http://informahealthcare.com/aan ISSN: 1939-6368 (print), 1939-6376 (electronic) Syst Biol Reprod Med, 2014; 60(1): 35–42 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/19396368.2013.855837
RESEARCH ARTICLE
Analysis of inter-examination differences in sperm nuclear vacuoles among male patients with infertility Akira Komiya*, Akihiko Watanabe, Yoko Kawauchi, and Hideki Fuse
Abstract
Keywords
We analyzed the inter-examination differences in sperm nuclear vacuoles among male patients with infertility. We enrolled 56 male patients with infertility who underwent multiple semen analyses and high-magnification observation of the sperm head. A total of 162 ejaculates were evaluated. The average patient age was 34.5 years. Following the conventional semen analysis, the nuclear vacuoles in motile spermatozoa were evaluated at 3700–6150 magnification on an inverted microscope equipped with differential interference contrast optics. A large sperm nuclear vacuole (LNV) was defined as one or more vacuoles with a maximum diameter exhibiting 450% width of the sperm head. We compared the differences in the proportion of spermatozoa with LNVs between two consecutive semen samples before treatment. Treatment-related differences in the number of LNVs were also analyzed. Student’s t-test was used to perform the statistical analyses. No differences were observed in any semen parameters between the first and second ejaculates. On high-magnification microscopy, the proportion of spermatozoa with LNVs was 23.5% and 29.4% (p ¼ 0.0220) in the first and second ejaculates, respectively in 33 patients. Among the 18 patients who underwent varicocele repair using a microsurgical subinguinal approach, the proportion of spermatozoa with LNVs at baseline, three, and six months after surgery was 27.7%, 12.0% (p ¼ 0.0132 versus baseline), and 10.3% (p ¼ 0.0226 versus baseline), respectively. After three months of medical treatment for male infertility in 28 patients, the proportion of spermatozoa with LNVs slightly decreased from 33.3% to 28.6% (p ¼ 0.1276); however, it was not statistically significant. In conclusion, when multiple ejaculates were obtained, in the subset of male patients with infertility, the proportion of spermatozoa with LNVs could be different. The number of LNVs decreased following varicocele repair.
Differential interference contrast optics, male infertility, semen analysis, sperm nuclear vacuole History Received 10 June 2013 Revised 15 August 2013 Accepted 19 August 2013 Published online 13 January 2014
Abbreviations: ART: assisted reproduction technology; IMSI: intracytoplasmic morphologically selected sperm injection; LNV: large nuclear vacuole; MSOME: motile sperm organelle morphology examination.
Introduction In the field of assisted reproduction technology (ART) Bartoov and colleagues developed a method, called the motile sperm organelle morphology examination (MSOME), to assess the detailed morphology of motile spermatozoa in real time at a magnification of up to 6300 [Bartoov et al. 2001]. The use of this method has yielded a definition of normal spermatozoa, and new abnormalities, including sperm head vacuoles, have been described [Bartoov et al. 2002]. The high-magnification observation method has been adapted to select the best spermatozoa for oocyte injection, introducing a new technique termed intracytoplasmic morphologically selected sperm injection (IMSI). Numerous vacuoles have *Address correspondence to Akira Komiya, M.D., Ph.D., Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama, Japan, Tel: +81-76-434-7372, E-mail: [email protected]
been identified inside the sperm nucleus using light microscopy at high-magnification, and significant increases in pregnancy rates and reductions in abortion rates have been obtained by excluding morphologically abnormal sperm from IMSI [Bartoov et al. 2003; Berkovitz et al. 2005, 2006]. With respect to the management of male infertility, we previously reported that large nuclear vacuoles (LNVs) in the sperm head are observed in the majority of ejaculates. In our previous study, the percentage of spermatozoa with LNVs was 28.0 22.14% (mean s.d.) among male patients with infertility. The percentage of spermatozoa with LNVs increased significantly as the semen quality decreased. The number of LNVs exhibited significant negative correlations with various parameters in the patients with male infertility in conventional semen and computer-assisted sperm analyses [Komiya et al. 2013]. Perdrix et al. [2012] also reported a strong correlation between a high relative vacuole area to the sperm head and poor sperm morphology. However, there is a lack of
20 14
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Toyama-shi, Japan
36
A. Komiya et al.
Syst Biol Reprod Med, 2014; 60(1): 35–42
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
information regarding inter-examination differences in LNV during the evaluation or treatment of male infertility. Thus far, only one study has reported the results of a comparison between multiple MSOME findings as an intervariation study of normal sperm and sperm with large nuclear vacuoles [Oliveira et al. 2010a]. A significant positive correlation and the absence of differences were observed with respect to normal morphology and LNV spermatozoa between two sperm samples obtained from each patient evaluated after a time interval. Moreover, no studies reporting treatmentrelated changes in sperm nuclear vacuoles have been published in the literature. In the present study, we report the analyses of interexamination differences in two consecutive semen samples during the evaluation of male infertility. We also evaluated the differences in the number of LNVs associated with surgical and/or medicinal treatment for male infertility.
with processed motile spermatozoa. The number of spermatozoa that could be evaluated for nuclear vacuoles was 57 65 (mean standard deviation/SD) per ejaculate with the available processed motile spermatozoa. This number is higher than that used in our previous report [Komiya et al. 2013]. A total of 7650 spermatozoa were evaluated for sperm nuclear vacuoles. Of these spermatozoa, 859 (11.2%) had LNVs. Comparison of semen parameters and sperm nuclear vacuoles in the first and second ejaculates during male infertility evaluation The mean values of the semen volume, sperm count, sperm motility, and normal sperm morphology in the first versus second ejaculates were 3.0 mL versus 3.1 mL, 31.8 106/mL versus 28.3 106/mL, 20.3% versus 23.5%, and 8.2% versus 10.2%, respectively, in the conventional semen analyses performed during the evaluation of male infertility (Table 2). There were no significant differences between the first and second ejaculates in the parameters evaluated in the conventional semen analyses. However, the percent of LNVs was significantly higher in the second ejaculate (29.4%) than in the first ejaculate (23.5%) in this cohort (p ¼ 0.0220, paired t-test). This finding was observed in the patients with varicocele, but not in those with idiopathic causes of male infertility (Table 2). However, the proportion of spermatozoa with LNVs (% LNVs) was not different between the patients with idiopathic causes of male infertility (29.7%) and palpable varicocele (30.8%; p ¼ 0.90501, unpaired t-test). The interval of the semen analysis was 28.8 28.8 days (mean SD). No correlations were found between the interval of the semen analysis and the change in % LNV.
Results Patient characteristics Among the 56 patients, high-magnification microscopy for LNVs was performed twice during the evaluation of male infertility before any treatment was administered in 33 patients (Table 1) and in 12 patients in whom no LNV data were available during or after treatment. The LNVs were evaluated before and three months after varicocele repair in 18 patients and before and three and six months after varicocele repair in 16 patients. The LNVs were also observed before and after medicinal therapy in 28 patients. The mean patient age was 34.5 years, and the mean duration of infertility was 40.3 months. The mean values of the semen volume, sperm count, sperm motility, and normal sperm morphology at baseline were 3.0 mL, 28.2 106/mL, 22.8%, and 8.3%, respectively (Table 1). These values were not significantly different between the patients with idiopathic causes of male infertility and those with varicocele. We evaluated the sperm nuclear vacuoles in 162 ejaculates using high-magnification microscopy as shown in Figure 1. No motile spermatozoa were found in 28 ejaculates (17.3%) following selection using PureCeptionÔ Determination Kits. At least one spermatozoon with a LNV was found in 130 of 134 ejaculates (97.0%)
Treatment-related changes in sperm nuclear vacuoles in the patients treated with varicocele repair Eighteen patients underwent varicocele repair using a microsurgical subinguinal approach. Among the conventional semen parameters, only sperm motility was significantly increased at three and six months after surgery (p ¼ 0.0003 and 0.0004, respectively, paired t-test). However, the semen
Table 1. Patient characteristics.
N Possible causes of male infertility Idiopathic Varicoceles Hormonal disorders Others Sum
Conventional semen analyses at baseline Semen volume (mL) Sperm count (106/mL) Sperm motility (%) Sperm normal morphology (%) SD: standard deviation
%
Age (years) mean SD
Duration of infertility (months) mean SD 41.4 24.0 38.1 30.6 65.7 25.0 31.2 16.3 40.3 26.6
24 23 3 6 56
42.9 41.1 5.4 10.7 100
34.0 4.2 34.8 3.8 35.3 2.5 34.7 4.1 34.5 3.9
All mean SD
Idiopathic mean SD
Varicoceles mean SD
Hormonal disorders mean SD
Others mean SD
3.0 1.4 28.2 43.6 22.8 17.9 8.3 9.6
3.1 1.3 28.3 41.9 24.0 18.5 9.2 10.3
2.9 1.3 24.0 44.6 22.1 14.3 5.8 6.4
2.5 0.4 19.0 34.5 10.5 9.7 3.8 5.0
2.2 1.9 35.7 35.1 28.9 16.6 14.8 5.9
Inter-examination differences in sperm vacuoles
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
DOI: 10.3109/19396368.2013.855837
37
Figure 1. High-magnification on an inverted microscope equipped with Nomarski differential interference contrast optics and a video system. The arrows indicate nuclear vacuoles observed through a 60 (1.42 numerical aperture) objective lens. (A) Spermatozoa with a nuclear vacuole exhibiting a 450% transverse diameter of the sperm nuclear area. The dotted lines indicate the width of the sperm head (3.1 mm) and the maximum diameter of a nuclear vacuole (1.8 mm). (B) Spermatozoa with a nuclear vacuole exhibiting a 50% transverse diameter of the sperm nuclear vacuole. (C) Spermatozoa without any nuclear vacuoles in the sperm nuclear area.
Table 2. Comparison of the 1st and 2nd ejaculates at evaluation for male infertility.
Conventional semen analysis Semen volume (mL) Sperm count (106/mL) Sperm motility (%) Normal sperm morphology (%)
MSOME All Idiopathic (n ¼ 16) Varicoceles (n ¼ 11) p Value**
1st ejaculate mean SD
2nd ejaculate mean SD
p Value*
3.02 1.5 31.8 49.8 20.3 17.3 8.2 8.6
3.1 1.5 28.3 42.7 23.5 19.7 10.2 11.9
0.2369 0.3059 0.1321 0.2956
1st ejaculate %LNV mean SD (%)
2nd ejaculate %LNV mean SD (%)
23.5 21.2 27.6 20.3 25.8 24.9 0.8549
29.4 35.6 29.4 20.6 32.4 27.1 0.7608
p Value* 0.0220# 0.8278 0.0254#
Interval of semen analysis mean SD(days)
Age mean SD (years)
28.8 28.8 18.9 6.5 38.9 38.9 0.1058
34.5 4.0 34.9 4.7 34.1 4.5 0.6409
Number of patients ¼ 33; n: number of patients who underwent an LNV evaluation by high-magnification microscopy; SD: standard deviation; *: paired t-test (1st versus 2nd ejaculate); #: statistically significant; MSOME: motile sperm organelle morphology examination; LNV: large sperm nuclear vacuole; **: unpaired t-test (Idiopathic versus Varicoceles).
volume, sperm count, and normal sperm morphology did not change significantly. The percent of LNV at baseline was 27.7%. At three and six months after surgery, the percent of LNV significantly decreased to 12.0% (p ¼ 0.0132 versus baseline) and 10.3% (p ¼ 0.0226 versus baseline), respectively, according to the paired t-test (Table 3). Sperm nuclear vacuoles in the patients treated with medicinal therapy Twenty-eight patients underwent medicinal therapy for male infertility: 14 were prescribed saikokaryukotsuboreito (2.5 grams three times daily, 30 minutes before meals), 15 were prescribed hochuekkito (2.5 grams three times daily,
30 minutes before meals), and six were prescribed other medicines. These patients consisted of 19 subjects with idiopathic male infertility, five subjects with varicocele, and four subjects with other causes of male infertility. Seven out of 28 patients underwent more than one cycle of medicinal treatment using alternative Japanese herbal medicines sequentially by saikokaryukotsuboreito and hochuekkito, in which the semen analysis conducted just before each cycle of medicinal therapy was used as the baseline. These patients had poorer semen quality than the other patients. The selection of the correct Japanese traditional herbal medicine was based on the pathogenic alteration. Saikokaryukotsuboreito is a Japanese herbal medicine that is given to male infertility patients with strong or fully active
38
A. Komiya et al.
Syst Biol Reprod Med, 2014; 60(1): 35–42
Table 3. Changes in the sperm nuclear vacuoles among the patients who underwent varicocele repair. Baseline (n ¼ 18) mean SD
3 months after surgery (n ¼ 18)
6 months after surgery (n ¼ 14)
mean SD
p Value*
mean SD
p Value*
2.8 1.1 23.4 49.6 17.3 11.8 3.1 2.9
2.9 1.3 36.5 31.7 30.3 18.1 5.6 8.0
0.4405 0.2888 0.0003# 0.1366
2.9 1.2 38.2 38.5 33.0 18.9 7.6 8.2
0.3300 0.2481 0.0016# 0.0620
27.7 32.7
12.0 16.3
0.0132#
11.4 11.9
0.0272#
Conventional semen analysis Semen volume (mL) Sperm count (106/mL) Sperm motility (%) Normal sperm morphology (%) MSOME %LNV
n: number of patients who underwent an LNV evaluation by high-magnification microscopy; SD: standard deviation; *: paired t-test (versus baseline); #: statistically significant; MSOME: motile sperm organelle morphology examination; LNV: large sperm nuclear vacuole
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
Table 4. The sperm nuclear vacuoles among the patients who underwent medicinal therapy. 3 months after Baseline medicinal therapy (n ¼ 35) (n ¼ 35) mean SD mean SD p Value* Conventional semen analysis Semen volume (mL) 2.8 1.3 Sperm count (106/mL) 18.5 29.1 Sperm motility (%) 14.1 11.0 6.4 7.1 Normal sperm morphology (%) MSOME %LNV 33.3 24.1
3.0 1.5 24.8 37.3 21.0 11.0 6.4 6.2
0.2026 0.2494 0.0098# 0.9951
28.6 26.4
0.1276
N: number of ejaculates evaluated for nuclear vacuoles by highmagnification microscopy; SD: standard deviation; *: paired t-test (baseline versus at 3 months); #: statistically significant; MSOME: motile sperm organelle morphology examination; %LNV: proportion of spermatozoa with large sperm nuclear vacuole
repairing responses (alternatively ‘‘excess’’). Hochuekkito is a Japanese herbal medicine that is given to male infertility patients with weak or hollow repairing responses (alternatively ‘insufficiency’) [Amano et al. 2010; Furuya et al. 2004; Komiya et al. 2011]. The conventional semen parameters did not change significantly at three months except for sperm motility (Table 4). The improvement in sperm motility was significant following the administration of hochuekkito (17.4 13.9% to 29.7 26.2%, p ¼ 0.0292, paired t-test) but not following the administration of saikokaryukotsuboreito (12.6 13.3% to 13.2 7.7, p ¼ 0.8498, paired t-test), when analyzed separately. The percentage of LNVs at baseline was 33.3%. After three months of medicinal therapy, the percentage of LNVs slightly decreased to 28.6% but this change was not significant (p ¼ 0.1276 versus baseline), according to the paired t-test (Table 4). No differences were found with respect to LNV according to the choice of medicine. Correlations between large nuclear vacuoles and conventional semen parameters The percentage of LNV increased significantly as sperm count (p ¼ 0.0039) and sperm motility (p ¼ 0.0086) decreased. The correlations were weak to moderate with sperm count (r ¼ 0.2508) and sperm motility (r ¼ 0.2295) and not significant with semen volume (r ¼ 0.0109,
p ¼ 0.9019), normal sperm morphology (r ¼ 0.0624, p ¼ 0. 4806), or age (r ¼ 0.0926, p ¼ 0.2930). These correlations are consistent with those reported in our previous study [Komiya et al. 2013].
Discussion In the present study, the inter-examination differences in sperm nuclear vacuoles were analyzed both during patient evaluation and treatment for male infertility. When multiple semen samples were evaluated for sperm nuclear vacuoles, the spermatozoa in the second ejaculate were shown to have more LNVs, especially among the patients with palpable varicocele. Surgical repair for palpable varicocele decreased the proportion of spermatozoa with LNVs. The effect of medicinal therapy on LNV was not significant. Numerous vacuoles have been identified inside the sperm nucleus using high-magnification light microscopy during MSOME procedures. Many studies have indicated that there is a positive relationship between sperm DNA fragmentation and large vacuoles in the sperm nuclear area [Oliveira et al. 2010b; Sakkas and Alvarez 2010; Skowronek et al. 2012; Utsuno et al. 2013]. Furthermore, several studies have reported that large nuclear vacuoles are linked with chromatin condensation failure [Boitrelle et al. 2011; Cassuto et al. 2012; Franco et al. 2012; Gopalkrishnan et al. 2000; Perdrix et al. 2011]. Therefore, the presence of sperm nuclear vacuoles on real-time optical microscopy without denaturation may indirectly indicate spermatozoa carrying possible DNA alterations, particularly chromatin condensation failure. We previously reported that the percentage of spermatozoa with LNVs increases significantly as the semen quality decreases, which was confirmed in the present study. The number of LNVs exhibit significant negative correlations with various parameters in conventional semen and computerassisted sperm analyses in patients with male infertility [Komiya et al. 2013]. Perdrix et al. [2012] reported similar findings. However, there is a lack of information regarding the inter-examination differences in sperm nuclear vacuoles in male patients with infertility. Sperm nuclear vacuoles have been reported in other species. Parkinson [2000] reported a case of a sterile Angus bull with sperm nuclear vacuolation. Revay et al. [2009] reported that macrocephaly in bull spermatozoa is associated with nuclear vacuoles, diploidy, and alteration of chromatin condensation. Moderate amounts of vacuolated spermatozoa
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
DOI: 10.3109/19396368.2013.855837
can develop as a short-term response to stress or thermal or chemical injury to the seminiferous epithelium in bulls [Coulter 1976; Courtens et al. 1980]. The percentage of vacuolated bovine spermatozoa bound to (43%) and penetrating the zona pellucida (34%) is lower than that observed in the inseminate (60%) [Thundathil et al. 1998]. In boar semen, Bonet et al. [1993] reported the presence of spermatozoa with expanded and vacuolated acrosomes. Czaker [1985] found that the presence of nuclear vacuoles is one of the morphological and cytochemical changes that occur in the nucleoli of spermatids in mice during spermiogenesis. Therefore, sperm nuclear vacuoles appear to be a common entity, at least in mammals, related to subfertility. However, the sperm vacuole dynamics as a function of treatment has not been previously described. Thus far only one study, conducted by Oliveira et al. [2010a], has reported the results of a comparison between multiple MSOME findings as an intervariation study of normal sperm and sperm with large nuclear vacuoles. In that study, a significant positive correlation and the absence of differences with respect to normal morphology and LNV spermatozoa were observed between two sperm samples evaluated after various time intervals. The mean time interval between the two semen evaluations was 119 102 days. The males with varicocele presented with an increased number of spermatozoa with LNVs. In contrast, in the present study, there was an inter-examination difference in the proportion of spermatozoa with LNVs, especially in the patients with palpable varicocele, indicating a progressive nature of this disease [Komiya et al. 2012]. In addition, LNVs were more frequently found in spermatozoa among the patients with idiopathic causes of male infertility than in those with palpable varicocele. Oliveira’s study included a much larger number of patients (n ¼ 240) than our study. The number of spermatozoa was also greater (at least 200 spermatozoa per patient) than that observed in our study. In addition, the mean time interval between the two semen evaluations was longer than that observed in our study. Moreover, the patient characteristics were different. In the above-mentioned study, 31.7% (76/240) of the patients had fathered at least one child (or a pregnancy that ended in miscarriage), and male infertility was present in only 44.2% (106/240) of the couples. In contrast, the present study solely included patients with male infertility. Therefore, the results of these two studies cannot be directly compared. The state of nuclear vacuoles during the preparation or incubation of spermatozoa has been investigated. Monqaut et al. [2011] compared the quality of sperm samples obtained following density-gradient centrifugation and swim-up by performing a nuclear structural analysis with high-magnification microscopy at 8000. Swim-up produced samples with less vacuolization, although the recovery rate was also lower. Boitrelle et al. [2012] reported that cryopreservation of human spermatozoa decreased the number of motile normal spermatozoa and induced nuclear vacuolization and chromatin decondensation in infertile males. Montjean et al. [2012] demonstrated that induced acrosome reactions were not correlated with significant modification of sperm nuclear condensation or morphology (Bartoov’s criteria). The authors simultaneously observed a highly significant decrease in the
Inter-examination differences in sperm vacuoles
39
number of vacuoles following the induction of the acrosome reaction. Neyer et al. [2012] reported that the use of incubation temperatures (20 or 37 C) and/or the induction of oxidative stress did not lead to the formation of any new vacuoles. After inducing the acrosome reaction, no modifications were detected in the proportion of vacuolated spermatozoa. Therefore, these results suggest that sperm head vacuoles are produced at earlier stages of sperm maturation and that the acrosome reaction is more likely induced in spermatozoa without large nuclear vacuoles. The present study is the first to report treatment-related changes in sperm nuclear vacuoles among male patients with infertility. As mentioned above, LNVs are thought to be related to sperm DNA damage. Any treatment is beneficial in terms of DNA alterations if the proportion of spermatozoa with LNVs decreases, even if conventional semen parameters remain unchanged. In the present study, most of the patients with palpable varicocele were treated with varicocelectomy, which is associated with improvements in sperm motility and the proportion of spermatozoa with LNVs. The mechanisms underlying the impairment of potential fertility induced by varicocele remain unclear [Fuse et al. 2006]. Several theories concerning the pathophysiological conditions of this malfunction have been proposed, including the retrograde flow of adrenal or renal substances through the varicocele into the testes [Comhaire et al. 1999; Ito et al. 1982], impairment of the thermoregulatory system of the pampiniform venous plexus with remarkable elevation in the scrotal temperature [Saypol 1981], and peritubular blood stasis inducing anoxic tissue destruction or epididymal dysfunction [Swerdloff and Walsh 1975]. An association between the presence of vascular disorders and a decrease in seminal findings over time has been demonstrated [Chehval and Purcell 1992; Gorelick and Goldstein 1993]. In addition, the sperm function, including the acrosome reaction and membrane integrity, is disturbed in vascular disorders [Fuse et al. 1991, 2003]. As a consequence, males with varicocele have apparent sperm DNA damage, for which oxidative stress is considered to be one of the most important causative factors [Agarwal et al. 2009; Ishikawa et al. 2007; Shiraishi et al. 2012]. It has been reported that varicocelectomy is associated with improved sperm DNA integrity and chromatin compaction and fragmentation, and can improve spermatogenesis [Fuse et al. 2006; Smit et al. 2013; Zini et al. 2011; Zini and Dohle 2011]. Varicocelectomy induces a significant decrease in oxidative damage to sperm DNA, demonstrating that varicocele repair is an effective treatment for varicocele [Chen et al. 2008; Dada et al. 2010]. Therefore, varicocele repair-related changes in sperm LNVs may be associated with improvements in oxidative damage in sperm DNA. In this study, following treatment with medicinal treatment primarily consisting of Japanese herbal medicines, the number of sperm LNVs slightly improved, although the difference was not statistically significant, whereas sperm motility significantly increased. The baseline semen parameters were worse in the patients treated with medicinal therapy than in the remaining subjects. This may be due to the persistent poor semen quality observed in the patients treated with multiple cycles of medicinal therapy. Japanese herbal medicines are thought to affect cytokines in the serum or
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
40
A. Komiya et al.
seminal plasma [Furuya et al. 2004; Tsujimura et al. 2011]. Several cytokines have direct effects on the testicular cell function, and a number of cytokines are produced within the testis, even in the absence of inflammation or immune activation. There is compelling evidence that cytokines play an important regulatory role in the development and normal function of the testes [Hedger and Meinhardt 2003]. Based on evaluations of cytokines, significant increases have been found in interleukin (IL)- 8, IL-13, interferon-gamma, and tumor necrosis factor-alpha following the administration of saikokaryukotsuboreito [Tsujimura et al. 2008]. Furuya et al. [2004] reported an inverse correlation between the level of seminal plasma IL-6 and sperm concentration in idiopathic infertile patients. In addition, the level of seminal plasma sFas became significantly elevated following the administration of hochuekkito and was significantly correlated with the sperm concentration. Therefore, certain kinds of cytokine or apoptotic pathways may play important roles in improving semen quality and/or sperm nuclear vacuoles under treatment with Japanese herbal medicines. There are some limitations to the present study. The sample size was relatively small. The number of spermatozoa evaluated for nuclear vacuoles was also relatively small. Furthermore, the data collection was retrospective. If the study had been performed among a larger population and in a prospective fashion, the results may have been different. The present study did not evaluate data regarding pregnancy or birth rates after the medicinal and surgical therapies. Such information would be beneficial in clinical practice. We did not take into account lifestyle aspects, such as smoking, body mass index, and diet, or the potential negative impact such factors may have had on sperm vacuole development. These factors have not been previously explored in the literature. In conclusion, the proportion of spermatozoa with LNVs was high in the second ejaculate in a subset of male patients with infertility. The number of LNVs decreased following surgical treatment for male infertility. The observation of LNVs may provide an additional parameter for measuring semen quality.
Materials and Methods The purposes of the present study were: (1) to analyze any inter-examination differences in two consecutive semen samples during the evaluation of male infertility, (2) to evaluate the differences in the number of LNVs associated with surgical and/or medicinal treatment for male infertility, and (3) to confirm the associations between the percentage of LNVs and the conventional semen parameters. The Institutional Review Board of the University of Toyama approved this study. Ethical consent for the work to be carried out was provided, and signed informed consent was obtained from each patient evaluated in this study. The study conformed to the principles outlined in the Declaration of Helsinki. Sample collection We enrolled 56 Japanese patients with male infertility who underwent multiple semen analyses and high-magnification observation of the sperm head, either during the evaluation,
Syst Biol Reprod Med, 2014; 60(1): 35–42
treatment, or follow-up after varicocele repair or medicinal therapy. The age of the patients was 34.5 3.9 y (mean SD, Table 1), and the duration of infertility was 40.3 26.6 months. Neither the patient age or duration of infertility differed according to the etiology of male infertility (unpaired t-test). Among these patients, 162 ejaculates were analyzed (2.9 ejaculates per subject on average). The semen samples were obtained following masturbation from infertile male patients who visited the Male Infertility Clinic at Toyama University Hospital. The semen samples were obtained after at least five days of abstinence and evaluated within one hour of collection using manual conventional semen analyses [WHO 1999], which were performed as previously described [Akashi et al. 2010; Komiya et al. 2013]. All manual assessments were performed by a single experienced laboratory technician (YK), and the sperm concentrations were assessed using an improved Neubauer hemocytometer. The samples were diluted according to the instructions of the WHO laboratory manual [WHO 1999]. To determine sperm motility, a 10-mL sample was loaded onto a clear slide glass and covered with a 22 22-mm2 cover glass under a positive phase-contrast microscope at a total magnification of x400. According to the Guidebook for Reproductive Medicine 2010 edited by the Japan Society of Reproductive Medicine, if the semen analysis is performed twice, the mean value should be used [Iwamoto 2010]. This policy was applied to the baseline LNV observations. The definition of male infertility included the failure to conceive following twelve months of unprotected intercourse due to possible male factors. At least one parameter of a conventional semen analysis was abnormal in these patients. Male factors were generally screened based on a medical history, physical examinations, conventional semen analyses, blood tests, including assessments of sex hormones, measurement of the testicular volume using an orchidometer, scrotal ultrasonography, and transrectal ultrasonography. Varicocele was diagnosed during scrotal examinations with the patient in a standing position and was graded as previously described [Komiya et al. 2012]. The Two-Step Discontinuous PureCeptionÔ Gradient Technique was used to select motile spermatozoa according to the manufacturer’s manual of PureCeptionÔ Determination Kits (Nakamedical, Tokyo, Japan), as previously described [Komiya et al. 2013]. Observation of spermatozoa by a high-magnification microscope The spermatozoa were analyzed at 3700 or 6150 magnification using an inverted microscope equipped with Nomarski differential interference contrast optics (IX71, Olympus, Tokyo) and a video system (FX630, Olympus, Tokyo). A 60 or 100 (1.42 or 1.40 numerical aperture) objective lens was used with oil. The images of the spermatozoa were captured and stored using an image-filing software program, FlvFs (Flovel, Tokyo), on a video system. We spent 30 to 60 min capturing and analyzing the images of each ejaculate. Originally, 100 spermatozoa per ejaculate were evaluated using an MSOME [Bartoov et al. 2002, 2003]; however, this was not always possible due to the poor semen quality in this cohort. A nuclear vacuole was defined as ‘‘large’’ if the maximum diameter of the vacuole was more
DOI: 10.3109/19396368.2013.855837
than 50% of the width of the sperm head (Figure 1) [Komiya et al. 2013]. The proportion of spermatozoa with LNVs (% LNV) was calculated and compared between the first and second ejaculates before treatment. This parameter was also compared between the baseline and post-treatment values in the patients treated with varicocele repair using a microsurgical subinguinal approach or medicinal therapy. The associations between the % LNV and the conventional semen parameters were also analyzed.
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
Statistical analysis The statistical analysis of the data was carried out using the JMP 8.0.1 statistical software package (SAS Institute Japan, Tokyo). Paired and unpaired Student’s t-tests were used to compare the values between the groups. Spearman’s rank correlation coefficient was used to determine the correlations between the % LNV and the parameters of the conventional semen analyses. A value of p50.05 was defined as being statistically significant.
Declaration of interest The authors declare no conflicts of interest. The authors alone are responsible for the contents of this paper.
Author contributions Performed the patient recruitment and treatment:AK, AW, HF; Performed the conventional semen analyses: YK; Designed the study and performed the observation of the LNVs and the data analyses: AK. Principle investigator and final approval of the version of the manuscript to be published: HF; All authors have read and approved the final manuscript.
References Agarwal, A., Sharma, R.K., Desai, N.R., Prabakaran, S., Tavares, A., and Sabanegh, E. (2009) Role of oxidative stress in pathogenesis of varicocele and infertility. Urology 73:461–469. Akashi, T., Watanabe, A., Komiya, A., and Fuse, H. (2010) Evaluation of the sperm motility analyzer system (SMAS) for the assessment of sperm quality in infertile men. Syst Biol Reprod Med 56:473–477. Amano, T., Imao, T., and Takemae, K. (2010) Clinical efficacy of Japanese traditional herbal medicine (Kampo) in patients with lateonset hypogonadism. Aging Male 13:166–173. Bartoov, B., Berkovitz, A., and Eltes, F. (2001) Selection of spermatozoa with normal nuclei to improve the pregnancy rate with intracytoplasmic sperm injection. N Engl J Med 345:1067–1068. Bartoov, B., Berkovitz, A., Eltes, F., Kogosovsky, A., Yagoda, A., Lederman, H., et al. (2003) Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertil Steril 80:1413–1419. Bartoov, B., Berkovitz, A., Eltes, F., Kogosowski, A., Menezo, Y., and Barak, Y. (2002) Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome. J Androl 23:1–8. Berkovitz, A., Eltes, F., Ellenbogen, A., Peer, S., Feldberg, D., and Bartoov, B. (2006) Does the presence of nuclear vacuoles in human sperm selected for ICSI affect pregnancy outcome? Hum Reprod 21: 1787–1790. Berkovitz, A., Eltes, F., Yaari, S., Katz, N., Barr, I., Fishman, A., et al. (2005) The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. Hum Reprod 20:185–190. Boitrelle, F., Albert, M., Theillac, C., Ferfouri, F., Bergere, M., Vialard, F., et al. (2012) Cryopreservation of human spermatozoa decreases the
Inter-examination differences in sperm vacuoles
41
number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation. J Androl 33:1371–1378. Boitrelle, F., Ferfouri, F., Petit, J.M., Segretain, D., Tourain, C., Bergere, M., et al. (2011) Large human sperm vacuoles observed in motile spermatozoa under high magnification: Nuclear thumbprints linked to failure of chromatin condensation. Hum Reprod 26:1650–1658. Bonet, S., Briz, M., and Fradera, A. (1993) Ultrastructural abnormalities of boar spermatozoa. Theriogenology 40:383–396. Cassuto, N.G., Hazout, A., Hammoud, I., Balet, R., Bouret, D., Barak, Y., et al. (2012) Correlation between DNA defect and sperm-head morphology. Reprod Biomed Online 24:211–218. Chehval, M.J. and Purcell, M.H. (1992) Deterioration of semen parameters over time in men with untreated varicocele: Evidence of progressive testicular damage. Fertil Steril 57:174–177. Chen, S.S., Huang, W.J., Chang, L.S., and Wei, Y.H. (2008) Attenuation of oxidative stress after varicocelectomy in subfertile patients with varicocele. J Urol 179:639–642. Comhaire, F.H., Dhooge, W., Mahmoud, A., and Depuydt, C. (1999) A strategy for the prevention of male infertility. Verh K Acad Geneeskd Belg 61:441–452; discussion 453–445. Coulter, G.H. (1976) Effect of dexamethasone on the incidence of the ‘‘crater’’ defect of bovine sperm. Theriogenology 9:165–173. Courtens, J., Amir, D., and Durand, J. (1980) Abnormal spermiogenesis in bulls treated with ethylene bromide: An ultrastructural and ultracytochemical study. J Ultrastruct Res 71:103–115. Czaker, R. (1985) On the origin of nuclear vacuoles in spermatozoa a fine structural and cytochemical study in mice. Andrologia 17: 547–557. Dada, R., Shamsi, M.B., Venkatesh, S., Gupta, N.P., and Kumar, R. (2010) Attenuation of oxidative stress & DNA damage in varicocelectomy: Implications in infertility management. Indian J Med Res 132:728–730. Franco Jr., J.G., Mauri, A.L., Petersen, C.G., Massaro, F.C., Silva, L.F., Felipe, V., et al. (2012) Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa. Int J Androl 35:46–51. Furuya, Y., Akashi, T., and Fuse, H. (2004) Effect of Bu-zhong-yi-qitang on seminal plasma cytokine levels in patients with idiopathic male infertility. Arch Androl 50:11–14. Fuse, H., Akashi, T., Mizuno, I., Nozaki, T., and Watanabe, A. (2006) Postoperative changes of sperm chromatin heterogeneity, using acridine orange staining, in varicocele patients. Arch Androl 52:223–226. Fuse, H., Iwasaki, M., Mizuno, I., and Ikehara-Kawauchi, Y. (2003) Evaluation of acrosome reactivity using the Acrobeads test in varicocele patients: Findings before and after treatment. Arch Androl 49:1–6. Fuse, H., Kazama, T., and Katayama, T. (1991) Hypoosmotic swelling test in patients with varicocele. Arch Androl 27:149–154. Gopalkrishnan, K., Padwal, V., Meherji, P.K., Gokral, J.S., Shah, R., and Juneja, H.S. (2000) Poor quality of sperm as it affects repeated early pregnancy loss. Arch Androl 45:111–117. Gorelick, J.I. and Goldstein, M. (1993) Loss of fertility in men with varicocele. Fertil Steril 59:613–616. Hedger, M.P. and Meinhardt, A. (2003) Cytokines and the immunetesticular axis. J Reprod Immunol 58:1–26. Ishikawa, T., Fujioka, H., Ishimura, T., Takenaka, A., and Fujisawa, M. (2007) Increased testicular 8-hydroxy-20 -deoxyguanosine in patients with varicocele. BJU Int 100:863–866. Ito, H., Fuse, H., Minagawa, H., Kawamura, K., Murakami, M., and Shimazaki, J. (1982) Internal spermatic vein prostaglandins in varicocele patients. Fertil Steril 37:218–222. Iwamoto, T. (2010) Semen analysis. In KANAHARA & CO., LTD. Male foctors: Guidebook for reproductive medicine ed. Japan Society for Reproductive Medicine pp. 154–155. Komiya, A., Watanabe, A., and Fuse, H. (2011) Herbal medicine in Japan. J Mens Health 8:S15–S18. Komiya, A., Watanabe, A., Kawauchi, Y., and Fuse, H. (2013) Sperm with large nuclear vacuoles and semen quality in the evaluation of male infertility. Syst Biol Reprod Med 59:13–20. Komiya, A., Watanabe, A., Kawauchi, Y., and Fuse, H. (2012) Testicular volume discrepancy is associated with decreased semen quality in infertile Japanese males with varicoceles. Reprod Med Biol 11: 117–121. Monqaut, A.L., Zavaleta, C., Lopez, G., Lafuente, R., and Brassesco, M. (2011) Use of high-magnification microscopy for the assessment of
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
42
A. Komiya et al.
sperm recovered after two different sperm processing methods. Fertil Steril 95:277–280. Montjean, D., Belloc, S., Benkhalifa, M., Dalleac, A., and Menezo, Y. (2012) Sperm vacuoles are linked to capacitation and acrosomal status. Hum Reprod 27:2927–2932. Neyer, A., Vanderzwalmen, P., Bach, M., Stecher, A., Spitzer, D., and Zech, N. (2012) Sperm head vacuoles are not affected by in-vitro conditions, as analysed by a system of sperm-microcapture channels. Reprod Biomed Online 26:368–377. Oliveira, J.B., Petersen, C.G., Massaro, F.C., Baruffi, R.L., Mauri, A.L., Silva, L.F., et al. (2010a) Motile sperm organelle morphology examination (MSOME): Intervariation study of normal sperm and sperm with large nuclear vacuoles. Reprod Biol Endocrinol 8:56. Oliveira, J.B., Massaro, F.C., Baruffi, R.L., Mauri, A.L., Petersen, C.G., Silva, L.F., et al. (2010b) Correlation between semen analysis by motile sperm organelle morphology examination and sperm DNA damage. Fertil Steril 94:1937–1940. Parkinson, T.J. (2000) Nuclear vacuolation as a cause of sterility in an angus bull. Vet J 159:207–210. Perdrix, A., Saidi, R., Menard, J.F., Gruel, E., Milazzo, J.P., Mace, B., et al. (2012) Relationship between conventional sperm parameters and motile sperm organelle morphology examination (MSOME). Int J Androl 35:491–498. Perdrix, A., Travers, A., Chelli, M.H., Escalier, D., Do Rego, J.L., Milazzo, J.P., et al. (2011) Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles. Hum Reprod 26:47–58. Revay, T., Nagy, S., Kopp, C., Flyckt, A., Rens, W., Rath, D., et al. (2009) Macrocephaly in bull spermatozoa is associated with nuclear vacuoles, diploidy and alteration of chromatin condensation. Cytogenet Genome Res 126:202–209. Sakkas, D. and Alvarez, J.G. (2010) Sperm DNA fragmentation: Mechanisms of origin, impact on reproductive outcome, and analysis. Fertil Steril 93:1027–1036. Saypol, D.C. (1981) Varicocele. J Androl 2:61–71. Shiraishi, K., Matsuyama, H., and Takihara, H. (2012) Pathophysiology of varicocele in male infertility in the era of assisted reproductive technology. Int J Urol 19:538–550.
Syst Biol Reprod Med, 2014; 60(1): 35–42
Skowronek, F., Casanova, G., Alciaturi, J., Capurro, A., Cantu, L., Montes, J.M., et al. (2012) DNA sperm damage correlates with nuclear ultrastructural sperm defects in teratozoospermic men. Andrologia 44:59–65. Smit, M., Romijn, J.C., Wildhagen, M.F., Veldhoven, J.L., Weber, R.F., and Dohle, G.R. (2013) Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate. J Urol 189:S146–150. Swerdloff, R.S. and Walsh, P.C. (1975) Pituitary and gonadal hormones in patients with varicocele. Fertil Steril 26:1006–1012. Thundathil, J., Palasz, A.T., Barth, A.D., and Mapletoft, R.J. (1998) Fertilization characteristics and in vitro embryo production with bovine sperm containing multiple nuclear vacuoles. Mol Reprod Dev 50:328–333. Tsujimura, A., Miyagawa, Y., Okuda, H., Yamamoto, K., Fukuhara, S., Nakayama, J., et al. (2011) Change in cytokine levels after administration of saikokaryuukotsuboreito or testosterone in patients with symptoms of late-onset hypogonadism. Aging Male 14:76–81. Tsujimura, A., Takada, S., Matsuoka, Y., Nakayama, J., Takao, T., Miyagawa, Y., et al. (2008) Clinical trial of treatment with saikokaryukotsuboreito for eugonadal patients with late-inset hypogonadism-related symptoms. Aging Male 11:95–99. Utsuno, H., Oka, K., Yamamoto, A., and Shiozawa, T. (2013) Evaluation of sperm head shape at high magnification revealed correlation of sperm DNA fragmentation with aberrant head ellipticity and angularity. Fertil Steril 99:1573–1580. WHO (1999) World Health Organization laboratory manual for the examination of human semen and sperm-cervical mucus interaction. World Health Organization/Cambridge University Press; Cambridge, UK. Zini, A., Azhar, R., Baazeem, A., and Gabriel, M.S. (2011) Effect of microsurgical varicocelectomy on human sperm chromatin and DNA integrity: A prospective trial. Int J Androl 34:14–19. Zini, A. and Dohle, G. (2011) Are varicoceles associated with increased deoxyribonucleic acid fragmentation? Fertil Steril 96: 1283–1287.
RESEARCH ARTICLE
Analysis of inter-examination differences in sperm nuclear vacuoles among male patients with infertility Akira Komiya*, Akihiko Watanabe, Yoko Kawauchi, and Hideki Fuse
Abstract
Keywords
We analyzed the inter-examination differences in sperm nuclear vacuoles among male patients with infertility. We enrolled 56 male patients with infertility who underwent multiple semen analyses and high-magnification observation of the sperm head. A total of 162 ejaculates were evaluated. The average patient age was 34.5 years. Following the conventional semen analysis, the nuclear vacuoles in motile spermatozoa were evaluated at 3700–6150 magnification on an inverted microscope equipped with differential interference contrast optics. A large sperm nuclear vacuole (LNV) was defined as one or more vacuoles with a maximum diameter exhibiting 450% width of the sperm head. We compared the differences in the proportion of spermatozoa with LNVs between two consecutive semen samples before treatment. Treatment-related differences in the number of LNVs were also analyzed. Student’s t-test was used to perform the statistical analyses. No differences were observed in any semen parameters between the first and second ejaculates. On high-magnification microscopy, the proportion of spermatozoa with LNVs was 23.5% and 29.4% (p ¼ 0.0220) in the first and second ejaculates, respectively in 33 patients. Among the 18 patients who underwent varicocele repair using a microsurgical subinguinal approach, the proportion of spermatozoa with LNVs at baseline, three, and six months after surgery was 27.7%, 12.0% (p ¼ 0.0132 versus baseline), and 10.3% (p ¼ 0.0226 versus baseline), respectively. After three months of medical treatment for male infertility in 28 patients, the proportion of spermatozoa with LNVs slightly decreased from 33.3% to 28.6% (p ¼ 0.1276); however, it was not statistically significant. In conclusion, when multiple ejaculates were obtained, in the subset of male patients with infertility, the proportion of spermatozoa with LNVs could be different. The number of LNVs decreased following varicocele repair.
Differential interference contrast optics, male infertility, semen analysis, sperm nuclear vacuole History Received 10 June 2013 Revised 15 August 2013 Accepted 19 August 2013 Published online 13 January 2014
Abbreviations: ART: assisted reproduction technology; IMSI: intracytoplasmic morphologically selected sperm injection; LNV: large nuclear vacuole; MSOME: motile sperm organelle morphology examination.
Introduction In the field of assisted reproduction technology (ART) Bartoov and colleagues developed a method, called the motile sperm organelle morphology examination (MSOME), to assess the detailed morphology of motile spermatozoa in real time at a magnification of up to 6300 [Bartoov et al. 2001]. The use of this method has yielded a definition of normal spermatozoa, and new abnormalities, including sperm head vacuoles, have been described [Bartoov et al. 2002]. The high-magnification observation method has been adapted to select the best spermatozoa for oocyte injection, introducing a new technique termed intracytoplasmic morphologically selected sperm injection (IMSI). Numerous vacuoles have *Address correspondence to Akira Komiya, M.D., Ph.D., Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama, Japan, Tel: +81-76-434-7372, E-mail: [email protected]
been identified inside the sperm nucleus using light microscopy at high-magnification, and significant increases in pregnancy rates and reductions in abortion rates have been obtained by excluding morphologically abnormal sperm from IMSI [Bartoov et al. 2003; Berkovitz et al. 2005, 2006]. With respect to the management of male infertility, we previously reported that large nuclear vacuoles (LNVs) in the sperm head are observed in the majority of ejaculates. In our previous study, the percentage of spermatozoa with LNVs was 28.0 22.14% (mean s.d.) among male patients with infertility. The percentage of spermatozoa with LNVs increased significantly as the semen quality decreased. The number of LNVs exhibited significant negative correlations with various parameters in the patients with male infertility in conventional semen and computer-assisted sperm analyses [Komiya et al. 2013]. Perdrix et al. [2012] also reported a strong correlation between a high relative vacuole area to the sperm head and poor sperm morphology. However, there is a lack of
20 14
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Toyama-shi, Japan
36
A. Komiya et al.
Syst Biol Reprod Med, 2014; 60(1): 35–42
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
information regarding inter-examination differences in LNV during the evaluation or treatment of male infertility. Thus far, only one study has reported the results of a comparison between multiple MSOME findings as an intervariation study of normal sperm and sperm with large nuclear vacuoles [Oliveira et al. 2010a]. A significant positive correlation and the absence of differences were observed with respect to normal morphology and LNV spermatozoa between two sperm samples obtained from each patient evaluated after a time interval. Moreover, no studies reporting treatmentrelated changes in sperm nuclear vacuoles have been published in the literature. In the present study, we report the analyses of interexamination differences in two consecutive semen samples during the evaluation of male infertility. We also evaluated the differences in the number of LNVs associated with surgical and/or medicinal treatment for male infertility.
with processed motile spermatozoa. The number of spermatozoa that could be evaluated for nuclear vacuoles was 57 65 (mean standard deviation/SD) per ejaculate with the available processed motile spermatozoa. This number is higher than that used in our previous report [Komiya et al. 2013]. A total of 7650 spermatozoa were evaluated for sperm nuclear vacuoles. Of these spermatozoa, 859 (11.2%) had LNVs. Comparison of semen parameters and sperm nuclear vacuoles in the first and second ejaculates during male infertility evaluation The mean values of the semen volume, sperm count, sperm motility, and normal sperm morphology in the first versus second ejaculates were 3.0 mL versus 3.1 mL, 31.8 106/mL versus 28.3 106/mL, 20.3% versus 23.5%, and 8.2% versus 10.2%, respectively, in the conventional semen analyses performed during the evaluation of male infertility (Table 2). There were no significant differences between the first and second ejaculates in the parameters evaluated in the conventional semen analyses. However, the percent of LNVs was significantly higher in the second ejaculate (29.4%) than in the first ejaculate (23.5%) in this cohort (p ¼ 0.0220, paired t-test). This finding was observed in the patients with varicocele, but not in those with idiopathic causes of male infertility (Table 2). However, the proportion of spermatozoa with LNVs (% LNVs) was not different between the patients with idiopathic causes of male infertility (29.7%) and palpable varicocele (30.8%; p ¼ 0.90501, unpaired t-test). The interval of the semen analysis was 28.8 28.8 days (mean SD). No correlations were found between the interval of the semen analysis and the change in % LNV.
Results Patient characteristics Among the 56 patients, high-magnification microscopy for LNVs was performed twice during the evaluation of male infertility before any treatment was administered in 33 patients (Table 1) and in 12 patients in whom no LNV data were available during or after treatment. The LNVs were evaluated before and three months after varicocele repair in 18 patients and before and three and six months after varicocele repair in 16 patients. The LNVs were also observed before and after medicinal therapy in 28 patients. The mean patient age was 34.5 years, and the mean duration of infertility was 40.3 months. The mean values of the semen volume, sperm count, sperm motility, and normal sperm morphology at baseline were 3.0 mL, 28.2 106/mL, 22.8%, and 8.3%, respectively (Table 1). These values were not significantly different between the patients with idiopathic causes of male infertility and those with varicocele. We evaluated the sperm nuclear vacuoles in 162 ejaculates using high-magnification microscopy as shown in Figure 1. No motile spermatozoa were found in 28 ejaculates (17.3%) following selection using PureCeptionÔ Determination Kits. At least one spermatozoon with a LNV was found in 130 of 134 ejaculates (97.0%)
Treatment-related changes in sperm nuclear vacuoles in the patients treated with varicocele repair Eighteen patients underwent varicocele repair using a microsurgical subinguinal approach. Among the conventional semen parameters, only sperm motility was significantly increased at three and six months after surgery (p ¼ 0.0003 and 0.0004, respectively, paired t-test). However, the semen
Table 1. Patient characteristics.
N Possible causes of male infertility Idiopathic Varicoceles Hormonal disorders Others Sum
Conventional semen analyses at baseline Semen volume (mL) Sperm count (106/mL) Sperm motility (%) Sperm normal morphology (%) SD: standard deviation
%
Age (years) mean SD
Duration of infertility (months) mean SD 41.4 24.0 38.1 30.6 65.7 25.0 31.2 16.3 40.3 26.6
24 23 3 6 56
42.9 41.1 5.4 10.7 100
34.0 4.2 34.8 3.8 35.3 2.5 34.7 4.1 34.5 3.9
All mean SD
Idiopathic mean SD
Varicoceles mean SD
Hormonal disorders mean SD
Others mean SD
3.0 1.4 28.2 43.6 22.8 17.9 8.3 9.6
3.1 1.3 28.3 41.9 24.0 18.5 9.2 10.3
2.9 1.3 24.0 44.6 22.1 14.3 5.8 6.4
2.5 0.4 19.0 34.5 10.5 9.7 3.8 5.0
2.2 1.9 35.7 35.1 28.9 16.6 14.8 5.9
Inter-examination differences in sperm vacuoles
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
DOI: 10.3109/19396368.2013.855837
37
Figure 1. High-magnification on an inverted microscope equipped with Nomarski differential interference contrast optics and a video system. The arrows indicate nuclear vacuoles observed through a 60 (1.42 numerical aperture) objective lens. (A) Spermatozoa with a nuclear vacuole exhibiting a 450% transverse diameter of the sperm nuclear area. The dotted lines indicate the width of the sperm head (3.1 mm) and the maximum diameter of a nuclear vacuole (1.8 mm). (B) Spermatozoa with a nuclear vacuole exhibiting a 50% transverse diameter of the sperm nuclear vacuole. (C) Spermatozoa without any nuclear vacuoles in the sperm nuclear area.
Table 2. Comparison of the 1st and 2nd ejaculates at evaluation for male infertility.
Conventional semen analysis Semen volume (mL) Sperm count (106/mL) Sperm motility (%) Normal sperm morphology (%)
MSOME All Idiopathic (n ¼ 16) Varicoceles (n ¼ 11) p Value**
1st ejaculate mean SD
2nd ejaculate mean SD
p Value*
3.02 1.5 31.8 49.8 20.3 17.3 8.2 8.6
3.1 1.5 28.3 42.7 23.5 19.7 10.2 11.9
0.2369 0.3059 0.1321 0.2956
1st ejaculate %LNV mean SD (%)
2nd ejaculate %LNV mean SD (%)
23.5 21.2 27.6 20.3 25.8 24.9 0.8549
29.4 35.6 29.4 20.6 32.4 27.1 0.7608
p Value* 0.0220# 0.8278 0.0254#
Interval of semen analysis mean SD(days)
Age mean SD (years)
28.8 28.8 18.9 6.5 38.9 38.9 0.1058
34.5 4.0 34.9 4.7 34.1 4.5 0.6409
Number of patients ¼ 33; n: number of patients who underwent an LNV evaluation by high-magnification microscopy; SD: standard deviation; *: paired t-test (1st versus 2nd ejaculate); #: statistically significant; MSOME: motile sperm organelle morphology examination; LNV: large sperm nuclear vacuole; **: unpaired t-test (Idiopathic versus Varicoceles).
volume, sperm count, and normal sperm morphology did not change significantly. The percent of LNV at baseline was 27.7%. At three and six months after surgery, the percent of LNV significantly decreased to 12.0% (p ¼ 0.0132 versus baseline) and 10.3% (p ¼ 0.0226 versus baseline), respectively, according to the paired t-test (Table 3). Sperm nuclear vacuoles in the patients treated with medicinal therapy Twenty-eight patients underwent medicinal therapy for male infertility: 14 were prescribed saikokaryukotsuboreito (2.5 grams three times daily, 30 minutes before meals), 15 were prescribed hochuekkito (2.5 grams three times daily,
30 minutes before meals), and six were prescribed other medicines. These patients consisted of 19 subjects with idiopathic male infertility, five subjects with varicocele, and four subjects with other causes of male infertility. Seven out of 28 patients underwent more than one cycle of medicinal treatment using alternative Japanese herbal medicines sequentially by saikokaryukotsuboreito and hochuekkito, in which the semen analysis conducted just before each cycle of medicinal therapy was used as the baseline. These patients had poorer semen quality than the other patients. The selection of the correct Japanese traditional herbal medicine was based on the pathogenic alteration. Saikokaryukotsuboreito is a Japanese herbal medicine that is given to male infertility patients with strong or fully active
38
A. Komiya et al.
Syst Biol Reprod Med, 2014; 60(1): 35–42
Table 3. Changes in the sperm nuclear vacuoles among the patients who underwent varicocele repair. Baseline (n ¼ 18) mean SD
3 months after surgery (n ¼ 18)
6 months after surgery (n ¼ 14)
mean SD
p Value*
mean SD
p Value*
2.8 1.1 23.4 49.6 17.3 11.8 3.1 2.9
2.9 1.3 36.5 31.7 30.3 18.1 5.6 8.0
0.4405 0.2888 0.0003# 0.1366
2.9 1.2 38.2 38.5 33.0 18.9 7.6 8.2
0.3300 0.2481 0.0016# 0.0620
27.7 32.7
12.0 16.3
0.0132#
11.4 11.9
0.0272#
Conventional semen analysis Semen volume (mL) Sperm count (106/mL) Sperm motility (%) Normal sperm morphology (%) MSOME %LNV
n: number of patients who underwent an LNV evaluation by high-magnification microscopy; SD: standard deviation; *: paired t-test (versus baseline); #: statistically significant; MSOME: motile sperm organelle morphology examination; LNV: large sperm nuclear vacuole
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
Table 4. The sperm nuclear vacuoles among the patients who underwent medicinal therapy. 3 months after Baseline medicinal therapy (n ¼ 35) (n ¼ 35) mean SD mean SD p Value* Conventional semen analysis Semen volume (mL) 2.8 1.3 Sperm count (106/mL) 18.5 29.1 Sperm motility (%) 14.1 11.0 6.4 7.1 Normal sperm morphology (%) MSOME %LNV 33.3 24.1
3.0 1.5 24.8 37.3 21.0 11.0 6.4 6.2
0.2026 0.2494 0.0098# 0.9951
28.6 26.4
0.1276
N: number of ejaculates evaluated for nuclear vacuoles by highmagnification microscopy; SD: standard deviation; *: paired t-test (baseline versus at 3 months); #: statistically significant; MSOME: motile sperm organelle morphology examination; %LNV: proportion of spermatozoa with large sperm nuclear vacuole
repairing responses (alternatively ‘‘excess’’). Hochuekkito is a Japanese herbal medicine that is given to male infertility patients with weak or hollow repairing responses (alternatively ‘insufficiency’) [Amano et al. 2010; Furuya et al. 2004; Komiya et al. 2011]. The conventional semen parameters did not change significantly at three months except for sperm motility (Table 4). The improvement in sperm motility was significant following the administration of hochuekkito (17.4 13.9% to 29.7 26.2%, p ¼ 0.0292, paired t-test) but not following the administration of saikokaryukotsuboreito (12.6 13.3% to 13.2 7.7, p ¼ 0.8498, paired t-test), when analyzed separately. The percentage of LNVs at baseline was 33.3%. After three months of medicinal therapy, the percentage of LNVs slightly decreased to 28.6% but this change was not significant (p ¼ 0.1276 versus baseline), according to the paired t-test (Table 4). No differences were found with respect to LNV according to the choice of medicine. Correlations between large nuclear vacuoles and conventional semen parameters The percentage of LNV increased significantly as sperm count (p ¼ 0.0039) and sperm motility (p ¼ 0.0086) decreased. The correlations were weak to moderate with sperm count (r ¼ 0.2508) and sperm motility (r ¼ 0.2295) and not significant with semen volume (r ¼ 0.0109,
p ¼ 0.9019), normal sperm morphology (r ¼ 0.0624, p ¼ 0. 4806), or age (r ¼ 0.0926, p ¼ 0.2930). These correlations are consistent with those reported in our previous study [Komiya et al. 2013].
Discussion In the present study, the inter-examination differences in sperm nuclear vacuoles were analyzed both during patient evaluation and treatment for male infertility. When multiple semen samples were evaluated for sperm nuclear vacuoles, the spermatozoa in the second ejaculate were shown to have more LNVs, especially among the patients with palpable varicocele. Surgical repair for palpable varicocele decreased the proportion of spermatozoa with LNVs. The effect of medicinal therapy on LNV was not significant. Numerous vacuoles have been identified inside the sperm nucleus using high-magnification light microscopy during MSOME procedures. Many studies have indicated that there is a positive relationship between sperm DNA fragmentation and large vacuoles in the sperm nuclear area [Oliveira et al. 2010b; Sakkas and Alvarez 2010; Skowronek et al. 2012; Utsuno et al. 2013]. Furthermore, several studies have reported that large nuclear vacuoles are linked with chromatin condensation failure [Boitrelle et al. 2011; Cassuto et al. 2012; Franco et al. 2012; Gopalkrishnan et al. 2000; Perdrix et al. 2011]. Therefore, the presence of sperm nuclear vacuoles on real-time optical microscopy without denaturation may indirectly indicate spermatozoa carrying possible DNA alterations, particularly chromatin condensation failure. We previously reported that the percentage of spermatozoa with LNVs increases significantly as the semen quality decreases, which was confirmed in the present study. The number of LNVs exhibit significant negative correlations with various parameters in conventional semen and computerassisted sperm analyses in patients with male infertility [Komiya et al. 2013]. Perdrix et al. [2012] reported similar findings. However, there is a lack of information regarding the inter-examination differences in sperm nuclear vacuoles in male patients with infertility. Sperm nuclear vacuoles have been reported in other species. Parkinson [2000] reported a case of a sterile Angus bull with sperm nuclear vacuolation. Revay et al. [2009] reported that macrocephaly in bull spermatozoa is associated with nuclear vacuoles, diploidy, and alteration of chromatin condensation. Moderate amounts of vacuolated spermatozoa
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
DOI: 10.3109/19396368.2013.855837
can develop as a short-term response to stress or thermal or chemical injury to the seminiferous epithelium in bulls [Coulter 1976; Courtens et al. 1980]. The percentage of vacuolated bovine spermatozoa bound to (43%) and penetrating the zona pellucida (34%) is lower than that observed in the inseminate (60%) [Thundathil et al. 1998]. In boar semen, Bonet et al. [1993] reported the presence of spermatozoa with expanded and vacuolated acrosomes. Czaker [1985] found that the presence of nuclear vacuoles is one of the morphological and cytochemical changes that occur in the nucleoli of spermatids in mice during spermiogenesis. Therefore, sperm nuclear vacuoles appear to be a common entity, at least in mammals, related to subfertility. However, the sperm vacuole dynamics as a function of treatment has not been previously described. Thus far only one study, conducted by Oliveira et al. [2010a], has reported the results of a comparison between multiple MSOME findings as an intervariation study of normal sperm and sperm with large nuclear vacuoles. In that study, a significant positive correlation and the absence of differences with respect to normal morphology and LNV spermatozoa were observed between two sperm samples evaluated after various time intervals. The mean time interval between the two semen evaluations was 119 102 days. The males with varicocele presented with an increased number of spermatozoa with LNVs. In contrast, in the present study, there was an inter-examination difference in the proportion of spermatozoa with LNVs, especially in the patients with palpable varicocele, indicating a progressive nature of this disease [Komiya et al. 2012]. In addition, LNVs were more frequently found in spermatozoa among the patients with idiopathic causes of male infertility than in those with palpable varicocele. Oliveira’s study included a much larger number of patients (n ¼ 240) than our study. The number of spermatozoa was also greater (at least 200 spermatozoa per patient) than that observed in our study. In addition, the mean time interval between the two semen evaluations was longer than that observed in our study. Moreover, the patient characteristics were different. In the above-mentioned study, 31.7% (76/240) of the patients had fathered at least one child (or a pregnancy that ended in miscarriage), and male infertility was present in only 44.2% (106/240) of the couples. In contrast, the present study solely included patients with male infertility. Therefore, the results of these two studies cannot be directly compared. The state of nuclear vacuoles during the preparation or incubation of spermatozoa has been investigated. Monqaut et al. [2011] compared the quality of sperm samples obtained following density-gradient centrifugation and swim-up by performing a nuclear structural analysis with high-magnification microscopy at 8000. Swim-up produced samples with less vacuolization, although the recovery rate was also lower. Boitrelle et al. [2012] reported that cryopreservation of human spermatozoa decreased the number of motile normal spermatozoa and induced nuclear vacuolization and chromatin decondensation in infertile males. Montjean et al. [2012] demonstrated that induced acrosome reactions were not correlated with significant modification of sperm nuclear condensation or morphology (Bartoov’s criteria). The authors simultaneously observed a highly significant decrease in the
Inter-examination differences in sperm vacuoles
39
number of vacuoles following the induction of the acrosome reaction. Neyer et al. [2012] reported that the use of incubation temperatures (20 or 37 C) and/or the induction of oxidative stress did not lead to the formation of any new vacuoles. After inducing the acrosome reaction, no modifications were detected in the proportion of vacuolated spermatozoa. Therefore, these results suggest that sperm head vacuoles are produced at earlier stages of sperm maturation and that the acrosome reaction is more likely induced in spermatozoa without large nuclear vacuoles. The present study is the first to report treatment-related changes in sperm nuclear vacuoles among male patients with infertility. As mentioned above, LNVs are thought to be related to sperm DNA damage. Any treatment is beneficial in terms of DNA alterations if the proportion of spermatozoa with LNVs decreases, even if conventional semen parameters remain unchanged. In the present study, most of the patients with palpable varicocele were treated with varicocelectomy, which is associated with improvements in sperm motility and the proportion of spermatozoa with LNVs. The mechanisms underlying the impairment of potential fertility induced by varicocele remain unclear [Fuse et al. 2006]. Several theories concerning the pathophysiological conditions of this malfunction have been proposed, including the retrograde flow of adrenal or renal substances through the varicocele into the testes [Comhaire et al. 1999; Ito et al. 1982], impairment of the thermoregulatory system of the pampiniform venous plexus with remarkable elevation in the scrotal temperature [Saypol 1981], and peritubular blood stasis inducing anoxic tissue destruction or epididymal dysfunction [Swerdloff and Walsh 1975]. An association between the presence of vascular disorders and a decrease in seminal findings over time has been demonstrated [Chehval and Purcell 1992; Gorelick and Goldstein 1993]. In addition, the sperm function, including the acrosome reaction and membrane integrity, is disturbed in vascular disorders [Fuse et al. 1991, 2003]. As a consequence, males with varicocele have apparent sperm DNA damage, for which oxidative stress is considered to be one of the most important causative factors [Agarwal et al. 2009; Ishikawa et al. 2007; Shiraishi et al. 2012]. It has been reported that varicocelectomy is associated with improved sperm DNA integrity and chromatin compaction and fragmentation, and can improve spermatogenesis [Fuse et al. 2006; Smit et al. 2013; Zini et al. 2011; Zini and Dohle 2011]. Varicocelectomy induces a significant decrease in oxidative damage to sperm DNA, demonstrating that varicocele repair is an effective treatment for varicocele [Chen et al. 2008; Dada et al. 2010]. Therefore, varicocele repair-related changes in sperm LNVs may be associated with improvements in oxidative damage in sperm DNA. In this study, following treatment with medicinal treatment primarily consisting of Japanese herbal medicines, the number of sperm LNVs slightly improved, although the difference was not statistically significant, whereas sperm motility significantly increased. The baseline semen parameters were worse in the patients treated with medicinal therapy than in the remaining subjects. This may be due to the persistent poor semen quality observed in the patients treated with multiple cycles of medicinal therapy. Japanese herbal medicines are thought to affect cytokines in the serum or
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
40
A. Komiya et al.
seminal plasma [Furuya et al. 2004; Tsujimura et al. 2011]. Several cytokines have direct effects on the testicular cell function, and a number of cytokines are produced within the testis, even in the absence of inflammation or immune activation. There is compelling evidence that cytokines play an important regulatory role in the development and normal function of the testes [Hedger and Meinhardt 2003]. Based on evaluations of cytokines, significant increases have been found in interleukin (IL)- 8, IL-13, interferon-gamma, and tumor necrosis factor-alpha following the administration of saikokaryukotsuboreito [Tsujimura et al. 2008]. Furuya et al. [2004] reported an inverse correlation between the level of seminal plasma IL-6 and sperm concentration in idiopathic infertile patients. In addition, the level of seminal plasma sFas became significantly elevated following the administration of hochuekkito and was significantly correlated with the sperm concentration. Therefore, certain kinds of cytokine or apoptotic pathways may play important roles in improving semen quality and/or sperm nuclear vacuoles under treatment with Japanese herbal medicines. There are some limitations to the present study. The sample size was relatively small. The number of spermatozoa evaluated for nuclear vacuoles was also relatively small. Furthermore, the data collection was retrospective. If the study had been performed among a larger population and in a prospective fashion, the results may have been different. The present study did not evaluate data regarding pregnancy or birth rates after the medicinal and surgical therapies. Such information would be beneficial in clinical practice. We did not take into account lifestyle aspects, such as smoking, body mass index, and diet, or the potential negative impact such factors may have had on sperm vacuole development. These factors have not been previously explored in the literature. In conclusion, the proportion of spermatozoa with LNVs was high in the second ejaculate in a subset of male patients with infertility. The number of LNVs decreased following surgical treatment for male infertility. The observation of LNVs may provide an additional parameter for measuring semen quality.
Materials and Methods The purposes of the present study were: (1) to analyze any inter-examination differences in two consecutive semen samples during the evaluation of male infertility, (2) to evaluate the differences in the number of LNVs associated with surgical and/or medicinal treatment for male infertility, and (3) to confirm the associations between the percentage of LNVs and the conventional semen parameters. The Institutional Review Board of the University of Toyama approved this study. Ethical consent for the work to be carried out was provided, and signed informed consent was obtained from each patient evaluated in this study. The study conformed to the principles outlined in the Declaration of Helsinki. Sample collection We enrolled 56 Japanese patients with male infertility who underwent multiple semen analyses and high-magnification observation of the sperm head, either during the evaluation,
Syst Biol Reprod Med, 2014; 60(1): 35–42
treatment, or follow-up after varicocele repair or medicinal therapy. The age of the patients was 34.5 3.9 y (mean SD, Table 1), and the duration of infertility was 40.3 26.6 months. Neither the patient age or duration of infertility differed according to the etiology of male infertility (unpaired t-test). Among these patients, 162 ejaculates were analyzed (2.9 ejaculates per subject on average). The semen samples were obtained following masturbation from infertile male patients who visited the Male Infertility Clinic at Toyama University Hospital. The semen samples were obtained after at least five days of abstinence and evaluated within one hour of collection using manual conventional semen analyses [WHO 1999], which were performed as previously described [Akashi et al. 2010; Komiya et al. 2013]. All manual assessments were performed by a single experienced laboratory technician (YK), and the sperm concentrations were assessed using an improved Neubauer hemocytometer. The samples were diluted according to the instructions of the WHO laboratory manual [WHO 1999]. To determine sperm motility, a 10-mL sample was loaded onto a clear slide glass and covered with a 22 22-mm2 cover glass under a positive phase-contrast microscope at a total magnification of x400. According to the Guidebook for Reproductive Medicine 2010 edited by the Japan Society of Reproductive Medicine, if the semen analysis is performed twice, the mean value should be used [Iwamoto 2010]. This policy was applied to the baseline LNV observations. The definition of male infertility included the failure to conceive following twelve months of unprotected intercourse due to possible male factors. At least one parameter of a conventional semen analysis was abnormal in these patients. Male factors were generally screened based on a medical history, physical examinations, conventional semen analyses, blood tests, including assessments of sex hormones, measurement of the testicular volume using an orchidometer, scrotal ultrasonography, and transrectal ultrasonography. Varicocele was diagnosed during scrotal examinations with the patient in a standing position and was graded as previously described [Komiya et al. 2012]. The Two-Step Discontinuous PureCeptionÔ Gradient Technique was used to select motile spermatozoa according to the manufacturer’s manual of PureCeptionÔ Determination Kits (Nakamedical, Tokyo, Japan), as previously described [Komiya et al. 2013]. Observation of spermatozoa by a high-magnification microscope The spermatozoa were analyzed at 3700 or 6150 magnification using an inverted microscope equipped with Nomarski differential interference contrast optics (IX71, Olympus, Tokyo) and a video system (FX630, Olympus, Tokyo). A 60 or 100 (1.42 or 1.40 numerical aperture) objective lens was used with oil. The images of the spermatozoa were captured and stored using an image-filing software program, FlvFs (Flovel, Tokyo), on a video system. We spent 30 to 60 min capturing and analyzing the images of each ejaculate. Originally, 100 spermatozoa per ejaculate were evaluated using an MSOME [Bartoov et al. 2002, 2003]; however, this was not always possible due to the poor semen quality in this cohort. A nuclear vacuole was defined as ‘‘large’’ if the maximum diameter of the vacuole was more
DOI: 10.3109/19396368.2013.855837
than 50% of the width of the sperm head (Figure 1) [Komiya et al. 2013]. The proportion of spermatozoa with LNVs (% LNV) was calculated and compared between the first and second ejaculates before treatment. This parameter was also compared between the baseline and post-treatment values in the patients treated with varicocele repair using a microsurgical subinguinal approach or medicinal therapy. The associations between the % LNV and the conventional semen parameters were also analyzed.
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
Statistical analysis The statistical analysis of the data was carried out using the JMP 8.0.1 statistical software package (SAS Institute Japan, Tokyo). Paired and unpaired Student’s t-tests were used to compare the values between the groups. Spearman’s rank correlation coefficient was used to determine the correlations between the % LNV and the parameters of the conventional semen analyses. A value of p50.05 was defined as being statistically significant.
Declaration of interest The authors declare no conflicts of interest. The authors alone are responsible for the contents of this paper.
Author contributions Performed the patient recruitment and treatment:AK, AW, HF; Performed the conventional semen analyses: YK; Designed the study and performed the observation of the LNVs and the data analyses: AK. Principle investigator and final approval of the version of the manuscript to be published: HF; All authors have read and approved the final manuscript.
References Agarwal, A., Sharma, R.K., Desai, N.R., Prabakaran, S., Tavares, A., and Sabanegh, E. (2009) Role of oxidative stress in pathogenesis of varicocele and infertility. Urology 73:461–469. Akashi, T., Watanabe, A., Komiya, A., and Fuse, H. (2010) Evaluation of the sperm motility analyzer system (SMAS) for the assessment of sperm quality in infertile men. Syst Biol Reprod Med 56:473–477. Amano, T., Imao, T., and Takemae, K. (2010) Clinical efficacy of Japanese traditional herbal medicine (Kampo) in patients with lateonset hypogonadism. Aging Male 13:166–173. Bartoov, B., Berkovitz, A., and Eltes, F. (2001) Selection of spermatozoa with normal nuclei to improve the pregnancy rate with intracytoplasmic sperm injection. N Engl J Med 345:1067–1068. Bartoov, B., Berkovitz, A., Eltes, F., Kogosovsky, A., Yagoda, A., Lederman, H., et al. (2003) Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertil Steril 80:1413–1419. Bartoov, B., Berkovitz, A., Eltes, F., Kogosowski, A., Menezo, Y., and Barak, Y. (2002) Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome. J Androl 23:1–8. Berkovitz, A., Eltes, F., Ellenbogen, A., Peer, S., Feldberg, D., and Bartoov, B. (2006) Does the presence of nuclear vacuoles in human sperm selected for ICSI affect pregnancy outcome? Hum Reprod 21: 1787–1790. Berkovitz, A., Eltes, F., Yaari, S., Katz, N., Barr, I., Fishman, A., et al. (2005) The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. Hum Reprod 20:185–190. Boitrelle, F., Albert, M., Theillac, C., Ferfouri, F., Bergere, M., Vialard, F., et al. (2012) Cryopreservation of human spermatozoa decreases the
Inter-examination differences in sperm vacuoles
41
number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation. J Androl 33:1371–1378. Boitrelle, F., Ferfouri, F., Petit, J.M., Segretain, D., Tourain, C., Bergere, M., et al. (2011) Large human sperm vacuoles observed in motile spermatozoa under high magnification: Nuclear thumbprints linked to failure of chromatin condensation. Hum Reprod 26:1650–1658. Bonet, S., Briz, M., and Fradera, A. (1993) Ultrastructural abnormalities of boar spermatozoa. Theriogenology 40:383–396. Cassuto, N.G., Hazout, A., Hammoud, I., Balet, R., Bouret, D., Barak, Y., et al. (2012) Correlation between DNA defect and sperm-head morphology. Reprod Biomed Online 24:211–218. Chehval, M.J. and Purcell, M.H. (1992) Deterioration of semen parameters over time in men with untreated varicocele: Evidence of progressive testicular damage. Fertil Steril 57:174–177. Chen, S.S., Huang, W.J., Chang, L.S., and Wei, Y.H. (2008) Attenuation of oxidative stress after varicocelectomy in subfertile patients with varicocele. J Urol 179:639–642. Comhaire, F.H., Dhooge, W., Mahmoud, A., and Depuydt, C. (1999) A strategy for the prevention of male infertility. Verh K Acad Geneeskd Belg 61:441–452; discussion 453–445. Coulter, G.H. (1976) Effect of dexamethasone on the incidence of the ‘‘crater’’ defect of bovine sperm. Theriogenology 9:165–173. Courtens, J., Amir, D., and Durand, J. (1980) Abnormal spermiogenesis in bulls treated with ethylene bromide: An ultrastructural and ultracytochemical study. J Ultrastruct Res 71:103–115. Czaker, R. (1985) On the origin of nuclear vacuoles in spermatozoa a fine structural and cytochemical study in mice. Andrologia 17: 547–557. Dada, R., Shamsi, M.B., Venkatesh, S., Gupta, N.P., and Kumar, R. (2010) Attenuation of oxidative stress & DNA damage in varicocelectomy: Implications in infertility management. Indian J Med Res 132:728–730. Franco Jr., J.G., Mauri, A.L., Petersen, C.G., Massaro, F.C., Silva, L.F., Felipe, V., et al. (2012) Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa. Int J Androl 35:46–51. Furuya, Y., Akashi, T., and Fuse, H. (2004) Effect of Bu-zhong-yi-qitang on seminal plasma cytokine levels in patients with idiopathic male infertility. Arch Androl 50:11–14. Fuse, H., Akashi, T., Mizuno, I., Nozaki, T., and Watanabe, A. (2006) Postoperative changes of sperm chromatin heterogeneity, using acridine orange staining, in varicocele patients. Arch Androl 52:223–226. Fuse, H., Iwasaki, M., Mizuno, I., and Ikehara-Kawauchi, Y. (2003) Evaluation of acrosome reactivity using the Acrobeads test in varicocele patients: Findings before and after treatment. Arch Androl 49:1–6. Fuse, H., Kazama, T., and Katayama, T. (1991) Hypoosmotic swelling test in patients with varicocele. Arch Androl 27:149–154. Gopalkrishnan, K., Padwal, V., Meherji, P.K., Gokral, J.S., Shah, R., and Juneja, H.S. (2000) Poor quality of sperm as it affects repeated early pregnancy loss. Arch Androl 45:111–117. Gorelick, J.I. and Goldstein, M. (1993) Loss of fertility in men with varicocele. Fertil Steril 59:613–616. Hedger, M.P. and Meinhardt, A. (2003) Cytokines and the immunetesticular axis. J Reprod Immunol 58:1–26. Ishikawa, T., Fujioka, H., Ishimura, T., Takenaka, A., and Fujisawa, M. (2007) Increased testicular 8-hydroxy-20 -deoxyguanosine in patients with varicocele. BJU Int 100:863–866. Ito, H., Fuse, H., Minagawa, H., Kawamura, K., Murakami, M., and Shimazaki, J. (1982) Internal spermatic vein prostaglandins in varicocele patients. Fertil Steril 37:218–222. Iwamoto, T. (2010) Semen analysis. In KANAHARA & CO., LTD. Male foctors: Guidebook for reproductive medicine ed. Japan Society for Reproductive Medicine pp. 154–155. Komiya, A., Watanabe, A., and Fuse, H. (2011) Herbal medicine in Japan. J Mens Health 8:S15–S18. Komiya, A., Watanabe, A., Kawauchi, Y., and Fuse, H. (2013) Sperm with large nuclear vacuoles and semen quality in the evaluation of male infertility. Syst Biol Reprod Med 59:13–20. Komiya, A., Watanabe, A., Kawauchi, Y., and Fuse, H. (2012) Testicular volume discrepancy is associated with decreased semen quality in infertile Japanese males with varicoceles. Reprod Med Biol 11: 117–121. Monqaut, A.L., Zavaleta, C., Lopez, G., Lafuente, R., and Brassesco, M. (2011) Use of high-magnification microscopy for the assessment of
Syst Biol Reprod Med Downloaded from informahealthcare.com by Gazi Univ. on 01/02/15 For personal use only.
42
A. Komiya et al.
sperm recovered after two different sperm processing methods. Fertil Steril 95:277–280. Montjean, D., Belloc, S., Benkhalifa, M., Dalleac, A., and Menezo, Y. (2012) Sperm vacuoles are linked to capacitation and acrosomal status. Hum Reprod 27:2927–2932. Neyer, A., Vanderzwalmen, P., Bach, M., Stecher, A., Spitzer, D., and Zech, N. (2012) Sperm head vacuoles are not affected by in-vitro conditions, as analysed by a system of sperm-microcapture channels. Reprod Biomed Online 26:368–377. Oliveira, J.B., Petersen, C.G., Massaro, F.C., Baruffi, R.L., Mauri, A.L., Silva, L.F., et al. (2010a) Motile sperm organelle morphology examination (MSOME): Intervariation study of normal sperm and sperm with large nuclear vacuoles. Reprod Biol Endocrinol 8:56. Oliveira, J.B., Massaro, F.C., Baruffi, R.L., Mauri, A.L., Petersen, C.G., Silva, L.F., et al. (2010b) Correlation between semen analysis by motile sperm organelle morphology examination and sperm DNA damage. Fertil Steril 94:1937–1940. Parkinson, T.J. (2000) Nuclear vacuolation as a cause of sterility in an angus bull. Vet J 159:207–210. Perdrix, A., Saidi, R., Menard, J.F., Gruel, E., Milazzo, J.P., Mace, B., et al. (2012) Relationship between conventional sperm parameters and motile sperm organelle morphology examination (MSOME). Int J Androl 35:491–498. Perdrix, A., Travers, A., Chelli, M.H., Escalier, D., Do Rego, J.L., Milazzo, J.P., et al. (2011) Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles. Hum Reprod 26:47–58. Revay, T., Nagy, S., Kopp, C., Flyckt, A., Rens, W., Rath, D., et al. (2009) Macrocephaly in bull spermatozoa is associated with nuclear vacuoles, diploidy and alteration of chromatin condensation. Cytogenet Genome Res 126:202–209. Sakkas, D. and Alvarez, J.G. (2010) Sperm DNA fragmentation: Mechanisms of origin, impact on reproductive outcome, and analysis. Fertil Steril 93:1027–1036. Saypol, D.C. (1981) Varicocele. J Androl 2:61–71. Shiraishi, K., Matsuyama, H., and Takihara, H. (2012) Pathophysiology of varicocele in male infertility in the era of assisted reproductive technology. Int J Urol 19:538–550.
Syst Biol Reprod Med, 2014; 60(1): 35–42
Skowronek, F., Casanova, G., Alciaturi, J., Capurro, A., Cantu, L., Montes, J.M., et al. (2012) DNA sperm damage correlates with nuclear ultrastructural sperm defects in teratozoospermic men. Andrologia 44:59–65. Smit, M., Romijn, J.C., Wildhagen, M.F., Veldhoven, J.L., Weber, R.F., and Dohle, G.R. (2013) Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate. J Urol 189:S146–150. Swerdloff, R.S. and Walsh, P.C. (1975) Pituitary and gonadal hormones in patients with varicocele. Fertil Steril 26:1006–1012. Thundathil, J., Palasz, A.T., Barth, A.D., and Mapletoft, R.J. (1998) Fertilization characteristics and in vitro embryo production with bovine sperm containing multiple nuclear vacuoles. Mol Reprod Dev 50:328–333. Tsujimura, A., Miyagawa, Y., Okuda, H., Yamamoto, K., Fukuhara, S., Nakayama, J., et al. (2011) Change in cytokine levels after administration of saikokaryuukotsuboreito or testosterone in patients with symptoms of late-onset hypogonadism. Aging Male 14:76–81. Tsujimura, A., Takada, S., Matsuoka, Y., Nakayama, J., Takao, T., Miyagawa, Y., et al. (2008) Clinical trial of treatment with saikokaryukotsuboreito for eugonadal patients with late-inset hypogonadism-related symptoms. Aging Male 11:95–99. Utsuno, H., Oka, K., Yamamoto, A., and Shiozawa, T. (2013) Evaluation of sperm head shape at high magnification revealed correlation of sperm DNA fragmentation with aberrant head ellipticity and angularity. Fertil Steril 99:1573–1580. WHO (1999) World Health Organization laboratory manual for the examination of human semen and sperm-cervical mucus interaction. World Health Organization/Cambridge University Press; Cambridge, UK. Zini, A., Azhar, R., Baazeem, A., and Gabriel, M.S. (2011) Effect of microsurgical varicocelectomy on human sperm chromatin and DNA integrity: A prospective trial. Int J Androl 34:14–19. Zini, A. and Dohle, G. (2011) Are varicoceles associated with increased deoxyribonucleic acid fragmentation? Fertil Steril 96: 1283–1287.
