Studies

#MaleHypofertility, #Homocysteine, #MTHFRSNP, #5MTHF, #OneCarbonCycle, #Folates

Clement 2023 – 5MTHF supplementation for HHcy

“Hyperhomocysteinemia in hypofertile male patients can be alleviated by supplementation with 5MTHF associated with one carbon cycle support”

Authors: A. Clement, E. Amar, P. Clement, É. Sedbon, C. Brami, S. Alvarez and Y. Menezo

Capsule: Homocysteine is known to be detrimental also to male infertility and a high rate of infertile male has high homocysteine. A large group of infertile males were tested for homocysteine before and after supplementation with Impryl. As seen in PCOS women (See Schiuma 2020) also in infertile men Impryl decreased homocysteine in all the treated patients, including those starting with a normal value. Many of the patients conceived after the treatment. Homocysteine testing and treatment is worth to consider in men suffering from infertility.

Clement 2023 – 5MTHF supplementation for HHcy

Abstract

Purpose:
Homocysteine (Hcy) is a cellular poison, side product of the
hydrolysis of S-Adenosyl Homocysteine, produced after the universal
methylation effector S -Adenosylmethionine liberates a methyl group to recipient targets. It inhibits the methylation processes and its rising is associated with multiple disease states and ultimately is both a cause and a consequence of oxidative stress, affecting male gametogenesis. We have determined hyper homocysteinhemia (HHcy) levels can be reliably reduced in hypofertile patients in order to decrease/avoid associated epigenetic problems and protect the health of future children, in consideration of the fact that treatment with high doses of folic acid is inappropriate.

Methods:
Homocysteine levels were screened in male patients consulting for
long-standing infertility associated with at least three failed Assisted
Reproductive Technology (ART) attempts and/or repeat miscarriages. Seventyseven patients with Hcy levels > 15 μM were treated for three months with a combination of micronutrients including 5- MethylTetraHydroFolate (5-MTHF), the compound downstream to the MTHFR enzyme, to support the one carbon cycle; re-testing was performed at the end of a 3 months treatment period. Genetic status for Methylenetetrahydrofolate Reductase (MTHFR) Single nucleotide polymorphisms (SNPs) 677CT (c.6777C > T) and 1298AC (c.1298A > C) was determined.

Results:
Micronutrients/5-MTHF were highly efficient in decreasing circulating
Hcy, from averages 27.4 to 10.7 μM, with a mean observed decrease of 16.7 μM. The MTHFR SNP 677TT (homozygous form) and combined heterozygous 677CT/1298AC status represent 77.9% of the patients with elevated Hcy.

Conclusions:
Estimation HHcy should not be overlooked in men suffering infertility
of long duration. MTHFR SNPs, especially 677TT, are a major cause of high homocysteinhemia (HHcy). In these hypofertile patients, treatment with micronutrients including 5-MTHF reduces Hcy and even allows spontaneous pregnancies post treatment. This type of therapy should be considered in order to ensure these patients’ quality of life and avoid future epigenetic problems in their descendants.

#Homocysteine, #PCOS, #FollicularFluid, #BlastocystRate, #Micronutrients

Kucuk 2023 – Follicular HCY in PCOS

“Follicular homocysteine as a marker of oocyte quality in PCOS and the role of micronutrients”

Authors: T. Kucuk, P. E. Horozal, A. Karakulak, E. Timucin and M. Dattilo

Capsule: Homocysteine is detrimental for fertility and some studies had shown that the amount of homocysteine in follicular fluids could be directly involved. Impryl had been already shown to decrease blood homocysteine in all treated women, thus it was investigated whether Impryl decreases homocysteine also in follicular fluid and whether this associate to pregnancies. PCOS ladies undergoing an ART cycle were tested for follicular homocysteine after Impryl for 3 months or no treatment. Follicular homocysteine was directly related to the pregnancies and its decrease with Impryl marked an improved clinical outcome. This is the first time a supplementation improves the clinical outcomes in ART cycles.

Kucuk 2023 – Follicular HCY in PCOS

Abstract

Purpose:
Does follicular homocysteine predict the reproductive potential of oocytes following FSH stimulation in PCOS women? Can it be modulated by dietary interventions?

Methods:
This was a prospective, randomized, interventional clinical study. Forty-eight PCOS women undergoing in vitro fertilization at a private fertility clinic were randomized for a dietary supplementation providing micronutrients involved in homocysteine clearance or no treatment. The supplement was assumed 2 months before stimulation until pick-up day. Monofollicular fluids were collected and frozen. After embryo transfer, the fluids from the follicles generating the transferred embryos were thawed and analyzed.

Results:
Follicular homocysteine showed a negative correlation with clinical pregnancy both in the whole population (r =  − 0.298; p = 0.041) and in controls (r =  − 0.447, p = 0.053). The support achieved a non-significantly lower concentration of follicular homocysteine (median [IQR]–7.6 [13.2] vs 24.3 [22.9]). Supplemented patients required far less FSH for stimulation (1650 [325] vs 2250 [337], p = 0.00002) with no differences in the number of oocytes collected, MII rate, and fertilization rate. Supplemented patients enjoyed higher blastocyst rate (55% [20.5] vs 32% [16.5]; p = 0.0009) and a trend for improved implantation rate (64% vs 32%; p = 0.0606). Clinical pregnancy rates were 58% vs 33% in controls (p = ns).

Conclusions:
Follicular homocysteine is a suitable reporter that might be investigated as a tool for oocyte-embryo selection. A diet enriched with methyl donors may be useful in PCOS and supplements may also help. These findings may be also true for non-PCOS women, which warrants investigation.

#Cysteine, #HydrogenSulfide, #LC-MS/MS, #L-Cystine, #Micronutrients, #Pyridoxal 5-Phosphate

Dattilo 2022 – Modulation of human H2S metabolism

“Modulation of Human Hydrogen Sulfide Metabolism by Micronutrients, Preliminary Data”

Authors: M. Dattilo, C. Fontanarosa, M. Spinelli, V. Bini and A. Amoresano

Capsule: A dietary supplement is not intended to cure diseases, nevertheless it is fair to demonstrate which is the actual metabolic effect. In the case of Redostim, that introduces a U-turn in metabolic modulation, it was even more important. Volunteers have been tested before and after taking Redostim to evaluate their metaboloma by complex mass spectrometry analyses. The results confirm that Redostim induces H2S.

Dattilo 2022 – Modulation of human H2S metabolism

Abstract

Purpose:
Hydrogen sulfide (H2S) is a pivotal gasotransmitter networking with nitric oxide (NO) and carbon monoxide (CO) to regulate basic homeostatic functions. It is released by the alternative pathways of transulfuration by the enzymes Cystathionine Beta Synthase (CBS) and Cystathionine Gamma Lyase (CSE), and by Cysteine AminoTransferase (CAT)/ 3-Mercaptopyruvate Sulfur Transferase (3MPST). A non-enzymatic, intravascular release is also in place. We retrospectively investigated the possibility to modulate the endogenous H2S release and signaling in humans by a dietary manipulation with supplemented micronutrients (L-cystine, Taurine and pyridoxal 5-phopsphate/P5P).

Methods:
Patients referring for antiaging purposes underwent a 10-day supplementation. Blood was collected at baseline and after treatment and the metabolome was investigated by mass spectrometry to monitor the changes in the metabolites reporting on H2S metabolism and related pathways.

Results:
Data were available from 6 middle aged subjects (2 women). Micronutrients increased 3-mercaptopyruvate (P = .03), reporting on the activity of CAT that provides the substrate for H2S release within mitochondria by 3MPST, decreased lanthionine (P = .024), reporting the release of H2S from CBS, and had no significant effect of H2S release from CSE. This is compatible with a homeostatic balancing. We also recorded a strong increase of reporters of H2S-induced pathways including 5-MethylTHF (P = .001) and SAME (P = .022), reporting on methylation capacity, and of BH4 (P = .021) and BH2 (P = .028) reporting on nitric oxide metabolism. These activations may be explained by the concomitant induction of non-enzymatic release of H2S.

Conclusions:
Although the current evidences are weak and will need to be confirmed, the effect of micronutrients was compatible with an increase of the H2S endogenous release and signaling within the control of homeostatic mechanisms, further endorsing the role of feeding in health and disease. These effects might result in a H2S boosting effect in case of defective activity of pathologic origin, which should be checked in duly designed clinical trials.

#Micronutrients, #OneCarbonCycle, #Oocytes, #Blastocyst, #OCM-Checkpoint

Golestanfar 2022 – Doubled Blastocyst Rate

“Metabolic enhancement of the one carbon metabolism (OCM) in bovine oocytes IVM increases the blastocyst rate: evidences for a OCM checkpoint”

Authors: A. Golestanfar, A. Niasari‑Naslaji, F. Jafarpour, S. Rouhollahi, N. Rezaei, Y. Menezo, M. Dattilo & M. H. Nasr‑Esfahani

Capsule: Top paper on top journal investigating the role of OCM support in the maturation and reproductive competence of oocytes. Immature bovine oocytes were cultured and matured in vitro for 24 hours with or without the ingredients of Impryl at a concentration resembling the oral administration in humans. Supported oocytes generated a double number of mature embryos and the effect was entirely due to the improved methylation of maternal DNA (from the support). This demonstrates that only oocytes well fed with micronutrients are able to generate an embryo. To achieve the same results in the clinical setting, we should treat the mothers-to-be while they are maturing the oocytes.

Golestanfar 2022 – Doubled Blastocyst Rate

Abstract

The one carbon metabolism (OCM) has a primary role in the process of oocyte maturation. In this study bovine oocytes were cultured for 24 h, up to MII stage, with standard medium supplemented or not with 8 metabolic enhancers of the OCM and the MII and blastocyst rate were compared. Additional analyses were performed on matured oocytes, cumulus cells, zygotes and blastocysts. The OCM supplementation increased the blastocyst rate derived from in vitro fertilization. The mitochondrial mass and DNMT3a protein expression were increased whereas DNA fragmentation decreased in matured oocytes. DNA methylation in female pronucleus of zygotes was increased. The supplementation did not directly affect the redox balance as ROS and GSH in matured oocytes and homocysteine in the spent medium were unchanged. The supplementation of the oocytes with metabolic enhancers of the OCM may increase the yield from the culture, likely due to improved DNA methylation and epigenetic programming. The lack of effects on MII rate with huge differences appearing at the blastocyst stage suggest the existence of a OCM metabolic check point that hampers oocytes progression.

#HydrogenSulfide, #SARS-Cov-2, #Covid 19, #HemeOxygenase1, #CarbonMonoxide

Dattilo 2020 – Host defences in Covid 19

“The role of host defences in Covid 19 and treatments thereof”

Author: M. Dattilo

Capsule: This is a review paper explaining how the virus SARS-CoV2 responsible for Covid 19 achieves virulence by hampering the H2S system. Those with constitutional low H2S reactivity are exposed to a serious disease. Treatments boosting the H2S signalling may be of help in Covid 19. This is already possible by micronutrients.

Dattilo 2022 – Modulation of human H2S metabolism

Abstract

Hydrogen sulfide (H2S) is a natural defence against the infections from enveloped RNA viruses and is likely involved
also in Covid 19. It was already shown to inhibit growth and pathogenic mechanisms of a variety of enveloped
RNA viruses and it was now found that circulating H2S is higher in Covid 19 survivors compared to fatal cases. H2S
release is triggered by carbon monoxide (CO) from the catabolism of heme by inducible heme oxygenase (HO-1)
and heme proteins possess catalytic activity necessary for the H2S signalling by protein persulfidation. Subjects with
a long promoter for the HMOX1 gene, coding for HO-1, are predicted for lower efficiency of this mechanism. SARScov-
2 exerts ability to attack the heme of hemoglobin and other heme-proteins thus hampering both release and
signalling of H2S. Lack of H2S-induced persulfidation of the KATP channels of leucocytes causes adhesion and release
of the inflammatory cytokines, lung infiltration and systemic endothelial damage with hyper-coagulability. These
events largely explain the sex and age distribution, clinical manifestations and co-morbidities of Covid-19. The
understanding of this mechanism may be of guidance in re-evaluating the ongoing therapeutic strategies, with
special attention to the interaction with mechanical ventilation, paracetamol and chloroquine use, and in the
individuation of genetic traits causing increased susceptibility to the disruption of these physiologic processes and
to a critical Covid 19. Finally, an array of therapeutic interventions with the potential to clinically modulate the HO-
1/CO/H2S axis is already available or under development. These include CO donors and H2S donors and a boost to
the endogenous production of H2S is also possible.

#ClinicalTrial, #MaleFertility, #Impryl, #Micronutrients

Smits 2020 – Idiopathic male infertility

“Impact of a nutritional supplement (Impryl) on male fertility: study protocol of a multicentre, randomised, double-blind, placebo-controlled clinical trial (SUppleMent Male fERtility, SUMMER trial)”

Authors: R. Smits, K. D’Hauwers, J. IntHout, D. Braat, K. Fleischer

Capsule: This is the official presentation and details disclosure of a huge clinical study with Impryl in male infertility. It is a prospective, randomized, double blind controlled vs placebo clinical trial ongoing at the Radbaud University, The Netherland. The study, started already in 2018, is planned to include 1200 patients to be treated for 6 months and followed-up for 15 months. Originally planned to conclude in December 2023, the study is now expected to take another year to complete the follow-up of treated patients. We are very proud to say that this is the largest and best quality ever study on male infertility.

Smits 2020 – Idiopathic male infertility

Abstract

Purpose:
Infertility is a worldwide problem and about 10%–15% of all couples will be affected by the inability to have children. In approximately 50% of infertile couples, a male factor is involved. Most of the male infertile cases are characterised as ‘idiopathic’, except for a small percentage of cases which are causative by a genetic aetiology. In the past decade, the role of oxidative stress related to sperm quality has been researched thoroughly and estimated to be the problem in 25%–87% of male infertility cases. Impryl is a nutritional supplement which works on the metabolic system and the regulation of oxidative stress by activating the 1-carbon cycle and therefore recycling of homocysteine. We hypothesise that the nutritional supplement Impryl in men of infertile couples might improve the ongoing pregnancy rate.

Methods:
We designed a multicentre, randomised, double-blind, placebo-controlled clinical trial. We aimed to include 1200 male adults aged 18–50 years, part of a couple that is diagnosed with infertility. The couple will either start or has already been started with fertility treatment, that is, expectative management (duration of 6 months), intrauterine insemination (IUI) with or without mild ovarian stimulation or ovulation induction, either in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) treatment. Male participants will be randomised in either the Impryl or the placebo group, with identical appearance of the tablets to be distributed (doses: one tablet each day), for a total duration of maximal 6 months. Patients can start directly with fertility treatment and/or natural conception. The primary outcome is the number of ongoing pregnancies confirmed by ultrasound at ≥10 to 12 weeks, and conceived in the time window between randomisation up to and including month 6 of intervention use. Secondary outcomes are change in semen parameters between baseline and after 3 months of intervention in the IUI/IVF/ICSI group, based on (prewash) total motile sperm count. Furthermore the number of pregnancies conceived in the optimal intervention time window (after full spermatogenesis of 72 days), overall number of pregnancies, time to pregnancy, embryo fertilisation rate in IVF/ICSI, embryo-utilisation rate in IVF/ICSI, number of miscarriages, live birth rate and adverse events are documented within the study period of 15 months.

Ethics and dissemination:
The protocol is approved by the local medical ethical review committee at the Radboud University Medical Centre and by the national Central Committee on Research Involving Human Subjects. Findings will be shared with the academic and medical community, funding and patient organisations in order to contribute to optimisation of medical care and quality of life for patients with infertility.

Trial registration numbers:
NCT03337360 and NTR6551.

#Homocysteine, #Micronutrients, #OneCarbonCycle, #PCOS 

Schiuma 2020 – Blood homocysteine in PCOS

“Micronutrients in support to the one carbon cycle for the modulation of blood fasting homocysteine in PCOS women”

Authors: N. Schiuma, A. Costantino, T. Bartolotti, M. Dattilo, V. Bini, M. C. Aglietti, M. Renga, A. Favilli, A. Falorni & S. Gerli

Capsule: PCOS women are known for a higher risk of increased homocysteine and this may be related to their fertility problems. Young PCOS women were randomized for supplementation with Impryl or no treatment and their homocysteine was controlled before and after the treatment. Impryl significantly reduced the mean blood homocysteine level. More relevant, all treated patients reduced homocysteine and the average decrease was significant also in the group starting from normal values.

Schiuma 2020 – Blood homocysteine in PCOS

Abstract

Purpose:
Fasting blood homocysteine is increased in PCOS women and is involved in several of its co-morbidities including cardiovascular disease and infertility. Corrective interventions based on the administration of supra-physiologic doses of folic acid work to a low extent. We aimed to test an alternative approach.

Methods:
This was a prospective, randomized, parallel group, open label, controlled versus no treatment clinical study. PCOS women aged > 18, free from systemic diseases and from pharmacological treatments were randomized with a 2:1 ratio for treatment with activated micronutrients in support to the carbon cycle (Impryl, Parthenogen, Switzerland—n = 22) or no treatment (n = 10) and followed-up for 3 months. Fasting blood homocysteine, AMH, testosterone, SHBGs, and the resulting FTI were tested before and at the end of the follow-up.

Results:
The mean baseline fasting blood homocysteine was above the normal limit of 12 μMol/L and inversely correlated with SHBG. AMH was also increased, whereas testosterone, SHBG, and FTI were within the normal limit. The treatment achieved a significant reduction of homocysteine, that did not change in the control group, independently of the starting value. The treatment also caused an increase of AMH and a decrease of SHBGs only in the subgroup with a normal homocysteine at baseline.

Conclusions:
In PCOS ladies, blood homocysteine is increased and inversely correlated with the SHBGs. Physiologic amounts of activated micronutrients in support to the carbon cycle achieve a reduction virtually in all exposed patients. Whether this is of clinical benefit remains to be established.

#Metabolism, #Micronutrients, #OneCarbonCycle, #Sperm 

Gallo 2018 – Metabolic enhancers affect sperm functionality

“Metabolic enhancers supporting 1-carbon cycle affect sperm functionality: an in vitro comparative study”

Authors: A. Gallo, Y. Menezo, B. Dale, G. Coppola, M. Dattilo, E. Tosti & R. Boni

Capsule: Top paper on top journal investigating the role of OCM support in sperm bioenergetics. Mitochondria produce ROS together with ATP (energy) and can keep on releasing ATP as long as able to neutralize the ROS generated. Human sperm mitochondria where shown to enjoy sustained bioenergetic activity over time when exposed to Impryl micronutrients at the concentration achieved after oral administration.

Gallo 2018 – Metabolic enhancers affect sperm functionality

Abstract

The sperm plasma membrane is a sensitive target to oxidative stress. The most representative reactive oxygen species (ROS) scavengers in the genital tract, hypotaurine and glutathione, require, for their synthesis, cysteine whose availability is associated with the 1-carbon cycle (1-CC). Human, bovine and ascidian spermatozoa were incubated with compounds supporting the 1-CC (Vitamin B6, Methylcobalamin, 5 Methyl Tetrahydrofolate, Zinc Bisglycinate and N-acetyl-cysteine) (TRT) and compared to the effects induced solely by N-acetyl-cysteine (NAC). In control groups (CNTRL), spermatozoa were incubated with medium alone. After 90 and 180 minutes of incubation, the mitochondrial membrane potential (ΔΨM) in TRT and NAC was significantly (P < 0.01) higher than in CNTRL. At H2DCFDA evaluation, ROS production differed between species whereas, at 2-OH Ethidium, it significantly decreased in bovine TRT group. Intracellular pH (pHi) did not significantly vary in relation to treatment. In ascidian spermatozoa, the NAC supplementation decreased external pH, which in turn brought to a pHi lowering. Buffering seawater with NaHCO3 reversed the beneficial effects of N-acetyl-cysteine supplementation. In conclusion, both fully supporting the 1-CC and treatment with N-acetyl-cysteine alone improved kinetics, ΔΨM and ROS production in mammalian sperm demonstrating for the first time the direct in vitro effects of these compounds on sperm functionality.

#WomanFertility, #Micronutrients, #OneCarbonCycle, #OvarianReserve 

Silvestris 2017 – One-Carbon Cycle restores ovarian reserve in subfertile women

“Supporting the One-Carbon Cycle restores ovarian reserve in subfertile women: absence of correlation with urinary Bisphenol A concentration”

Authors: E. Silvestris, M. Cohen, D. Cornet, L. Jacquesson-Fournols, P. Clement, J. Chouteau, M. Schneider, T. Besnard, and Y. Ménézo

Capsule: Fifty-five women with a history of 3–7 years of infertility and low AMH level were tested for AMH before and after 4 months of dietary support with micronutrients in support to the OCM. Their AMH value increased independently of the starting value and of age with a mean change from 1.34 to 1.88 (p = 0.0001). Eight patients spontaneously conceived during the 3-months follow-up of the study.

Silvestris 2017 – One-Carbon Cycle restores ovarian reserve in subfertile women

Abstract

Environmental endocrine disrupting chemicals (EDCs), including bisphenol A (BPA), induce DNA methylation errors and oxidative stress, and alter fertility. Animal studies have demonstrated that supporting the one-carbon cycle (1-CC) with appropriate dietary supplements can reduce the effects of EDCs. Anti-Mullerian hormone (AMH), a marker of ovarian functionality, has been tested in subfertile female patients, to control this hypothesis in humans. Fifty-five women with a history of 3–7 years of infertility, with at least two assisted reproductive technology (ART) treatment failures, and low serum levels of AMH were enrolled in the study. Before starting any further ART treatment, they were tested for AMH and for follicular count. A urinary control of BPA was proposed. Then a support of the 1-CC, already tested in other clinical studies, was initiated and continued for 4 months. At the end of this period, antral follicle count and serum AMH levels were re-evaluated. The AMH levels before and after treatment were compared using the Wilcoxon test (nonparametric test, non-Gaussian population). Out of the 55 patients, 35 accepted a BPA dosage in the urine. No correlation was found between BPA and serum AMH concentrations. Forty-nine patients followed the full treatment with 1-CC supplements, which resulted in increased AMH levels, independent of initial AMH levels and maternal age (in the range studied), p = 0.0001. Eight patients spontaneously conceived ongoing pregnancies within 3 months, at the end of the protocol. A support of the 1-CC can partly alleviate metabolic derangements induced by environment, as observed in animal models, and improve endocrine background in women.

#Sperm, #Oocyte, #OxidativeStress, #DNA, #Methylation, #Epigenetics

Dattilo 2016 – Feeding the antioxidants system improves gamete quality

“Improvement of gamete quality by stimulating and feeding the endogenous antioxidant system: mechanisms, clinical results, insights on gene-environment interactions and the role of diet”

Authors: M. Dattilo & G. D’Amato & E. Caroppo & Y. Ménézo

Capsule: The role of the OCM in male infertility is reviewed together with the positive effects from a supplementation with methyl donors. These effects appear directly related to the improvement of methylations leading to improved sperm maturation and decreased DNA damage. The rationale for Impryl, coverage of the full pathway and use of pre-activated micronutrients, is explained.

Dattilo 2016 – Feeding the antioxidants system improves gamete quality

Abstract

Oxidative damage triggers extensive repair in gametes and thereafter in the zygote but it results in clinically
relevant damage when affecting the maturation of the gametes
chromatin, i.e. padlocking and epigenetic marking. It associates with defective DNA methylation and/or with oxidation of
the methyl marks leading to derangement of gamete epigenetics, defects of chromatin condensation and aneuploidy. A
proper feed to the one carbon cycle has the potential to stimulate the endogenous antioxidant defences, i.e. gluthatione
synthesis, and to activate compensative homeostatic mechanisms restoring both the oxy-redox balance and DNA methylation, which are indeed strictly cross-regulated. This has
been shown to produce measurable clinical improvements of
male reproductive potential in pilot studies herein
summarised. However, the effects of dietary habits and of
supplementations are variable according to the individual genetic substrate, as genetic variants of several of the concerned
enzymes occur with high frequency. Individual risk assessments and personalised interventions are still difficult to implement, in the meantime, a very varied diet may facilitate
metabolic compensation in the majority of the cases. This
review aims to report on the mechanisms of damage, on the
opportunities to modulate the physiologic oxy-redox
homeostasis by means of a varied diet or dietary supplements
and on the open issues related to the genetic variability of the
population.

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