http://informahealthcare.com/gye ISSN: 0951-3590 (print), 1473-0766 (electronic) Gynecol Endocrinol, 2014; 30(5): 359–362 ! 2014 Informa UK Ltd. DOI: 10.3109/09513590.2013.879856
IVF
Oral melatonin supplementation improves oocyte and embryo quality in women undergoing in vitro fertilization-embryo transfer Takuji Nishihara1, Shu Hashimoto1, Keijiro Ito1, Yoshiharu Nakaoka1, Kazuya Matsumoto2, Yoshihiko Hosoi2, and Yoshiharu Morimoto1 IVF Namba Clinic, Osaka, Japan and 2Graduate School of Biology-Oriented Science and Technology, Kinki University, Wakayama, Japan
Abstract
Keywords
The aim of this study was to evaluate the efficacy of oral melatonin supplementation on oocyte and embryo quality in patients in an assisted reproductive technologies program. All patients were treated for at least 2 weeks with melatonin (3 mg/day). To evaluate the cumulative effect of melatonin supplementation, we compared cycle outcomes between the first (no supplementation) and second cycles (melatonin supplementation) of patients who completed two treatment cycles. There were no significant differences in maturation rates (p ¼ 0.50), blastocyst rates (p ¼ 0.75), and the rate of good quality blastocysts (p ¼ 0.59) between the first and second cycles. The fertilization rate of ICSI was higher in the second cycle than that in the first cycle (69.3 versus 77.5%). Being limited to patients with a low fertilization rate in the first cycle (560%), the fertilization rate dramatically increased after melatonin treatment (35.1 versus 68.2%). The rate of good quality embryos also increased (48.0 versus 65.6%). An important finding in our study was that oral melatonin supplementation can have a beneficial effect on the improvement of fertilization and embryo quality and this may have occurred due to a reduction in oxidative damage.
Assisted reproductive technology, melatonin, oxidative stress, reactive oxygen species
Introduction Assisted reproductive technologies (ART) are widely accepted procedures for the treatment of infertility. Unfortunately, the success rate of in vitro fertilization and embryo transfer (IVF-ET) still remains limited to 30–40%. Reactive oxygen species (ROS) have been considered to play multiple roles in female reproduction [1,2]. ROS are generated especially during the ovulatory process. Some studies have indicated that oxidative stress may be a cause of poor oocyte and embryo quality [3,4]. ROS have adverse effects leading to DNA damage, lipid peroxidation, and protein damage. Besides their adverse effects, accumulating data have shown that controlled and adequate ROS concentrations exert physiological functions [5]. Oxidative stress is caused by an imbalance between ROS generation and antioxidant capacity. Melatonin, or N-acetyl-5-methoxytryptamine, is a hormone produced in the pineal gland at the base of the brain and is also an important regulator of seasonal reproduction and circadian rhythms. Melatonin has the ability to powerfully scavenge the free radicals that cause oxidative damage, and its metabolites are potent direct free radical scavengers [6] and also indirect antioxidants by virtue of their ability to modulate gene transcription for antioxidant enzymes [7]. Recent studies have demonstrated that oxidative stress has toxic effects on oocyte quality and fertilization rate from oxidative stress [8], and melatonin oral supplementation increases intra-follicular melatonin concentrations, reduces intra-follicular oxidative damage, and improves fertilization rate, pregnancy rates [9], and oocyte and embryo
Address for correspondence: Takuji Nishihara, IVF Namba Clinic, Osaka, Japan. E-mail: [email protected]
History Received 16 January 2013 Revised 9 October 2013 Accepted 30 December 2013 Published online 17 March 2014
quality [10]. Human preovulatory follicular fluids contain higher concentrations of melatonin than that of plasma, and it has been shown that melatonin concentrations in follicular fluids increased depending on follicular growth [11]. Thus, melatonin in preovulatory follicular fluids seems to have an important role in oocyte maturation and embryo development. However, few data exist regarding the cumulative effect of melatonin on cycle outcomes with and without melatonin supplementation. The aim of this study was to assess the potential benefit of oral melatonin supplementation for poor fertilization and embryo quality patients in an ART program.
Materials and methods Study population
20 14
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1
Patients with a poor oocyte and embryo quality in a previous IVF cycle were included in the study. They had experienced in a previous IVF cycle, which had obtained less than 60% fertilization rate, had not obtained good quality embryos, and had not reached to the blastocyst stage. Exclusion criteria were patients over the age of 42 years and patients who received melatonin at any time before enrollment. Approval for this study was obtained from the local Ethics Committee of the IVF Japan group and expressed informed consent was obtained from couples before IVF-ET treatment. Follicle stimulation protocol This study was designed for two consecutive IVF cycles. All patients began taking 3 mg of melatonin (Pure Encapsulations, MA, USA) orally, once a day, for at least 2 weeks, ending on the day of human chorionic gonadotropin (hCG) injection at the
360
T. Nishihara et al.
Gynecol Endocrinol, 2014; 30(5): 359–362
second cycle. All patients were stimulated with a standard IVF protocol. Patients were administered the GnRH agonist buserelin (Suprecur nasal solution 0.15%; Sanofi-Aventis, Tokyo, Japan) at a daily dose of 600 mg starting in the mid-luteal phase of the preceding cycle. All patients were treated with injections of 300 IU recombinant FSH on the third day of the cycle (Gonal-FÕ, EMD Serono, Inc., Merck Serono, Darmstadt, Germany). The dose of the GnRH-agonist was continued until the day of hCG injection (human chorionic gonadotropin for injection; Fuji Pharma., Tokyo, Japan).
Gynecol Endocrinol Downloaded from informahealthcare.com by University of Laval on 06/19/14 For personal use only.
IVF procedure Oocyte retrieval was performed by transvaginal aspiration 36 h after hCG injection with an 18 gauge single lumen needle (Smiths Medical, MN, USA). All oocytes were inseminated in GM-HTF (Gynemed GmbH & Co. KG, Lensahn, Germany) or injected with a sperm using the standard ICSI technique. Fertilization was confirmed 14–16 h after insemination or ICSI by the presence of two pronuclei and extrusion of the second polar body. Fertilized oocytes were cultured in groups of a maximum of 4 oocytes in 1 mL of globalÕ totalÔ (LifeGlobal, Ontario, Canada) until day 5. Embryo and blastocyst quality classification The embryos were classified according to the criteria proposed by Veeck’s criteria. A good quality embryo was defined as one that had reached the four-cell stage on day 2, and reached the seven-cell stage on day 3, and had less than 20% of its volume filled with fragments. A good quality blastocyst was defined as full blastocyst stage onward on day 5 and not including the inner cell mass and the trophectoderm with very few cells. Study end-points To evaluate the cumulative effect of melatonin supplementation during the study period, we compared cycle outcomes between the first (no supplementation) and second cycles (melatonin supplementation) of patients who completed two treatment cycles. Primary outcome measures were rates of maturation, fertilization, good quality embryos, blastocyst, and good quality blastocysts. Statistical analysis Stat view Version 5.0 (SAS Institute Inc., NC, USA) was used for all statistical analyses. Continuous variables were presented as
mean and SD and analyzed by Student’s paired t-test. All tests were two tailed and a p value of 50.05 was considered significant.
Results This study was conducted between May 2009 and December 2012 at the IVF Namba Clinic. To evaluate the cumulative effect of melatonin, we compared cycle outcomes between the first and second cycles of patients. This study used data from a total of 194 cycles in 97 patients. Of those, 78 were inseminated by ICSI and 19 by conventional IVF (cIVF) in the no supplementation group. In the melatonin supplementation group, 83 cycles were inseminated by ICSI and 14 by cIVF. The method of insemination was changed from cIVF in the first cycle to ICSI in the second cycle for 9 patients because of the lack of adequate motile sperm, and changed from ICSI in the first cycle to cIVF in the second cycle for 4 patients. These 13 patients were excluded in the analysis of fertilization. There were no significant differences in estradiol levels on the day of hCG (p ¼ 0.16) or in the number of retrieved oocytes (p ¼ 0.10) between the first and second cycles (Table 1). There were no significant differences between the first and second cycles in the maturation rate on the day of oocyte retrieval in the ICSI cycles (p ¼ 0.50) (Table 2). The fertilization rate of ICSI was higher in the second cycle than that in the first cycle (control: 69.3 ± 29.4 versus melatonin: 77.5 ± 21.8%, respectively; p50.05). The fertilization rate of cIVF was no significant difference between the first and second cycles (p ¼ 0.25). Being limited to patients with a low fertilization rate in the first cycle (560%) to evaluate the effect of melatonin in poor fertilization cases, the fertilization rate dramatically increased after melatonin treatment (control: 35.1 ± 21.5 versus melatonin: 68.2 ± 22.0%, respectively; p50.01). The rate of good quality embryos also increased (control: 48.0 ± 30.3 versus melatonin: 65.6 ± 26.1%, respectively; p50.01). Thirty-eight patients were excluded in the analysis of good quality embryo because oocytes were cryopreserved at the pronuclear stage in the first and/or second cycle. There were no significant differences between the first and second cycles in blastocyst rates (p ¼ 0.75) or the rate of good-quality blastocysts (p ¼ 0.59). Twenty-eight patients were excluded in the analysis of blastocyst rate because embryos were cryopreserved, and also three patients were excluded in the analysis of the rate of good
Table 1. Characteristics of the two consecutive cycles.
Inseminations (cycles) E2 on the day of hCG (pg/ml) Number of retrieved oocytes
First cycle (n ¼ 97)
Second cycle (n ¼ 97)
p Value
ICSI 78 cIVF 19 2387.5 ± 2180.8 9.7 ± 7.5
ICSI 83 cIVF 14 2143.1 ± 1741.1 9.0 ± 6.1
0.29 0.22
Values are means ± SEM. cIVF, conventional IVF; E2, estradiol. Table 2. Comparison of the primary outcomes between the two consecutive cycles.
Maturation rate (%) (n ¼ 74) Fertilization rate of ICSI (%) (n ¼ 74) Fertilization rate of cIVF (%) (n ¼ 10) Fertilization rate (%) (560% at first cycle) (n ¼ 25) Rate of good quality embryos (%) (n ¼ 46) Blastocyst rate (%) (n ¼ 18) Rate of good quality blastocysts (%) (n ¼ 15) Values are means ± SEM.
First cycle
Second cycle
p Value
79.9 ± 34.5 69.3 ± 29.4 68.1 ± 28.9 35.1 ± 21.5 48.0 ± 30.3 44.9 ± 26.2 16.7 ± 27.6
82.6 ± 24.6 77.5 ± 21.8 80.8 ± 19.4 68.2 ± 22.0 65.6 ± 26.1 43.1 ± 15.9 21.2 ± 25.5
0.50 50.05 0.25 50.01 50.01 0.75 0.59
Melatonin in IVF
DOI: 10.3109/09513590.2013.879856
blastocyst because the embryos had not reached blastocyst stage in the first and/or second cycle.
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Discussion In this study, we demonstrated the potential benefit of melatonin supplementation for poor fertilization and embryo quality patients. Regarding pre-treatment with melatonin, there were no significant differences in maturation rate between the first and second cycles. Oocytes within ovarian follicles begin meiosis during embryogenesis, but then underwent arrest at prophase of meiosis I until transient elevations in luteinizing hormone. During the growth phase, oocytes synthesize and accumulate RNA, proteins, and organelles (e.g. mitochondria), as well as oxidative stress [12]. Melatonin was also reported to enhance meiotic maturation of porcine [13], buffalo [14], and mouse oocytes in vitro [15,16]. Larger studies are needed to confirm the effectiveness of melatonin supplementation on oocyte maturation rate. The fertilization rate of ICSI increased after melatonin treatment. Similar results on the clinical usefulness of melatonin administration have been previously reported [8]. They concluded that in patients who failed to become pregnant in the previous IVF-ET cycle with a low fertilization rate (550%), the fertilization rate was markedly higher than that in the previous IVF-ET cycle with melatonin administration. In their other study, intra-follicular concentrations of eight-hydoxy-20 -deoxyguanosine (8-OHdG), which is a sensitive indicator of DNA damage as the result of oxidative stress, was found to be reduced with melatonin administration [17]. We also previously demonstrated that low molecular antioxidant levels were found to be lower and 8-OHdG was found to be higher in patients who had low fertilization rates (manuscript in preparation). For these reasons, decreasing oxidative stress may be an important factor in determining oocyte quality in the environment of preovulatory follicular fluids. Our study also demonstrated a significant improvement in embryo quality. Evidence for potentially promoting oocyte and embryo quality in other species has been previously reported. Tian et al. [18] demonstrated the beneficial effects of melatonin on in vitro development of 2-cell mouse embryos cultured in HTF medium. Rodriguez-Osorio et al. [19] also demonstrated that melatonin has a positive effect on porcine embryo cleavage rates and blastocyst cell numbers. Taken together, these data suggest that melatonin supplementation may have a beneficial effect on oocyte and embryo development. Poor oocyte quality is the cause of infertility in women and results in polyspermy, arrested embryonic development, or spontaneous abortion. Thus, melatonin supplementation is likely to become a treatment for improving oocyte quality in women who cannot become pregnant because of poor oocyte quality. There was no significant difference in blastocyst quality between the first and second cycles. Few data exist regarding the effect of melatonin on blastocyst development. In a study on culture mediums, Papis et al. demonstrated that a relatively short period (48 h) of in vitro culture with melatonin may have significant effects on the final development rate and, to some extent, on the quality (cell number) of bovine embryos. They concluded that beneficial or harmful effects of melatonin on bovine embryo development in vitro were observed depending on oxygen tension during treatment [20]. Hence, under physiological oxygen tension (7%), embryos cultured with melatonin were presumably suppressed below an optimum level necessary for blastocyst culture. An important finding in our study was the fact that melatonin supplementation can have a beneficial effect on the improvement of fertilization and embryo quality and this may have occurred
361
due to a reduction in oxidative damage. These results are consistent with recent studies [9,10,21,22]. Carr et al. demonstrated the long-term safety of melatonin use. They reported open label follow-up data of 41 cases of the safety of melatonin use (mean duration of 4.3 year) in 50 children. They concluded that there were no reported late-onset adverse events [23]. Melatonin should be considered for poor fertilization and embryo quality patients due to its safety and simplicity of use. Additional studies are needed to confirm these results.
Declaration of interest The authors report no declarations of interest.
References 1. Fujii J, Iuchi Y, Okada F. Fundamental roles of reactive oxygen species and protective mechanisms in the female reproductive system. Reprod Biol Endocrinol 2005;3:43–52. 2. Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol 2005;3:28. 3. Guerin P, El Mouatassim S, Menezo Y. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update 2001;7:175–89. 4. Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 2003;79:829–43. 5. Rizzo A, Roscino M, Binetti F, Sciorsci R. Roles of reactive oxygen species in female reproduction. Reprod Domest Anim 2012;47:344–52. 6. Zavodnik IB, Domanski AV, Lapshina EA, et al. Melatonin directly scavenges free radicals generated in red blood cells and a cell-free system: chemiluminescence measurements and theoretical calculations. Life Sci 2006;79:391–400. 7. Tomas-Zapico C, Coto-Montes A. A proposed mechanism to explain the stimulatory effect of melatonin on antioxidative enzymes. J Pineal Res 2005;39:99–104. 8. Tamura H, Takasaki A, Miwa I, et al. Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate. J Pineal Res 2008;44: 280–7. 9. Tamura H, Takasaki A, Taketani T, et al. The role of melatonin as an antioxidant in the follicle. J Ovarian Res 2012;5:5. 10. Batıog˘lu AS, Sahin U, Gu¨rlek B, et al. The efficacy of melatonin administration on oocyte quality. Gynecol Endocrinol 2012;28:91–3. 11. Nakamura Y, Tamura H, Takayama H, Kato H. Increased endogenous level of melatonin in preovulatory human follicles does not directly influence progesterone production. Fertil Steril 2003;80: 1012–16. 12. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod 2006; 74:585–92. 13. Kang JT, Koo OJ, Kwon DK, et al. Effects of melatonin on preimplantation development of porcine parthenogenetic embryos. Reprod Fertil Dev 2009;22:327–8. 14. Manjunatha BM, Devaraj M, Gupta PSP, et al. Effect of taurine and melatonin in the culture medium on buffalo in vitro embryo development. Reprod Domest Anim 2009;44:12–16. 15. Ahn HJ, Bae IH. Effects of melatonin on the meiotic maturation of mouse oocytes in vitro. Korean J Fertil Steril 2004;31: 155–68. 16. Na K, Kim J, Lee J, et al. Effect of melatonin on the maturation of mouse GV oocytes and apoptosis of cumulus cells in vitro. Fertil Steril 2005;84:S103Abst. 17. Tamura H, Takasaki A, Miwa I, et al. Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate. J Pineal Res 2007;44: 280–7. 18. Tian XZ, Wen Q, Shi JM, et al. Effects of melatonin on in vitro development of mouse two-cell embryos cultured in HTF medium. Endocr Res 2010;35:17–23. 19. Rodriguez-Osorio N, Kim IJ, Wang H, et al. Melatonin increases cleavage rate of porcine preimplantation embryos in vitro. J Pineal Res 2007;43:283–8.
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20. Papis K, Poleszczuk O, Wenta-Muchalska E, Modlinski JA. Melatonin effect on bovine embryo development in vitro in relation to oxygen concentration. J Pineal Res 2007;43:321–6. 21. Unfer V, Raffone E, Rizzo P, Buffo S. Effect of a supplementation with myo-inositol plus melatonin on oocyte quality in women who failed to conceive in previous in vitro fertilization cycles for poor oocyte quality: a prospective, longitudinal, cohort study. Gynecol Endocrinol 2011;27:857–61.
Gynecol Endocrinol, 2014; 30(5): 359–362
22. Eryilmaz OG, Devran A, Sarikaya E, et al. Melatonin improves the oocyte and the embryo in IVF patients with sleep disturbances, but does not improve the sleeping problems. J Assist Reprod Genet 2011;28:815–20. 23. Carr R, Wasdell MB, Hamilton D, et al. Long-term effectiveness outcome of melatonin therapy in children with treatmentresistant circadian rhythm sleep disorders. J Pineal Res 2007;43: 351–9.
IVF
Oral melatonin supplementation improves oocyte and embryo quality in women undergoing in vitro fertilization-embryo transfer Takuji Nishihara1, Shu Hashimoto1, Keijiro Ito1, Yoshiharu Nakaoka1, Kazuya Matsumoto2, Yoshihiko Hosoi2, and Yoshiharu Morimoto1 IVF Namba Clinic, Osaka, Japan and 2Graduate School of Biology-Oriented Science and Technology, Kinki University, Wakayama, Japan
Abstract
Keywords
The aim of this study was to evaluate the efficacy of oral melatonin supplementation on oocyte and embryo quality in patients in an assisted reproductive technologies program. All patients were treated for at least 2 weeks with melatonin (3 mg/day). To evaluate the cumulative effect of melatonin supplementation, we compared cycle outcomes between the first (no supplementation) and second cycles (melatonin supplementation) of patients who completed two treatment cycles. There were no significant differences in maturation rates (p ¼ 0.50), blastocyst rates (p ¼ 0.75), and the rate of good quality blastocysts (p ¼ 0.59) between the first and second cycles. The fertilization rate of ICSI was higher in the second cycle than that in the first cycle (69.3 versus 77.5%). Being limited to patients with a low fertilization rate in the first cycle (560%), the fertilization rate dramatically increased after melatonin treatment (35.1 versus 68.2%). The rate of good quality embryos also increased (48.0 versus 65.6%). An important finding in our study was that oral melatonin supplementation can have a beneficial effect on the improvement of fertilization and embryo quality and this may have occurred due to a reduction in oxidative damage.
Assisted reproductive technology, melatonin, oxidative stress, reactive oxygen species
Introduction Assisted reproductive technologies (ART) are widely accepted procedures for the treatment of infertility. Unfortunately, the success rate of in vitro fertilization and embryo transfer (IVF-ET) still remains limited to 30–40%. Reactive oxygen species (ROS) have been considered to play multiple roles in female reproduction [1,2]. ROS are generated especially during the ovulatory process. Some studies have indicated that oxidative stress may be a cause of poor oocyte and embryo quality [3,4]. ROS have adverse effects leading to DNA damage, lipid peroxidation, and protein damage. Besides their adverse effects, accumulating data have shown that controlled and adequate ROS concentrations exert physiological functions [5]. Oxidative stress is caused by an imbalance between ROS generation and antioxidant capacity. Melatonin, or N-acetyl-5-methoxytryptamine, is a hormone produced in the pineal gland at the base of the brain and is also an important regulator of seasonal reproduction and circadian rhythms. Melatonin has the ability to powerfully scavenge the free radicals that cause oxidative damage, and its metabolites are potent direct free radical scavengers [6] and also indirect antioxidants by virtue of their ability to modulate gene transcription for antioxidant enzymes [7]. Recent studies have demonstrated that oxidative stress has toxic effects on oocyte quality and fertilization rate from oxidative stress [8], and melatonin oral supplementation increases intra-follicular melatonin concentrations, reduces intra-follicular oxidative damage, and improves fertilization rate, pregnancy rates [9], and oocyte and embryo
Address for correspondence: Takuji Nishihara, IVF Namba Clinic, Osaka, Japan. E-mail: [email protected]
History Received 16 January 2013 Revised 9 October 2013 Accepted 30 December 2013 Published online 17 March 2014
quality [10]. Human preovulatory follicular fluids contain higher concentrations of melatonin than that of plasma, and it has been shown that melatonin concentrations in follicular fluids increased depending on follicular growth [11]. Thus, melatonin in preovulatory follicular fluids seems to have an important role in oocyte maturation and embryo development. However, few data exist regarding the cumulative effect of melatonin on cycle outcomes with and without melatonin supplementation. The aim of this study was to assess the potential benefit of oral melatonin supplementation for poor fertilization and embryo quality patients in an ART program.
Materials and methods Study population
20 14
Gynecol Endocrinol Downloaded from informahealthcare.com by University of Laval on 06/19/14 For personal use only.
1
Patients with a poor oocyte and embryo quality in a previous IVF cycle were included in the study. They had experienced in a previous IVF cycle, which had obtained less than 60% fertilization rate, had not obtained good quality embryos, and had not reached to the blastocyst stage. Exclusion criteria were patients over the age of 42 years and patients who received melatonin at any time before enrollment. Approval for this study was obtained from the local Ethics Committee of the IVF Japan group and expressed informed consent was obtained from couples before IVF-ET treatment. Follicle stimulation protocol This study was designed for two consecutive IVF cycles. All patients began taking 3 mg of melatonin (Pure Encapsulations, MA, USA) orally, once a day, for at least 2 weeks, ending on the day of human chorionic gonadotropin (hCG) injection at the
360
T. Nishihara et al.
Gynecol Endocrinol, 2014; 30(5): 359–362
second cycle. All patients were stimulated with a standard IVF protocol. Patients were administered the GnRH agonist buserelin (Suprecur nasal solution 0.15%; Sanofi-Aventis, Tokyo, Japan) at a daily dose of 600 mg starting in the mid-luteal phase of the preceding cycle. All patients were treated with injections of 300 IU recombinant FSH on the third day of the cycle (Gonal-FÕ, EMD Serono, Inc., Merck Serono, Darmstadt, Germany). The dose of the GnRH-agonist was continued until the day of hCG injection (human chorionic gonadotropin for injection; Fuji Pharma., Tokyo, Japan).
Gynecol Endocrinol Downloaded from informahealthcare.com by University of Laval on 06/19/14 For personal use only.
IVF procedure Oocyte retrieval was performed by transvaginal aspiration 36 h after hCG injection with an 18 gauge single lumen needle (Smiths Medical, MN, USA). All oocytes were inseminated in GM-HTF (Gynemed GmbH & Co. KG, Lensahn, Germany) or injected with a sperm using the standard ICSI technique. Fertilization was confirmed 14–16 h after insemination or ICSI by the presence of two pronuclei and extrusion of the second polar body. Fertilized oocytes were cultured in groups of a maximum of 4 oocytes in 1 mL of globalÕ totalÔ (LifeGlobal, Ontario, Canada) until day 5. Embryo and blastocyst quality classification The embryos were classified according to the criteria proposed by Veeck’s criteria. A good quality embryo was defined as one that had reached the four-cell stage on day 2, and reached the seven-cell stage on day 3, and had less than 20% of its volume filled with fragments. A good quality blastocyst was defined as full blastocyst stage onward on day 5 and not including the inner cell mass and the trophectoderm with very few cells. Study end-points To evaluate the cumulative effect of melatonin supplementation during the study period, we compared cycle outcomes between the first (no supplementation) and second cycles (melatonin supplementation) of patients who completed two treatment cycles. Primary outcome measures were rates of maturation, fertilization, good quality embryos, blastocyst, and good quality blastocysts. Statistical analysis Stat view Version 5.0 (SAS Institute Inc., NC, USA) was used for all statistical analyses. Continuous variables were presented as
mean and SD and analyzed by Student’s paired t-test. All tests were two tailed and a p value of 50.05 was considered significant.
Results This study was conducted between May 2009 and December 2012 at the IVF Namba Clinic. To evaluate the cumulative effect of melatonin, we compared cycle outcomes between the first and second cycles of patients. This study used data from a total of 194 cycles in 97 patients. Of those, 78 were inseminated by ICSI and 19 by conventional IVF (cIVF) in the no supplementation group. In the melatonin supplementation group, 83 cycles were inseminated by ICSI and 14 by cIVF. The method of insemination was changed from cIVF in the first cycle to ICSI in the second cycle for 9 patients because of the lack of adequate motile sperm, and changed from ICSI in the first cycle to cIVF in the second cycle for 4 patients. These 13 patients were excluded in the analysis of fertilization. There were no significant differences in estradiol levels on the day of hCG (p ¼ 0.16) or in the number of retrieved oocytes (p ¼ 0.10) between the first and second cycles (Table 1). There were no significant differences between the first and second cycles in the maturation rate on the day of oocyte retrieval in the ICSI cycles (p ¼ 0.50) (Table 2). The fertilization rate of ICSI was higher in the second cycle than that in the first cycle (control: 69.3 ± 29.4 versus melatonin: 77.5 ± 21.8%, respectively; p50.05). The fertilization rate of cIVF was no significant difference between the first and second cycles (p ¼ 0.25). Being limited to patients with a low fertilization rate in the first cycle (560%) to evaluate the effect of melatonin in poor fertilization cases, the fertilization rate dramatically increased after melatonin treatment (control: 35.1 ± 21.5 versus melatonin: 68.2 ± 22.0%, respectively; p50.01). The rate of good quality embryos also increased (control: 48.0 ± 30.3 versus melatonin: 65.6 ± 26.1%, respectively; p50.01). Thirty-eight patients were excluded in the analysis of good quality embryo because oocytes were cryopreserved at the pronuclear stage in the first and/or second cycle. There were no significant differences between the first and second cycles in blastocyst rates (p ¼ 0.75) or the rate of good-quality blastocysts (p ¼ 0.59). Twenty-eight patients were excluded in the analysis of blastocyst rate because embryos were cryopreserved, and also three patients were excluded in the analysis of the rate of good
Table 1. Characteristics of the two consecutive cycles.
Inseminations (cycles) E2 on the day of hCG (pg/ml) Number of retrieved oocytes
First cycle (n ¼ 97)
Second cycle (n ¼ 97)
p Value
ICSI 78 cIVF 19 2387.5 ± 2180.8 9.7 ± 7.5
ICSI 83 cIVF 14 2143.1 ± 1741.1 9.0 ± 6.1
0.29 0.22
Values are means ± SEM. cIVF, conventional IVF; E2, estradiol. Table 2. Comparison of the primary outcomes between the two consecutive cycles.
Maturation rate (%) (n ¼ 74) Fertilization rate of ICSI (%) (n ¼ 74) Fertilization rate of cIVF (%) (n ¼ 10) Fertilization rate (%) (560% at first cycle) (n ¼ 25) Rate of good quality embryos (%) (n ¼ 46) Blastocyst rate (%) (n ¼ 18) Rate of good quality blastocysts (%) (n ¼ 15) Values are means ± SEM.
First cycle
Second cycle
p Value
79.9 ± 34.5 69.3 ± 29.4 68.1 ± 28.9 35.1 ± 21.5 48.0 ± 30.3 44.9 ± 26.2 16.7 ± 27.6
82.6 ± 24.6 77.5 ± 21.8 80.8 ± 19.4 68.2 ± 22.0 65.6 ± 26.1 43.1 ± 15.9 21.2 ± 25.5
0.50 50.05 0.25 50.01 50.01 0.75 0.59
Melatonin in IVF
DOI: 10.3109/09513590.2013.879856
blastocyst because the embryos had not reached blastocyst stage in the first and/or second cycle.
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Discussion In this study, we demonstrated the potential benefit of melatonin supplementation for poor fertilization and embryo quality patients. Regarding pre-treatment with melatonin, there were no significant differences in maturation rate between the first and second cycles. Oocytes within ovarian follicles begin meiosis during embryogenesis, but then underwent arrest at prophase of meiosis I until transient elevations in luteinizing hormone. During the growth phase, oocytes synthesize and accumulate RNA, proteins, and organelles (e.g. mitochondria), as well as oxidative stress [12]. Melatonin was also reported to enhance meiotic maturation of porcine [13], buffalo [14], and mouse oocytes in vitro [15,16]. Larger studies are needed to confirm the effectiveness of melatonin supplementation on oocyte maturation rate. The fertilization rate of ICSI increased after melatonin treatment. Similar results on the clinical usefulness of melatonin administration have been previously reported [8]. They concluded that in patients who failed to become pregnant in the previous IVF-ET cycle with a low fertilization rate (550%), the fertilization rate was markedly higher than that in the previous IVF-ET cycle with melatonin administration. In their other study, intra-follicular concentrations of eight-hydoxy-20 -deoxyguanosine (8-OHdG), which is a sensitive indicator of DNA damage as the result of oxidative stress, was found to be reduced with melatonin administration [17]. We also previously demonstrated that low molecular antioxidant levels were found to be lower and 8-OHdG was found to be higher in patients who had low fertilization rates (manuscript in preparation). For these reasons, decreasing oxidative stress may be an important factor in determining oocyte quality in the environment of preovulatory follicular fluids. Our study also demonstrated a significant improvement in embryo quality. Evidence for potentially promoting oocyte and embryo quality in other species has been previously reported. Tian et al. [18] demonstrated the beneficial effects of melatonin on in vitro development of 2-cell mouse embryos cultured in HTF medium. Rodriguez-Osorio et al. [19] also demonstrated that melatonin has a positive effect on porcine embryo cleavage rates and blastocyst cell numbers. Taken together, these data suggest that melatonin supplementation may have a beneficial effect on oocyte and embryo development. Poor oocyte quality is the cause of infertility in women and results in polyspermy, arrested embryonic development, or spontaneous abortion. Thus, melatonin supplementation is likely to become a treatment for improving oocyte quality in women who cannot become pregnant because of poor oocyte quality. There was no significant difference in blastocyst quality between the first and second cycles. Few data exist regarding the effect of melatonin on blastocyst development. In a study on culture mediums, Papis et al. demonstrated that a relatively short period (48 h) of in vitro culture with melatonin may have significant effects on the final development rate and, to some extent, on the quality (cell number) of bovine embryos. They concluded that beneficial or harmful effects of melatonin on bovine embryo development in vitro were observed depending on oxygen tension during treatment [20]. Hence, under physiological oxygen tension (7%), embryos cultured with melatonin were presumably suppressed below an optimum level necessary for blastocyst culture. An important finding in our study was the fact that melatonin supplementation can have a beneficial effect on the improvement of fertilization and embryo quality and this may have occurred
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due to a reduction in oxidative damage. These results are consistent with recent studies [9,10,21,22]. Carr et al. demonstrated the long-term safety of melatonin use. They reported open label follow-up data of 41 cases of the safety of melatonin use (mean duration of 4.3 year) in 50 children. They concluded that there were no reported late-onset adverse events [23]. Melatonin should be considered for poor fertilization and embryo quality patients due to its safety and simplicity of use. Additional studies are needed to confirm these results.
Declaration of interest The authors report no declarations of interest.
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