Oral Antioxidant Treatment of Men Significantly Improves the Reproductive Outcome of IVF Cycles
doi.org/10.3390/jcm10153254
Main article: Treatment of Asthenozoospermia
Background
Highly reactive oxidizing agents, known as reactive oxygen species (ROS), are normally present in different parts of the human body, including seminal fluid. They are in fact vital for the proper functioning of sperm cells and have a natural balance of enzyme and non-enzyme related antioxidants.
However in some males, due to clinical disorders, environmental factors or improper lifestyle and nutritional choices, this equilibrium is disturbed and leads to an abnormal concentration of ROS, described as ‘oxidative stress.’
A study in 2008 reported that elevated ROS in seminal fluid had a negative impact on fertilization rate, embryo development and pregnancy outcomes in IVF/ICSI (in vitro fertilisation / intracytoplasmic sperm injection) patients. Since then, several more studies have provided further evidence to support the theory that elevated accumulation of ROS has a detrimental effect of sperm parameters.
It is therefore quite rational that experts are looking into various ways of reducing ROS, especially through dietary supplementation. Unfortunately, to date very few studies have analysed the effect of antioxidant therapy on ICSI clinical outcomes.
Aim
To study oral antioxidant supplementation, in men with previously low fertilisation rates (ICSI), and its effect on embryological, clinical, and neonatal outcomes.
Methodology
A multicenter, longitudinal, prospective study was conducted in Roma, Italy. Couples were included as per the following criteria:
- Female partner was less than 38 years old
- History of ICSI with less than 60% unexplained fertilisation rate, using fresh oocytes and ejaculated sperm
- More than 6 inseminated oocytes
- Male sperm count was greater than 1×106/mL
- No drug abuse
With the following exclusion criteria:
- Men suffering from severe oligospermia, azoospermia or any known cause of infertility
- Women with diminished ovarian reserve, i.e. <6 retrieved oocytes in a previous IVF cycle, or raised early follicular phase follicle-stimulation hormone (FSH), or <1 ng/mL Anti-Mullerian Hormone concentration
- Any male or females suffering from metabolic, autoimmune, endocrine disorders or tumours
The selected males were prescribed Gametogen tablets (2 per day), to be taken orally, for a duration of 90 days.
2 Gametogen tablets contained:
- Myo-inositol (1000mg)
- Alpha-lipoic acid (800mg)
- Folic acid (400mg)
- Coenzyme Q10 (200mg)
- Zinc (15mg)
- Selenium (83μg)
- Vitamin B2 (2.8mg)
- Vitamin B6 (2.8mg)
- Vitamin B12 (5μg)
Semen parameters was measured at baseline (T0), and at the end of treatment (T90), following 3 days of sexual abstinence. The ejaculates were then stored at room temperature for 30 minutes to liquefy, before being processed via a two-layer density gradient method, followed by conventional sperm analysis, according to WHO guidelines.
Sperm DNA fragmentation was measured via SCD (Sperm Chromatin Dispersion) using a Halo-sperm kit (Halotech DNA SL, Spain).
After antioxidant treatment, a second ICSI cycle was performed by each participating couple. Controlled ovarian hyperstimulation, oocyte recovery, ICSI, embryo culture and assessment was carried out according to established protocols.
Embryo transfer (ET) was normally performed 72 hours (3 days) after the collection of oocytes except in cases where:
- The patient had only 1-2 fertilised oocytes, with ET done on day 2.
- The patient had at least 4 good quality embryos on day 3, then transfer at blastocyst stage (day 5) was considered.
Embryos that developed up to the blastocyst but were surplus to the requirements, were cryopreserved by vitrification.
Primary study outcome was clinical pregnancy rate. Fertilization rate, cleavage rate, quality of embryos, blastocyst development rate, implantation rate, live-birth rate, cumulative clinical pregnancy rate per couple, cumulative live-birth rate per couple and any adverse events were measured as secondary outcomes. Newborn birthweights were also analysed and compared with Italian neonatal charts.
Results
Of the 86 males, 77 completed the study, with 9 lost to follow-up or non-compliance.
Baseline measurements revealed 74% (57/77) of males had one or more abnormal sperm parameters according to WHO 2010 criteria. Among these 74% of males, 51% were diagnosed with oligospermia and asthenospermia combined, 19% with asthenospermia and 4% with oligospermia.
Analysis of semen parameters, at baseline (T0) and after 90 days (T90), revealed statistically significant improvements in sperm count, concentration, progressive motility and total motile sperm count.
| T0 | T90 | |
| Sperm concentration (×106/mL) | 27.2 | 27.5 |
| Number of sperm (×106 /mL) | 58.3 | 63.5 |
| Progressive motility (%) | 27.6 | 34.6 |
| Total motile sperm count (×106) | 26.8 | 33.1 |
Antioxidant therapy also seemed to have a positive effect on DNA fragmentation with a 42% decrease in fragmentation, from 28.3% (T0) to 16.3% (T90). However, due to the low numbers of paired data, the trend was not statistically significant (P=0.08).
Initial analysis of ICSI outcomes confirmed the number of mature oocytes was similar to previous attempts (9.7 vs. 10.1), however the fertilization rate was significantly higher (72.9 vs. 39.8 %). This resulted in a significantly higher number of cleavage stage embryos per patient (3.3 vs. 2.1) and proportion of top quality embryos (82.7 vs. 64.8 %), with 47% of patients going on to develop at least one blastocyst compared to only 5% previously.
| T0 | T90 | |
| Top quality embryos (%) | 64.8 | 82.7 |
| Patients who developed at least 1 blastocyst (n) | 4 | 36 |
| Number of developed blastocysts per patient | 0.2 | 1.1 |
There were 2 spontaneous abortions from attempts at T0 following clinical pregnancy, however attempts at T90 following treatment resulted in a total of 25 successful singleton clinical pregnancies.
In comparison with baseline (T0), ICSI cycles done after antioxidant treatment showed significantly higher cumulative clinical pregnancy (38 vs. 1 %) and live birth rates (30 vs. 0 %) per couple.
| T0 | T90 | |
| Cycles with day 2-3 ET | 65/68 (96%) | 45/74 (61%) |
| Implantation | 2/124 (2%) | 15/85 (18%) |
| Pregnancy | 2/65 (3%) | 15/45 (33%) |
| Live-birth rate | 0/124 (0%) | 13/83 (16%) |
| Miscarriage | 2/2 (100%) | 0/15 (0%) |
| Cycles with day 5 ET | 3/68 (4%) | 29/73 (40%) |
| Implantation | 0/4 (0%) | 10/32 (31%) |
| Pregnancy | 0/3 (0%) | 10/29 (34%) |
| Live-birth rate | 0/4 (0%) | 10/32 (31%) |
| Miscarriage | 0 | 0/10 (0%) |
| No. ET in freeze-thaw cycles | 3 | 9 |
| Implantation | 1/4 (25%) | 4/9 (44%) |
| Pregnancy | 1/3 (3%) | 4/9 (44%) |
| Live-birth rate | 0/1 (0%) | #/9 |
| Miscarriage | 1/1 (100%) | 1/4 (25%) |
| Cumulative clinical pregnancy rate per couple | 1/77 (1%) | 29/77 (38%) |
| Cumulative live-birth rate per couple | 0/77 (0%) | 23/77 (30%) |
Perinatal characteristics of newborns with available data (22/23) confirmed that they were of appropriate weight and there was no preterms, stillbirths or malformations.
No side effects from the oral intake of Gametogen was reported by participants.
Limitations
- Small study size.
- Observed results could be either from a single component or a specific mixture of antioxidants.
- Effect of treatment on semen oxidative status was not analysed.
Funding
This study was funded by the manufacturer of Gametogen (Laborest).
Glossary
Asthenospermia
Reduced sperm motility.
Azoospermia
A complete absence of sperm in ejaculated semen.
Blastocyst
A fertilised embryo that has developed an inner cell mass and outer layer (trophoblast) some time from day 4 onwards.
Cumulative
An increasing total after successive additions.
Longitudinal
Long term study of outcomes.
n
Sample size.
Neonate
Newborn child.
Oligospermia
Low sperm count.
Oocyte
Unfertilised immature / mature egg.
P-value
The probability that a result occurred by random chance.
Perinatal
Period of time from birth of baby up to 1 years of age.
Vitrification
Rapid freezing an embryo to avoid formation of ice crystals.
Similar studies
Gambera L, et al. (2019). Effects of antioxidant treatment on seminal parameters in patients undergoing in vitro fertilization. https://doi.org/10.4081/aiua.2019.3.187
Canepa P, et al. (2018). Combined treatment with myo-inositol, alpha-lipoic acid, folic acid and vitamins significantly improves sperm parameters of sub-fertile men: A multi-centric study. https://doi.org/10.26355/eurrev_201810_16180
Korosi T, et al. (2017). Physiological Intra-Cytoplasmic Sperm Injection (PICSI) outcomes after oral pretreatment and semen incubation with myo-inositol in oligoasthenoteratozoospermic men: results from a prospective, randomized controlled trial. https://pubmed.ncbi.nlm.nih.gov/28724183/
Rago R, et al. (2017). Controlled, prospective, observational study on the efficiency and tolerability of a combination of potential Nrf2-inducing antioxidants and micronutrients as pre-treatment for ICSI in dyspermic patients with previous failure. https://pubmed.ncbi.nlm.nih.gov/28429340/
Geva E, et al. (1996). The effect of antioxidant treatment on human spermatozoa and fertilization rate in an in vitro fertilization program. https://doi.org/10.1016/s0015-0282(16)58514-8
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