance of the follitropin delta dosing algorithm. STUDY FUNDING/COMPETING INTEREST(S): No formal funding has been received for this study. SI is funded by a UK Medical Research Council skills development fellowship (MR/N015177/1). SMN has received speakers fees, travel to meetings and participated in advisory Boards for Beckman Coulter, IBSA, Ferring Pharmaecuticals, Finox, Merck Serono, Merck and Roche Diagnostics. SMN has received research support from Ansh laboratories, Beckman Coulter, Ferring Pharmaceuticals and Roche Diagnostics. TRIAL REGISTRATION NUMBER: N/A.

Clinical utility of different anti-Müllerian hormone - AMH assays for the purpose of pregnancy prediction.

PURPOSE: Comparison of outcomes of IVF cycles where the AMH levels was measured with five different AMH kits: Immunotech (IOT), Beckman Coulter II Gen. RUO, Beckman Coulter II Gen. IVD (BC II IVD), Ansh Labs ultrasensitive (Ansh) and the automated Elecsys Roche assay. METHODS: Retrospective analysis of clinical data for 3693 cycles. RESULTS: In women < 35 years with low (<0.6 ng/ml) and high (>1.4 ng/ml) AMH concentrations, and in those > 39 years with medium (≥0.6 and ≤1.4 ng/ml) and high AMH concentrations the clinical pregnancy rate differed significantly among groups of patients whose AMH level was measured with different kits. In those subgroups, the highest rates were recorded for the BC II IVD and Ansh groups, while the lowest in the IOT group. AMH concentrations differed significantly between different kits in all age groups (the highest in each age group was for the IOT kit and the lowest for BC II IVD). AMH correlates positively with antral follicle count, MII and number of oocytes retrieved. CONCLUSIONS: This study demonstrated that we could expect very different pregnancy rates with the same AMH results depending on the AMH kit used. That would means, different values of AMH could similarly lead to misleading clinical decisions in IVF.

New AMH assay allows rapid point of care measurements of ovarian reserve.

In this study, we compare two commercial automated immunoassays used to evaluate serum anti-Müllerian hormone (AMH) levels as a prognostic value for ovarian response and pregnancy outcome in assisted reproductive technology cycles. Serum AMH was measured for 193 women. We performed a simultaneous measurement in serum AMH with the two alternative kits VIDAS® and Elecsys® AMH assay. For all women undergoing in vitro fertilization cycle, we collected data on their antral follicle count (AFC) and numbers of retrieved cumulus oocyte complexes (OC) and metaphase II oocytes and pregnancy outcome. The AMH values provided by VIDAS® were correlated with the values obtained with Elecsys® (0.977 for fresh and 0.971 for the frozen samples). For both assays AMH exhibited a moderate positive correlation with AFC, OC and MII oocytes (0.612, 0.674, 0.605 for VIDAS® and 0.570, 0.617, 0.530 for Elecsys®, respectively). AMH prediction of biochemical and clinical pregnancy was similar. The present results suggest that the VIDAS® AMH assay is broadly comparable to the Elecsys-AMH assay in terms of technical performance for clinical or epidemiological use. Both automated assays performed in a similar way and the choice of assay can be made depending on the technical configuration of each laboratory.

Alternative Oral Agents in Prophylaxis and Therapy of Uterine Fibroids-An Up-to-Date Review.

Uterine fibroids (UFs) are the most common tumors of the female genital tract. The effect of UFs on the quality of life and the overall cost of treatment are significant issues worldwide. Tumor size and location are the two specific factors which influence the occurrence of symptoms, the need for, and method of, treatment (some tumors require surgery while some can be treated with selected drugs). Primary prevention and treatment of early UF disease are worthy goals that might have a great impact on health care systems. Several treatments and prophylactic methods can be used in this endeavor. This publication presents current data about lesser-known substances which may have a beneficial effect on the treatment or prophylaxis of UFs and can be administered orally, serving as an alternative to (or complement of) surgery or selective progesterone receptor modulators (SPRMs). Early prevention and treatment of UFs in women from high-risk groups should be our priority. Innovative forms of UF management are under intensive investigation and may be promising options in the near future. Many of them evaluated vitamin D, paricalcitol, epigallocatechin gallate (EGCG), elagolix, aromatase inhibitors (AIs), and cabergoline and deemed them to be safe and effective. The next step in such projects should be properly constructed randomized control trials (RCTs), carried out by successive phases.