Supplementation of biotin to sperm preparation medium increases the motility and longevity in cryopreserved human spermatozoa
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- Kalthur, G., Salian, S.R., Keyvanifard, F. et al. J Assist Reprod Genet (2012) 29: 631. doi:10.1007/s10815-012-9760-8
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To study the effect of supplementing biotin to sperm preparation medium on the motility of frozen-thawed spermatozoa.
Semen samples of men attending the University infertility clinic (n = 105) were cryopreserved using glycerol-egg yolk-citrate buffered cryoprotective medium in liquid nitrogen. After a period of two weeks, the semen samples were thawed and the motile spermatozoa were extracted by swim-up technique using Earle’s balanced salt solution (EBSS) medium supplemented with either biotin (10 nM) or pentoxifylline (1 mM). The post-wash motility was observed up to 4 h after incubation.
Both biotin and pentoxifylline supplementation resulted in significant increase in total motility (p < 0.05), progressive motility (p < 0.001) and rapid progressive motility (p < 0.05 v/s biotin and p < 0.01 v/s pentoxifylline) compared to the control at 1 h post-incubation period. Significantly higher percentage of total (p < 0.01, p < 0.05 in biotin and pentoxifylline respectively), progressive (p < 0.001) and rapid progressive motilities (p < 0.01) were observed in these two groups even at 2 h compared to the control. In the control group at 4 h after incubation, ~11% decline in total motility and ~8% decline in progressive motility was observed. However, in both biotin and pentoxifylline group the motility was significantly higher than control (p < 0.001). No significant difference in the motility was observed between biotin and pentoxifylline groups at any of the time intervals studied.
Biotin can enhance the sperm motility and prolong the survival of frozen-thawed semen samples which may have potential benefit in assisted reproductive technology field.
Poor structural and functional characteristics of spermatozoa are common consequences of human semen cryopreservation. Efforts have been made to increase the post-thaw motility and sperm function by using pharmacological agents, vitamins and antioxidants. Among these, pentoxifylline is currently the most widely used pharmacological agent, which has been shown to enhance the motility and longevity of spermatozoa under in vitro conditions . However, the studies suggesting toxic effect of pentoxifylline on embryo development [2–4] have raised concern about its application in human assisted reproductive technology (ART) set up. Therefore, the search for safe alternate agents that can enhance the sperm function without exerting any toxic effect on gametes and embryos is of clinical significance.
Biotin (vitamin B7 or vitamin H), is an essential micronutrient required for the normal growth and development of the body. It acts as a coenzyme in all carboxylation reactions involved in the biosynthesis of fatty acids, gluconeogenesis, metabolism of the branched-chain amino acids and de novo synthesis of purine nucleotides. Studies have demonstrated that deficiency of biotin during the gestation period leads to teratogenicity in mouse  and hamster  indicating its role in reproduction and development. However, its effect on sperm function was not known until now. In the present investigation, we report for the first time that biotin can act as a promising sperm motility enhancer under in vitro conditions.
Materials and methods
The semen samples were collected from 105 men (mean age of 33.48 ± 2.1 years), who visited the University infertility clinic for semen analysis between 2008 and 2010. The study was approved by the Institutional Ethical Committee. The subjects were explained about the study, and a written consent was taken from them.
Men with sexual abstinence of 3–5 days were asked to provide their semen samples in a sterile container by masturbation. Out of 105 samples, 70 were normozoospermic subjects and 35 men were with abnormal semen parameters. After liquefaction of the ejaculate, sperm density was determined using Makler’s counting chamber (Sefi Instruments, Israel). Sperm motility and morphology were assessed according to WHO criteria .
Effect of different concentration of biotin supplemented to sperm preparation medium on progressive motility in normozoospermic semen samples (n = 10)
Incubation time (h)
Percentage progressive motility (Mean ± SE)
Concentration of biotin (nM)
86.25 ± 4.33
86.25 ± 6.81
91.25 ± 2.77
91.63 ± 3.34
91.63 ± 2.31
90.50 ± 3.21
86.75 ± 4.72
93.33 ± 2.17*
83.25 ± 2.74
85.88 ± 1.78
89.00 ± 4.55
88.38 ± 3.77
87.38 ± 1.83
85.00 ± 4.22
84.38 ± 3.59
86.67 ± 3.44**
80.38 ± 3.04
82.50 ± 2.20
88.50 ± 1.82*
86.75 ± 2.10**
83.63 ± 2.35
82.88 ± 1.92
79.88 ± 2.03
73.00 ± 4.91
During cryopreservation, semen samples were mixed with equal volume of glycerol- egg yolk-citrate buffered freezing medium and stored in liquid nitrogen as described elsewhere . To avoid observer bias, all the cryo-vials were coded by a person who was not aware of the study. After two weeks, the frozen samples were rapidly thawed at 37°C and divided into three equal parts. The cryoprotectants were completely removed by mixing the sperm suspension with 2 ml of EBSS supplemented with HSA and by centrifuging at 1800 rpm for 8 min. The motile spermatozoa were extracted from the pellet by swim-up technique . Briefly, the sperm pellet was carefully overlaid with 200–300 μl of EBSS medium supplemented with HSA, EBSS with 10 nM biotin (Fluka, Cat. No. 14400) or EBSS with 1 mM pentoxifylline (Sigma, Cat. No. P1784).
The influence of biotin supplementation to sperm preparation medium was assessed by motility evaluation at 1, 2 and 4 hours after incubation at 37°C and 5% CO2 (Hera Cell 150i, Germany). About 10 μl of sperm suspension was placed on a clean glass slide, and a cover slip was placed carefully over it. A total of 200 spermatozoa was scored for the motility evaluation using the light microscope (Olympus, India) under 400X magnification, and graded as rapid progressive (grade a), slow or sluggish (grade b), non-progressive (grade c) and immotile (grade d) spermatozoa .
The motility data are presented as Mean±SE and analyzed by one-way ANOVA using GraphPadInstat 3.0 software (USA). Multiple comparisons were made using Bonferroni procedure and the values with P < 0.05 were considered as statistically significant.
Out of the 105 study subjects, 70 were normozoospermic and 35 were with abnormal semen parameters (25 asthenozoospermic, 1 oligozoospermic, 6 oligoasthenozoospermic, 2 asthenoteratozoospermic and 1 oligoasthenoteratozoospermic). The study group had a mean sperm density of 49.56 ± 3.28 Millions/ml, 59.51 ± 2.09% total motility, 42.54 ± 2.2% progressive motility, 60.54 ± 2.48% viability and 26.29 ± 1.62% spermatozoa with normal morphology. The post-wash findings in frozen-thawed spermatozoa processed in the presence of biotin or pentoxifylline did not show any significant difference between normozoospermic and semen samples with abnormal parameters. Hence, the results are discussed below taking both normozoospermic and abnormal group together.
Decrease in motility and poor survival of frozen-thawed spermatozoa has been consistently observed in earlier studies [11, 12]. Improving the motility in frozen-thawed spermatozoa and maintaining their survival till fertilization is a real challenge in assisted reproduction. In the present investigation, we observed a time-dependent decline in rapid progressive motility as well as total motility in frozen-thawed spermatozoa under in vitro conditions.
Pentoxifylline has been used extensively as a sperm motility enhancer in fresh ejaculates, post-thaw semen samples and testicular sperm [10, 13, 14]. The clinical experience with pentoxifylline has contradictory reports in the literature. Even though a majority of the studies found a significant improvement in the motility after pentoxifylline treatment [10, 13–15], few have observed either no improvement  or adverse effect . However, our observations confirm that pentoxifylline can significantly enhance the motility of spermatozoa subjected to freeze-thaw process, both in normozoospermic and abnormal semen samples. The enhanced motility induced by pentoxifylline is due to its inhibitory effect on phosphodiesterase, which in turn helps in maintaining a high intracellular cyclic adenosine monophosphate (cAMP) level in the spermatozoa . A marginal decline in motility with the increase in the incubation time observed in pentoxifylline group may be due to the failure of maintaining an elevated level of cAMP in the cytoplasm of the frozen-thawed spermatozoa.
In the present study we observed that supplementation of biotin to sperm preparation medium not only resulted in significant enhancement in the sperm motility but also maintained the motility until 4 hours of incubation. In addition, the biotin supplemented group maintained significantly higher percentage of sperm with rapid progressive motility even at 4 h after incubation. The motility was marginally higher in the biotin group compared to pentoxifylline group at all the time intervals studied, which indicates its potential application as sperm motility enhancer.
From this preliminary data, it is difficult to elucidate the exact mechanism of action of biotin. Nonetheless, using an indirect approach, we made an attempt to know whether the effect is mediated through inhibition of phosphodiesterase enzyme activity. The sperm motility was inhibited by addition of different concentration of phosphodiesterase IV to the sperm suspension extracted by swim-up technique. The motility did not improve even after adding different concentrations of biotin to phosphodiesterase-treated sperm suspension (unpublished data). This suggests that biotin-induced motility enhancement may not be mediated through the inhibition of phosphodiesterase enzyme. However, further studies are required to elucidate the exact molecular mechanism behind biotin-induced sperm motility enhancement.
The nutritional value of biotin has been well established. Earlier studies have shown that biotin deficiency can cause teratogenesis in lower species of animals  and in humans [19, 20]. Reduced activity of the biotin-dependent enzymes alters the lipid metabolism and can lead to biotin-responsive inborn errors of lipid metabolism [21, 22]. Now it is clearly evident that biotin has a significant role to play in regulation of chromatin structure , heme synthesis  and prevention of DNA damage . Zempleni and Mock  suggest that teratogenic effect of biotin deficiency in mice and humans is due to the genomic instability caused by decrease in biotinylated histones. Incidence of the birth defects was found to reduce when the women were given multivitamin supplements (including biotin) prior to and during pregnancy . These evidences clearly emphasize the nutritional importance of biotin during pregnancy or fetal development. However, its effect on sperm function enhancement was not known until now.
In conclusion, our novel finding has demonstrated that supplementing biotin to sperm preparation medium can significantly enhance the post-thaw sperm motility and their longevity in vitro. In our view, since biotin is an essential micronutrient it may not exert any toxic effect on sperm or the developing embryo at the tested concentration (10 nM). Therefore, biotin may be a promising and safe alternative agent to enhance the post-thaw sperm quality in an ART set up.
Authors kindly acknowledge the technical support provided by Mrs. Jalyalaxmi Pai, Mrs. Kirthi Patil, and Ms. Anita Poojary. Authors thank Dr. Prashanth Naik and Mr. Naveenchandra Kumar, Mangalore University for editing the language of the manuscript.