Scholarly article on topic 'Effect of metabolic and antioxidant supplementation on sperm parameters in oligo-astheno-teratozoospermia, with and without varicocele: A double-blind placebo-controlled study'

Effect of metabolic and antioxidant supplementation on sperm parameters in oligo-astheno-teratozoospermia, with and without varicocele: A double-blind placebo-controlled study Academic research paper on "Veterinary science"

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Academic research paper on topic "Effect of metabolic and antioxidant supplementation on sperm parameters in oligo-astheno-teratozoospermia, with and without varicocele: A double-blind placebo-controlled study"

Accepted: 25 September 2017

DOI: 10.1111/and.12927

ORIGINAL ARTICLE

aNDROLOGia

Effect of metabolic and antioxidant supplementation on sperm parameters in oligo-astheno-teratozoospermia, with and without varicocele: A double-blind placebo-controlled study

G. M. Busetto1 S | A. Agarwal4 D | A. Virmani2 | G. Antonini1 | G. Ragonesi1 | F. Del Giudice1 | S. Micic3 | V. Gentile1 | E. De Berardinis1 0

1Urology Department, Sapienza Rome University, Rome, Italy

2Sigma-Tau HealthScience, Utrecht, The Netherlands

3Andrology Department, Uromedica Polyclinic, Belgrade, Serbia

4Andrology Center, American Center for Reproductive Medicine, Cleveland Clinic, OH, USA

Correspondence

Gian Maria Busetto, Urology Department, Sapienza Rome University, Rome, Italy. Email: gianmaria.busetto@uniroma1.it

Funding information

Sigma-Tau Health Science

Summary

Since sperm require high energy levels to perform their specialised function, it is vital that essential nutrients are available for spermatozoa when they develop, capacitate and acquire motility. However, they are vulnerable to a lack of energy and excess amounts of reactive oxygen species, which can impair sperm function, lead to immotil-ity, acrosomal reaction impairment, DNA fragmentation and cell death. This monocen-tric, randomised, double-blind, placebo-controlled trial investigated the effect of 6 months of supplementation with L-carnitine, acetyl-L-carnitine and other micronutri-ents on sperm quality in 104 subjects with oligo- and/or astheno- and/or teratozoo-spermia with or without varicocele. In 94 patients who completed the study, sperm concentration was significantly increased in supplemented patients compared to the placebo (p = .0186). Total sperm count also increased significantly (p = .0117) in the supplemented group as compared to the placebo group. Both, progressive and total motility were higher in supplemented patients (p = .0088 and p = .0120, respectively). Although pregnancy rate was not an endpoint of the study, of the 12 pregnancies that occurred during the follow-up, 10 were reported in the supplementation group. In general, all these changes were more evident in varicocele patients. In conclusion, supplementation with metabolic and antioxidant compounds could be efficacious when included in strategies to improve fertility.

KEYWORDS

antioxidant, oligo-astheno-teratozoospermia, sperm, spermiogram, varicocele

1 | INTRODUCTION

Infertility is the inability of a sexually active, noncontracepting couple to achieve a spontaneous pregnancy within one year. Worldwide, the incidence of infertility is about 15%, of which, in general, 50% can be attributed to a male-associated factor. This can be reported with or without abnormal semen parameters (WHO, 2000). Male fertility can be affected by many factors ranging from congenital, endocrine,

immunologic, infectious or lifestyle factors as well as various malignancies. On the other hand, in 30%-40% of the cases, no obvious male infertility-associated factor is found (idiopathic male infertility; Nieschlag, Behre, & Nieschlag, 2010).

Varicocele is defined as an abnormal dilatation of scrotal veins, and various studies report a general prevalence of 15% in the healthy male population, whereas it is 40% in infertile men (Nagler, Luntz, & Martinis, 1997). Although the pathophysiologic mechanisms are not

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2018 The Authors. Andrologia Published by Blackwell Verlag GmbH

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yet completely known, varicocele has adverse effects on spermatogenesis and, to date, is considered as most common among the known causes of male infertility (Practice Committee of American Society for Reproductive Medicine and the Society for Male Reproduction and Urology, 2014).

Since sperm functions such as capacitation and motility are all highly energy-dependent (Talwar & Hayatnagarkar, 2015), spermatozoa have very high energy requirements. Many factors that negatively affect semen quality act through decreasing energy availability by mitochondrial dysfunction (Amaral, Lourengo, Marques, & Ramalho-Santos, 2013). Spermatozoa are also vulnerable to reactive oxygen species (ROS) because their plasma membranes and cytoplasm are rich in polyunsaturated fatty acids (Agarwal et al., 2014). In particular, elevated ROS exposure leads to membrane damage, membrane instability and functional alterations causing cell death (Agarwal et al., 2014). Latest evidence demonstrates an association between high ROS levels and increased mitochondrial DNA (mtDNA) copy number with decreased mtDNA integrity (Bonanno et al., 2016). Oxidative stress (OS) occurs when there is an imbalance between oxidants and antioxidants (Agarwal, Hamada, & Esteves, 2012). However, for normal sperm cell function including chroma-tin compaction in maturing spermatozoa during epididymal transit, a delicate redox balance between reduction and oxidation is required (Wright, Milne, & Leeson, 2014). In general, an oxidative milieu may lead to cellular degeneration by apoptosis or necrosis, and a reducing milieu could favour cell survival (Durackova, 2014). Thus, a therapeutic strategy would need to use supplements to increase sperm energy metabolism, minimise free radical damage to sperm and improve the cellular processes connected with the formation and maturation of sperm.

L-Carnitine and acetyl-L-carnitine play an important role in spermatozoa energy metabolism (Agarwal & Said, 2004; Zhou, Liu, & Zhai, 2007). Many clinical studies have shown that oral administration to asthenozoospermic subjects increases the percentage of mobile spermatozoa, progressive rapid motility, average speed and linearity of sperm motility (Balercia et al., 2005; Lenzi et al., 2004). Selenium is an essential component of several major metabolic pathways: antioxidant defence systems, thyroid hormone metabolism and immune function (Brown & Arthur, 2001). Coenzyme Q10 (CoQ10) is concentrated in the mitochondria located in the midpiece of sperm, and the levels of this compound show a significant correlation with sperm count and motility. Furthermore, CoQ10 may be deficient in varicocele leading to higher sensitivity to oxidative damage (Balercia et al., 2004). Fructose, citric acid, vitamin C, vitamin B12 and zinc are related to increased damage to the sperm genetic material, synthesis of coenzymes, metabolism and energy production (Chia, Ong, Chua, Ho, & Tay, 2000; Dawson, Harris, Teter, & Powell, 1992; Moslemi & Tavanbakhsh, 2011).

Thus, the objective of this trial was to evaluate sperm quality after supplementation of oligo- and/or astheno- and or teratozoospermic subjects with or without varicocele with selected naturally occurring antioxidative compounds in a randomised, double-blind, placebo-controlled setting.

2 | MATERIALS AND METHODS

Between December 2014 and June 2015, 104 infertile patients with oligo- and/or astheno- and/or teratozoospermia with an average age of 32.5 years (range 18-48) were enrolled in this single-centre, randomised, double-blind, placebo-controlled trial to determine the effect of antioxidant supplementation on semen quality. All participants were enrolled from our Andrology Clinic at the Department of Gynecological-Obstetric Sciences and Urological Sciences, "Sapienza" Rome University. The block randomisation method was used to randomise subjects into groups resulting in equal sample sizes to ensure a balance across the groups over time. At the commencement of the study, 52 patients with varicocele grade I-III (confirmed with Doppler ultrasound) and 52 patients without varicocele were divided into two groups each consisting of the supplementation and a placebo subgroup. Ten patients dropped out from the study leaving 45 patients with varicocele and 49 without varicocele.

The supplementation formulation (Proxeed Plus from Sigma-Tau HealthScience, Utrecht, the Netherlands) consisted of 1,000 mg L-carnitine, 725 mg fumarate, 500 mg acetyl-L-carnitine, 1,000 mg fructose, 20 mg CoQ10, 90 mg vitamin C, 10 mg zinc, 200 |ig folic acid and 1.5 ig vitamin B12. The placebo was provided from the same company and was made with excipients (sucrose, silica (anti-caking), lemon flavour, acesulfame K (E950) sweetener) of the supplementation without the active compounds.

Subjects received supplements or placebo (two sachets daily for 6 months) according to the randomisation schedule (nQuery Advisor nTerim 2.0 (2012) program) and were instructed of the method of use. One evaluation of a spermiogram was carried out at the beginning of the treatment (V1) to examine semen parameters in each patient. At the end of the 6-month treatment (V2), a consecutive semen sample was collected. Together with the semen analyses, before and after the treatment, we collected demographic data (age, weight, height), physical examination, blood pressure, medical history and intake of previous/concomitant therapies.

Semen samples were collected after 3-5 days of sexual abstinence. Ejaculate volume, total sperm count, total and progressive motility, as well as normal sperm morphology were evaluated according to WHO guidelines (2010; 5th edition guidelines).

Subjects included in our trial were men between 18 and 50 years of age with oligo-, astheno- and/or teratozoospermia, with or without varicocele, and having a history of infertility for more than 12 months. The varicocele patients were not surgically treated before and during the treatment. Patients without varicocele were suffering from idiopathic male infertility, and no other previous history of diseases affecting fertility. Every patient underwent a complete check-up to exclude any other cause of infertility (history, examination, complete ultrasound and Doppler, hormones and genetic tests) with no difference between varicocele and nonvaricocele patients. Fertile female partners were required with regular menstrual cycles, age <40 and couples not looking for fertility-related procedures such as in vitro fertilization (IVF) or artificial insemination (AI), or intracy-toplasmic sperm injection (ICSI) for the next 90 days.

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Subjects with known hypersensitivity to any of the treatment compounds, history of undescended testes or cancer, endocrine disorders, history of post-pubertal mumps, genitourinary surgery, obstructive azoospermia or obstructive pathology of the urogenital system, autoimmune disease, cystic fibrosis, history of taking any therapy affecting fertility within last 3 months, excessive consumption of alcohol or regular use of illicit or "recreational" drugs, positive serology for HIV, subjects following any special diet, any condition which in the opinion of the investigator might put the subject at risk by participating in this study and subjects involved in any other clinical trials were excluded from the trial. Endpoints of the study were sperm concentration, semen volume, total sperm count, total motility, progressive motility and percentage of normal sperm morphology.

The Ethical Committee of the Department of Gynecological-Obstetric Sciences and Urological Sciences, "Sapienza" Rome University, approved the study protocol (Institute Ethical Approval Number PXP-001A). The study was conducted in line with European Urology and Good Clinical Practice guidelines, with ethical principles laid down in the latest version of the Declaration of Helsinki. Every patient signed an informed consent to participate in the study.

2.1 | Sample size

Planning to carry out the analysis of covariance in a factorial design with two groups (Proxeed or Placebo, with and without varicocele), defined f = am/a = 0.25, a correlation coefficient (R2) between the baseline and final equal to 0.50, an a = .05 (significance) and p = .20 (power of 80%), made it necessary that at least 88 patients equally distributed in 22 units for each subgroup had to be enrolled. However, in anticipation of having about 15% of dropout, 104 patients (52 per arm) were enrolled.

2.2 | Statistical analysis

All continuous variables have been reported as mean, median, standard deviations, minimum and maximum values. Discrete and nominal variables have been reported as frequency and percentage in contingency tables. The basal homogeneity of groups has been tested, on the continuous variables, by the analysis of variance (ANOVA) with two levels (drug and varicocele). The Shapiro-Wilk test was adopted for checking the normal distribution of the data. In the present analysis, no discrete variables were considered for testing the homogeneity of groups.

All the study endpoints considered in the present analysis were evaluated, on the complete sample, by the analysis of covariance for a model with two classification levels. The independent variable was the value detected at the baseline visit, while the dependent variable was the value detected at the end of treatment. The Wilcoxon rank-sum test was adopted for comparing the two groups at baseline, while the Wilcoxon signed rank test was used in the comparisons before/after by group. A "Responder," was defined as, a patient whose parameters improved in comparison to the values before the treatment at the

final visit. A responder analysis was also carried out. The Chi-squared test was adopted for detecting possible differences between the two treatment groups. All the above analyses (apart from the ANCOVA) were repeated for the comparison of the two groups separately by the presence of varicocele. Considering the low power, due to the small size, the responses of the tests presented separately for the presence of the varicocele have to be evaluated accordingly. SAS® Vers. 9.4 was used for performing all the analyses.

3 | RESULTS

In total, 94 (of 104) patients completed the study. Table 1 summarises demographic and baseline characteristics of the population by treatment group. The results of the homogeneity tests show that the two groups were well-balanced. The descriptive analyses show that at baseline, all sperm parameters in patients suffering from varicocele were lower when compared to the non-varicocele group.

Adverse events (Table 2) that did not lead to stop the therapy, occurred only in the treatment group. All events were not serious: four patients had nausea and three vertigo or headache.

The results of the inferential analyses of the semen parameters are presented in Table 3. As for the ANCOVA, the p-values refer to the intention-to-treat population (ITT). The last observation carried forward (LOCF) method was used for replacing the missing data. The analyses are also presented for varicocele patients (Table 4) and non-varicocele patients (Table 5). The Wilcoxon rank-sum test was adopted for calculating the p-values. In analyzing the comparisons before/after in the placebo group, a significant difference in some parameters was observed. Therefore, the results of all the tests were also included in both the tables and the text. The responder analysis for all the parameters was carried out for all the groups and included in Table 6.

3.1 | Sperm concentration

The overall results for the sperm concentration in all subjects are summarised in Table 3. In the placebo group, sperm concentration was 41.4 ± 17.9 x 106/ml at baseline and 43.7 ± 13.6 x 106/ ml at the final visit. In the supplemented group, sperm concentration was 40.8 ± 18.2 x 106/ml at baseline and 51.4 ± 13.9 x 106/ml at final visit. While for the placebo group no change was observed (p = .5244), the increase in sperm concentration for the treatment group was significant (p = .0026). Before the treatment, the sperm concentration in the placebo group did not differ from that in the treatment group (p = .8453). At the end of the trial, the difference between both groups was significant (p = .0186) in favour of the supplemented group.

In varicocele patients (Table 4), the mean sperm concentrations in the placebo group was 38.7 ± 18.1 x 106/ml at baseline and 39.9 ± 17.2 x 106/ml at the final visit (p = .7572). In the supplemented

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Parameter Statistics Placebo Supplemented Total

Age (years) N 52 52 104

Missing 0 0 0

Mean 32.5 32.5 32.5 (p = .9792)

Std. Deviation 6.7 6.7 6.7

Median 33.0 32.0 32.3

Range 19.0-48.6 18.8-48.4 18.8-48.6

Height (cm) N 52 52 104

Missing 0 0 0

Mean 178.6 177.2 177.9 (p = .2487)

Std. Deviation 6.0 6.0 6.0

Median 180.0 177.0 178.0

Range 168.0-190.0 163.0-192.0 163.0-192.0

Weight (kg) N 52 52 104

Missing 0 0 0

Mean 76.4 75.1 75.8 (p = .4242)

Std. Deviation 8.7 7.7 8.2

Median 75.0 75.0 75.0

Range 62.0-94.0 62.0-93.0 62.0-94.0

HR (b/min) N 52 52 104

Missing 0 0 0

Mean 70.8 70.4 70.6 (p = .6295)

Std. Deviation 4.5 3.5 4.0

Median 70.0 70.0 70.0

Range 60-80 60-78 60-80

SBP (mmHg) N 52 52 104

Missing 0 0 0

Mean 119.4 117.2 118.3 (p = .0961)

Std. Deviation 7.3 6.3 6.9

Median 120.0 120.0 120.0

Range 100-130 110-130 100-130

DBP (mmHg) N 52 52 104

Missing 0 0 0

Mean 72.4 73.2 72.8 (p = .4707)

Std. Deviation 5.8 5.5 5.7

Median 70.0 70.0 70.0

Range 60-90 60-85 60-90

TABLE 1 Baseline characteristics

Results from ANOVA with two levels (drug and varicocele).

group, sperm concentration significantly (p = .0403) increased from the supplemented group a mean sperm concentration of

visit. Before the treatment, the placebo and treatment groups showed no difference (p = .9708). The comparison of the changes from baseline baseline between the two groups showed nonsignificant difference (p = .1391) in favour of the supplemented group.

The results of sperm concentration at baseline in non-varico-cele patients (Table 5) in the placebo group were 44.1 ± 17.5 x 106/ ml and 47.2 ± 7.8 x 106/ml at the final visit (p = .5318). In

ml at the final visit (p = .0354). Before the treatment, the placebo and treatment groups showed no difference (p = .8048). The comparison of the changes from baseline between the two groups showed no difference (p = .2460) in favour of the supplemented group.

The responder analysis (Table 6) showed that 73.3% of supplemented patients versus 51.0% of the patients in the placebo group increased from baseline (p = .0262).

38.5 ± 19.0 x 106/ml at baseline to 50.2 ± 17.9 x 107ml at the final

43.2 ± 17.3 x 106/ml was found at baseline and of 52.5 ± 9.4 x 106/

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TABLE 2 Listing of adverse events

Treatment group Id. no. Age Description (PT term) Seriousness Relationship Action taken

Supplemented 36 32 Nausea Not serious Probable None

Gastro-oesophageal reflux disease Not serious Probable None

67 27 Nausea Not serious Possible None

Vertigo Not serious Possible None

68 21 Headache Not serious Possible None

Nausea Not serious Possible None

85 28 Headache Not serious Possible None

Nausea Not serious Possible None

3.2 | Semen volume

Overall results for the semen volume are summarised in Table 3. While the mean semen volume in the placebo group was 3.0 ± 1.0 ml at baseline, it was 2.9 ± 1.0 ml at the final visit. In the supplemented group, 3.1 ± 1.2 ml were ejaculated at baseline and 3.2 ± 0.9 ml at the final visit. In both groups, placebo and treatment group, no changes (p = .6787) and (p = .6271) were observed. There were also no differences for the comparison between the two groups at baseline (p = .7499) and at the final visit (p = .1313).

In varicocele patients (Table 4), the mean semen volume at baseline in the placebo group was 2.7 ± 0.7 ml and 2.4 ± 1.1 ml after the treatment. In the treatment group, semen volume was 2.9 ± 1.2 ml at baseline and 3.2 ± 1.2 ml at the final visit. No difference before/ after was observed in both the placebo (p = .2250) and the supplemented group (p = .3632). Comparing the two groups at baseline (p = .8761) and at the end of the study showed also no difference (p = .1273).

As for non-varicocele patients (Table 5), the semen volume in the placebo group was 3.2 ± 1.1 ml before and 3.3 ± 0.8 ml after the treatment. In the supplemented group it was 3.4 ± 1.2 ml at baseline and 3.3 ± 0.6 ml at the final visit. Furthermore, for non-varicocele patients, no difference before/after was observed in both the placebo group (p = .7711) and the supplemented group (p = .8753). The data at baseline (p = .6144) and at the end of the study (p = .5026) also did not differ.

The responder analysis (Table 6) did not show a difference between the two groups; 48.9% of supplemented patients versus 46.9% in the placebo group were considered as responders at final visit (p = .8500).

3.3 | Total sperm count

The overall results for the total sperm count in all subjects are summarised in Table 3. In the placebo group, 113.1 ± 37.4 x 106 at baseline and 127.8 ± 61.4 x 106 at the final visit, while the total sperm count in the supplemented group, was 114.2 ± 37.8 x 106 at baseline and 163.5 ± 64.3 x 106 at the final visit. While for the placebo group no change was observed (p = .2030), the increase in the supplemented group was highly significant (p < .0001). In contrast,

no difference (p = .8658) was observed between the two groups at baseline. At the end of the study, the two groups differed in favour of the supplemented group as was confirmed by the inferential analysis with p = .0117.

In the varicocele group (Table 4), total sperm count in the placebo was 100.5 ± 41.9 x 106 at baseline and 102.4 ± 77.2 x 106 at the final visit (p = .5749). The supplemented group had a sperm concentration of 96.3 ± 36.1 x 106 at baseline and of 158.8 ± 90.1 x 106 at the final visit (p = .0009). While both groups did not differ at baseline (p = .8764), the values in the supplemented group were significantly higher at the final visit (p = .0066).

In non-varicocele patients (Table 5), the total sperm count for the group was 125.6 ± 27.7 x 106 at baseline and 152.1 ± 23.9 x 106 after the treatment (p = .0022) and increased significantly (p = .0005), from 132.0 ± 30.9 x 106 at baseline to 167.6 ± 28.5 x 106 at final visit in the supplemented groups. No significant difference between the two groups was observed at baseline (p = .4259) and final visit (p = .2460).

The responder analysis (Table 6) showed that 82.2% of supplemented patients versus 55.1% in the placebo group increased from baseline (p = .0048).

3.4 | Progressive motility

For the progressive motility, the overall results are summarised in Table 3. In the placebo group, progressive motility was 23.0 ± 7.8% at baseline and 24.5 ± 7.2% at the final visit (p = .1567), while it was 23.4 ± 6.1% and 28.6 ± 8.2% at baseline final visit (p = .0012). The two groups did not differ (p = .6701), before the treatment. However, at the end of the study, a significant (p = .0088) difference in favour of the supplemented group was observed.

In varicocele patients (Table 4), progressive motility was 21.8 ± 6.2% at baseline and 23.6 ± 6.8% at the final visit in the placebo group (p = .1570) while it was 23.1 ± 5.2% and 27.4 ± 7.2%, respectively visit in the treated group (p = .0149). No difference between the two groups was observed at baseline (p = .3096) and at study end (p = .1686).

In the non-varicocele group (Table 5), progressive motility was 24.2 ± 9.1% before and 25.4 ± 7.7% after treatment in the placebo arm (p = .4866) and 23.7 ± 7.0% at baseline and 29.6 ± 9.0% at the final visit in the treatment arm (p = .0311). Also for this comparison,

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the test was not statistically significant for both baseline (p = .8907) and final visit (p = .2040).

The responder analysis (Table 6) showed that 73.3% of supplemented patients responded to the treatment whereas it was only 51.0% in the placebo group (p = .0262).

3.5 | Total motility

The summary results for total motility are depicted in Table 3. In the placebo group, the means were 32.6 ± 9.2% before and 34.6 ± 7.1% after the treatment (p = .1483). In the supplemented group, baseline and

TABLE 3 Sperm parameters. Absolute values of baseline and final

p-Values

Parameter Groups Statistics Baseline Final before/after**

Sperm concentration (106 ml)

Placebo

Supplemented

Missing Mean

Std. Deviation Median Range N

Missing Mean

Std. Deviation

Median

p-Values by visit*

52 0 41.4 17.9 38.3

11.0; 79.0 52 0 40.8 18.2 39.0

12.3; 77.0 .8453

49 3 43.7 13.6 44.0

16.0; 79.0 45 7 51.4 13.9 49.0 28.0; 86.0 .0186

Volume of ejaculate (ml) Placebo

Supplemented

N 52 49

Missing 0 3

Mean 3.0 2.9 .6787

Std. Deviation 1.0 1.0

Median 2.8 3.0

Range 1.3; 6.3 1.1; 5.1

N 52 45

Missing 0 7

Mean 3.1 3.2 .6271

Std. Deviation 1.2 0.9

Median 2.8 3.2

Range 1.4; 6.0 1.1; 5.5

p-Values by visit* .7499 .1313

Total sperm count (106)

Placebo

Supplemented

Missing Mean

Std. Deviation Median Range N

Missing Mean

Std. Deviation

Median

p-Values by visit*

113.1 37.4

30.0; 197.6 52 0

114.2 37.8

43.2; 205.8 .8658

127.8 61.4 136.7

24.0; 270.0 45 7

163.5 64.3 158.4

48.4; 369.6 .0117

<.0001

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TABLE 3 (Continued)

p-Values

Parameter Groups Statistics Baseline Final before/after**

Progressive motility (%) Placebo N 52 49

Missing 0 3

Mean 23.0 24.5 .1567

Std. Deviation 7.8 7.2

Median 22.3 23.0

Range 5.9; 43.2 8.1; 44.0

Supplemented N 52 45

Missing 0 7

Mean 23.4 28.6 .0012

Std. Deviation 6.1 8.2

Median 23.2 27.0

Range 12.0; 40.0 15.0; 57.9

p-Values by visit* .6701 .0088

Total motility (%) Placebo N 52 49

Missing 0 3

Mean 32.6 34.6 .1483

Std. Deviation 9.2 7.1

Median 32.0 35.0

Range 8.0; 55.0 12.0; 49.2

Supplemented N 52 45

Missing 0 7

Mean 31.7 39.0 <.0001

Std. Deviation 8.2 8.0

Median 31.3 37.5

Range 18.9; 48.0 29.0; 65.3

p-Values by visit* .5239 .0120

Sperm morphology—typical (%) Placebo N 52 49

Missing 0 3

Mean 21.1 15.7 .0146

Std. Deviation 16.2 9.4

Median 15.0 15.0

Range 3.0; 59.0 3.0; 52.0

Supplemented N 52 44

Missing 0 8

Mean 23.5 17.7 .0055

Std. Deviation 14.6 15.2

Median 20.0 13.5

Range 5.0; 64.0 3.0; 77.0

p-Values by visit* .2062 .3791

(Continues)

values obtained at the final visit differed significantly (p < .0001). While the two groups were balanced at baseline (p = .5239), a significant (p = .0120) difference between the two groups at the end of the treatment was evident in favour of the supplemented group.

In varicocele patients (Table 4), total sperm motility in subjects who received the placebo did not change from baseline to the final visit (p = .1214). However, in the treated group, values increased from 31.5 ± 8.1% at baseline to 37.5 ± 7.1% after the treatment (p = .0065).

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p-Values

Parameter Groups Statistics Baseline Final before/after**

Sperm morphology—atypical (%)

Placebo

Supplemented

Missing Mean

Std. Deviation Median Range N

Missing Mean

Std. Deviation

Median

p-Values by visit*

52 0 78.9

41.0; 97.0 52 0 80.2

16.6 85.0

22.0; 96.0 .5379

84.1 9.3

48.0; 97.0 45 7 82.5

15.2 86.0

23.0; 100.0 .5081

*The p-values for the Baseline visit are derived from the comparison between the two groups with the Wilcoxon rank-sum test. The p-values for the final visit are derived from the ANCOVA on the ITT population.

**The p-values before/after by treatment group are derived from the Wilcoxon signed rank test.

Yet, no difference was observed at baseline (p = .7836) and at the end of the study (p = .3164).

As for the non-varicocele group (Table 5), total motility was 33.9 ± 10.2% before and 34.7 ± 7.5% after the treatment in the placebo group (p = .5604) and 31.8 ± 8.4% and 40.2 ± 8.7% at baseline and final visit respectively, in the treated group (p = .0028). Both groups were balanced at baseline (p = .5396). However a statistical difference in favour of the supplemented group was evident (p = .0257).

The responder analysis (Table 6) showed that 68.9% of supplemented patients versus 53.1% in the placebo group increased from baseline (p = .1166).

3.6 | Normal sperm morphology

Analyzing sperm morphology (Table 3), results indicate that in the placebo group 21.1 ± 16.2% sperm at baseline and 15.7 ± 9.4% sperm after the treatment (p = .0146) has normal morphology. In the treatment group, normal sperm morphology was 23.5 ± 14.6% before and 17.7 ± 15.2% after the (p = .0055) treatment. No difference between the two groups was observed at baseline (p = .2062) and at study end (p = .3791).

Looking at atypical morphology (Table 3) the placebo group showed significantly (p = .0105) higher values at the final visit, while baseline and final values in the supplemented group did not differ (p = .1310). Further, two groups did not differ at baseline (p = .5379) and at the end of study (p = .5081).

3.7 | Pregnancy rate

Twelve pregnancies occurred during the follow-up time: 10 in the supplementation group (nine non-varicocele and one varicocele) and

two in the placebo group (one non-varicocele and one varicocele). One spontaneous abortion was reported in the placebo arm.

4 | DISCUSSION

Male infertility is a medical condition and a relevant social problem that has a strong impact on well-being. From various studies, it has emerged that seminal oxidative stress and sperm DNA damage must be taken into account as critical factors in the aetiology of semen alterations and infertility (Saalu, 2010; Vessey et al., 2016).

When levels of free radicals, and in particular ROS, are increased and antioxidant levels are decreased, OS occurs (Agarwal, Roychoudhury, Bjugstad, & Chou, 2016). OS has negative effects on sperm quality parameters and was shown to impact the DNA carried by these specialised cells. Human mitochondrial DNA gene alterations were associated with many pathological conditions, and this damage per se is also a recognized cause of poor sperm quality. Thus, targeting OS is a strategy to increase fertility and spermatozoa number and quality (Agarwal et al., 2016).

Varicocele is associated with an increase in ROS production and seminal OS leading to sperm dysfunction. Mitochondria are a key source of ROS production especially when they are damaged or dysfunctional due to lack of proper substrates and cofactors. In addition, mitochondrial gene mutations can also affect the respiratory electron transfer chain. These DNA variants probably underlie mitochondrial dysfunction leading to impaired ATP synthesis and ultimately interfere with sperm motility and fertility status (Heidari et al., 2016).

Non-enzymatic antioxidants including vitamins (mainly vitamins A, B, C, E), glutathione as well as metabolic coenzymes such as pantothenic acid, Co Q10, carnitines (i_-carnitine and acetyl-l-carnitine)

and micronutrients (zinc, selenium, copper) are often deficient , hence causing a general diminution in the antioxidant status as well as mi-tochondrial dysfunction (Jeulin & Lewin, 1996; Virmani, Ali, Pinto, Zerelli, & Binienda, 2016). Nutrients such as zinc, folic acid, vitamin B12, L-carnitine and acetyl-L-carnitine are also associated with sperm

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production and maturation (Adams et al., 1998; Ebisch, Thomas, Peters, Braat, & Steegers-Theunissen, 2007; Jeulin & Lewin, 1996; Watanabe et al., 2003).

Studies demonstrate that using these substances has a beneficial effect on fertility, in particular on sperm quality and are therefore

TABLE 4 Sperm parameters. Patients with varicocele. Absolute values of baseline and final visits

p-Values

Parameter Groups Statistics Baseline Final before/after**

Sperm concentration (106 ml)

Placebo

Supplemented

Missing Mean

Std. Deviation Median Range N

Missing Mean

Std. Deviation

Median

p-Values by visit*

26 0 38.7 18.1 32.0

17.5; 76.0 26 0 38.5 19.0 32.5

12.3; 76.0 .9708

24 2 39.9 17.2

16.0; 79.0 21 5 50.2

17.9 45.0 28.0; 86.0

Volume of ejaculate (ml) Placebo

Supplemented

N 26 24

Missing 0 2

Mean 2.7 2.4 .2250

Std. Deviation 0.7 1.1

Median 2.7 2.2

Range 1.3; 4.1 1.1; 5.0

N 26 21

Missing 0 5

Mean 2.9 3.2 .3632

Std. Deviation 1.2 1.2

Median 2.6 3.2

Range 1.4; 5.6 1.1; 5.5

p-Values by visit* .8761 .1273

Total sperm count (106)

Placebo

Supplemented

Missing Mean

Std. Deviation Median Range N

Missing Mean

Std. Deviation

Median

p-Values by visit*

100.5 41.9 94.9

30.0; 197.6 26 0 96.3 36.1 96.1

43.2; 190.6 .8764

102.4 77.2 77.7

24.0; 270.0 21 5

158.8 90.1 126.0

48.4; 369.6 .0066

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TABLE 4 (Continued)

BUSETTO et al.

Parameter Groups Statistics Baseline Final p-Values before/after**

Progressive motility (%) Placebo N 26 24

Missing 0 2

Mean 21.8 23.6 .1570

Std. Deviation 6.2 6.8

Median 22.3 22.0

Range 10.0; 40.0 15.0; 44.0

Supplemented N 26 21

Missing 0 5

Mean 23.1 27.4 .0149

Std. Deviation 5.2 7.2

Median 23.4 27.0

Range 13.0; 33.3 15.0; 41.7

p-Values by visit* .3096 .1686

Total motility (%)

Placebo

Supplemented

Missing Mean

Std. Deviation Median Range N

Missing Mean

Std. Deviation

Median

p-Values by visit*

31.3 8.2

8.0; 45.0 26 0 31.5 8.1

21.0; 46.7 .7836

24 2 34.5 6.9 35.0

18.0; 49.0 21 5 37.5 7.1 36.0

29.0; 55.0 .3164

*The p-values for the Baseline visit are derived from the comparison between the actual baseline values of the two groups with the Wilcoxon rank-sum test. The p-values for the final visit are derived from the comparison on the differences before/after between the two groups with the Wilcoxon rank-sum test.

**The p-values before/after by treatment group are derived from the Wilcoxon signed rank test.

recommended as potentially effective therapy for the treatment of male infertility (Walczak-J edrzejowska, Wolski, & Slowikowska-Hilczer, 2013).

L-Carnitine together with acetyl-L-carnitine is a safe treatment commonly used because of their capacity in improving sperm quality and pregnancy rate in males suffering from astheno-teratozoospermia (Wang et al., 2010). Selenium is essential for testis development, spermatogenesis and final sperm quality. It acts via a positive antioxidant action through glutathione peroxidase enzymes (Moslemi & Tavanbakhsh, 2011). Both vitamin E and zinc play a role in antioxidant balance regulation and are able to improve sperm concentration, percentage of progressively motile sperm and consequently pregnancy rate (Ajina, Sallem, Haouas, & Mehdi, 2016; Chen et al., 2012). Coenzyme Q10 levels show a significant correlation with sperm count and with sperm motility (Festa et al., 2014; Mancini et al., 1994). Administration of coenzyme Q10 to men with idiopathic asthenozoo-spermia results in an increase in sperm motility (Balercia et al., 2004).

The real association between varicocele and fertility status is still not completely clarified, but a recent meta-analysis showed a significant improvement in semen parameters in patients after varicocelectomy (Agarwal et al., 2007). Furthermore, after surgical treatment, a reversal in the sperm DNA damage was evidenced (Zini & Dohle, 2011). Gual-Frau et al. (2015) confirmed a beneficial effect of antioxidant compounds in patients suffering from grade I varicocele. In their study, patients showed an average relative reduction of 22.1% in sperm DNA fragmentation (p = .02) with 31.3% fewer highly degraded sperm cells (p = .07). The total number of sperm was also significantly increased after 3 months of treatment. Another recent trial conducted on patients with high-grade varicocele, and randomised for surgical treatment or L-carnitine supplementation, reported good results in all sperm parameters: motility changed from 21.7% to 35.4% (vs. 33.9%-47.5% in L-carnitine group), normal sperm morphology changed from 46.3% to 60% (vs. 56.6%-69.7% in the l-carnitine group) and seminal volume changed

from 3.5 to 4.2 ml (vs. 2.9-4.3 ml in the L-carnitine group). The authors concluded that supplementary treatment was as effective as varicocelectomy in improving semen parameters and can therefore be used as an alternative to surgery (Sofimajidpour, Ghaderi, & Ganji, 2016).

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Lastly, in a Cochrane analysis, men taking oral dietary supplementation for infertility were able to obtain better live birth rates in couples undergoing assisted reproductive techniques (Showell et al., 2014).

Comparing the effectiveness of varicocelectomy and medical therapy to treat these cases of infertility is difficult, and trials are

TABLE 5 Sperm parameters. Patients without varicocele. Absolute values of baseline and final visits

p-Values

Parameter Groups Statistics Baseline Final before/after**

Sperm concentration (106 ml) Placebo N 26 25

Missing 0 1

Mean 44.1 47.2 .5318

Std. Deviation 17.5 7.8

Median 42.3 48.5

Range 11.0; 79.0 30.0; 65.0

Supplemented N 26 24

Missing 0 2

Mean 43.2 52.5 .0354

Std. Deviation 17.3 9.4

Median 42.4 50.5

Range 19.0; 77.0 37.7; 78.0

p-Values by visit* .8048 .2460

Volume of ejaculate(ml) Placebo N 26 25

Missing 0 1

Mean 3.2 3.3 .7711

Std. Deviation 1.1 0.8

Median 3.0 3.1

Range 1.5; 6.3 2.0; 5.1

Supplemented N 26 24

Missing 0 2

Mean 3.4 3.3 .8753

Std. Deviation 1.2 0.6

Median 3.3 3.2

Range 1.9; 6.0 2.0; 4.5

p-Values by visit* .6144 .5026

Total sperm count (106) Placebo N 26 25

Missing 0 1

Mean 125.6 152.1 .0022

Std. Deviation 27.7 23.9

Median 118.0 154.0

Range 69.3; 178.6 107.5; 200.9

Supplemented N 26 24

Missing 0 2

Mean 132.0 167.6 .0005

Std. Deviation 30.9 28.5

Median 132.2 163.8

Range 68.4; 205.8 107.5; 220.8

p-Values by visit* .4259 .2460

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TABLE 5 (Continued)

BUSETTO et al.

Parameter Groups Statistics Baseline Final p-Values before/after**

Progressive motility (%) Placebo N 26 25

Missing o l

Mean 24.2 25.4 .4866

Std. Deviation 9.l ??

Median 23.9 25.o

Range 5.9; 43.2 8.1; 4o.o

Supplemented N 26 24

Missing o 2

Mean 23.? 29.6 .o3ll

Std. Deviation ?.o 9.o

Median 23.2 2?.5

Range l2.o; 4o.o l5.o; 5?.9

p-Values by visit* .89o? .2o4o

Total motility (%)

Placebo

Supplemented

Missing Mean

Std. Deviation Median Range N

Missing Mean

Std. Deviation

Median

p-Values by visit*

lo.2 32.o

15.5; 55.o 26 o

31.8 8.4 31.4

l8.9; 48.o .5396

?.5 35.o

l2.o; 49.2 24 2 4o.2 8.? 3?.8

29.o; 65.3 .o25?

*The p-values for the Baseline visit are derived from the comparison between the actual baseline values of the two groups with the Wilcoxon rank-sum test. The p-values for the final visit are derived from the comparison on the differences before/after between the two groups with the Wilcoxon rank-sum test.

**The p-values before/after by treatment group are derived from the Wilcoxon signed rank test.

limited by small case studies and non-randomisation. There is only one report with a direct comparison between L-carnitine and varicocelectomy in patients with grade II/III varicocele. The authors describe a statistically significant improvement in sperm count, mo-tility and morphology after treatment, and results are not different between different treatment methods. The main limitations of the study are the inclusion criteria, small sample size. In addition, this study is not a randomized, double-blind placebo-controlled (DBPC) study (Sofimajidpour et al., 2016).

Our trial evaluated the utilisation of a combination of metabolic substances, antioxidants and micronutrients to improve sperm parameters. For a better understanding of the action of the supplementation, we applied a DBPC system and very specific inclusion and exclusion criteria. Furthermore, in consideration of the still not clear effect of varicocele on male fertility, we divided our cohort into infertile varicocele patients and idiopathic infertile non-varicocele patients. At the end of the trial, we observed a marked increase in

sperm count and concentration together with increases in motility, progressive motility and morphology. All differences between treatment and placebo groups were statistically significant in both varico-cele and non-varicocele patients. A small difference (not statistically significant) was also observed in the semen volume in favour of the experimental group. In general, differences were more evident in those patients suffering from varicocele, which can probably be explained with the major OS and ROS-mediated damage that is usually associated with this condition. Unfortunately, at the moment, it is not possible to conclude whether the medical treatment is inferior or superior to varicocelectomy in those men with varicocele. Affirmation whether or not oral supplementation can replace surgery has yet to be properly established. Nevertheless, it is important to take into consideration that the role of oral supplements in clinical practice in the two groups is completely different, and one could possibly rather speak about an association between surgery and oral supplementation be more appropriate.

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TABLE 6 Responder analysis

Placebo Supplemented

Parameter Non responders Responders Non responders Responders p-Values

Sperm concentration (106 ml) 24 (49.0%) 25 (51.0%) 12 (26.7%) 33 (73.3%) .0262

W varicocele 12 (50.0%) 12 (50.0%) 6 (28.6%) 15 (71.4%) .1432

W/O varicocele 12 (48.0%) 13 (52.0%) 6 (25.0%) 18 (75.0%) .0950

Volume of ejaculate (ml) 26 (53.1%) 23 (46.9%) 23 (51.1%) 22 (48.9%) .8500

W varicocele 16 (66.7%) 8 (33.3%) 9 (42.9%) 12 (57.1%) .1088

W/O varicocele 10 (40.0%) 15 (60.0%) 14 (58.3%) 10 (41.7%) .1994

Total sperm count (106) 22 (44.9%) 27 (55.1%) 8 (17.8%) 37 (82.2%) .0048

W varicocele 15 (62.5%) 9 (37.5%) 4 (19.0%) 17 (81.0%) .0032

W/O varicocele 7 (28.0%) 18 (72.0%) 4 (16.7%) 20 (83.3%) .3419

Progressive motility (%) 24 (49.0%) 25 (51.0%) 12 (26.7%) 33 (73.3%) .0262

W varicocele 12 (50.0%) 12 (50.0%) 5 (23.8%) 16 (76.2%) .0706

W/O varicocele 12 (48.0%) 13 (52.0%) 7 (29.2%) 17 (70.8%) .1762

Total motility (%) 23 (46.9%) 26 (53.1%) 14 (31.1%) 31 (68.9%) .1166

W varicocele 11 (45.8%) 13 (54.2%) 7 (33.3%) 14 (66.7%) .3932

W/O varicocele 12 (48.0%) 13 (52.0%) 7 (29.2%) 17 (70.8%) .1762

Although pregnancy rate was not an endpoint of the study, it is interesting to note that of the 12 pregnancies that occurred during the follow-up time, 10 were reported in the supplementation group.

The safety of the formulation was assured by its composition, and tolerability was confirmed by the almost total absence of adverse effects during the treatment. We did not compare the effect of this treatment with surgical treatment of varicocele, and we did not evaluate DNA fragmentation and the levels of ROS. Furthermore, latest evidences revealed that evaluating OS can be a diagnostic tool in predicting the best responders to supplementation (Vessey et al., 2016). Oxidative stress is a cause of male infertility with significant negative effect on semen parameters, and varicocele is not only causing OS, but also an additional cause of poor sperm quality. The use of carnitines and other functional substances can form part of an efficacious strategy to manage and treat male infertility in both non-varicocele and varicocele subjects. Indeed, we plan future studies to examine the role of energy metabolism, OS and ROS in particular to gain a better understanding of underlying mechanisms and thereby to determine the best strategies for male infertility treatment.

G. M. Busetto IB http://orcid.org/0000-0002-7291-0316 A. Agarwal 3 http://orcid.org/0000-0003-0585-1026 E. De Berardinis 3 https://orcid.org/0000-0003-1498-2810

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How to cite this article: Busetto GM, Agarwal A, Virmani A, et al. Effect of metabolic and antioxidant supplementation on sperm parameters in oligo-astheno-teratozoospermia, with and without varicocele: A double-blind placebo-controlled study. Andrologia. 2018;e12927. https://doi.org/10.1111/and.12927