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Effect of Spermatozoa Apoptosis on the Clinical Outcomes with Human in vitro Fertilization
Journal of Reproduction & Contraception 2012 Mar.; 23(1):41-50
http://www.RandC.cn [email protected]
Effect of Spermatozoa Apoptosis on the Clinical Outcomes with Human in vitro Fertilization Yu-bao WANG1, Lei-wen ZHAO2, Zu-qiong XIANG2, Xiu-zhuang ZHOU3 , You-lun GUI1, Chang-hai HE1 1. Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China 2. Shanghai Renji Hospital, Shanghai 200001, China 3. Zoneking Software Co., Ltd., Beijing 100190, China
Objective To analyze the effect of spermatozoa apoptosis on the clinical outcomes during in vitro Fertilization (IVF-ET). Methods A prospective analysis was carried out, 519 infertile couples were divided into 3 groups according to the clinical outcomes with IVF, including pregnancy and birth (liveborn group), pregnancy but abortion (miscarriage group), failure to pregnancy (failure group). Spermatozoa collected from 519 male partners were processed through density gradient centrifugation (DGC) and swim-up and the apoptosis of the spermatozoa, in ejaculated / processed semen, were evaluated using flow cytometry by determining the levels of disruption of mitochondrial membrane potential (d-MMP) and activation of caspase-3 (CP3). Results For ejaculated semen, apoptosis was significantly different among 3 groups individually, the result was as follows: liveborn group < failure group < miscarriage group (P<0.05). For processed semen, miscarriage had the highest apoptosis level (P<0.05), and there was no significant difference between liveborn group and failure group (P>0.05). Compared with the d-MMP, the activation of CP3, either in ejaculated or processed semen, showed more powerful predictive value for miscarriage other than liveborn and failure. The cutoff value of CP3 in ejaculated semen was 42.0%, with sensitivity: 0.931, specificity: 0.804. Conclusion The level of apoptosis of spermatozoa played a strongly impact on the clinical outcomes with IVF and the activation of CP3 in ejaculated semen possessed a high predictive value for miscarriage.
This work was supported by a grant from STCSM (Subject No: 074207002) Corresponding author: Yu-bao WANG; Tel: +86-21-64049215; E-mail: [email protected] 41
Key words: sperm apoptosis; caspase-3; mitochondrial membrane potential; miscarriage
It is well known that spermatozoa apoptosis plays an important role in spermiogenesis[1] and correlated with motility loss and abnormal morphology[2-5], causes DNA fragment[6], damages penetration capacity[7] and chromosomal decondensation after fertilized[8]. Reactive oxygen species (ROS) released from mitochondria and activation of caspase-3 (CP3) were considered as typical of apoptosis in ejaculated human spermatozoa[9-11]. And ROS contributed strongly to spermatozoa DNA fragmentation which frequently encountered in subfertile males [12-14]. The mechanism of ROS leading to the DNA damage was still unsolved but it was partly correlated with the disruption of mitochondrial membrane potential (d-MMP). On the other hand, the negative effects of DAN damage on the clinical outcomes, such as high rates of miscarriage and defected birth, were proved by series of studies[15-17]. Due to the special physical architecture of the spermatozoa (mitochondria and nucleus are in different cellular compartments), apoptosis can not precipitate immediate DNA cleavage typical of performance in somatic cells. As a consequence of this impediment to complete apoptosis, the spermatozoa with imcomplete apoptosis would not loss the ability to fertilize the oocyte[18]. So it is very possible for apoptosis spermatozoa, possessing high levels of DNA damage, to finish the fertilization. Because of the association between apoptosis and DNA damage, it is necessary to evaluate the impact of apoptosis on the clinical outcomes with IVF, and we also hypothesis that the levels of spermatozoa apoptosis may be one of the effective indicators for clinical outcomes. In this study, d-MMP and CP3 were calculated as the indicator of levels of spermatozoa apoptosis. The aim of this study was to compare the difference of apoptosis levels among 3 groups divided according to the clinical outcomes, including pregnancy and birth (liveborn group), pregnancy but abortion (miscarriage group) and failure to pregnancy (failure group), and evaluated the predictive values of spermatozoa apoptosis for clinical outcomes.
Materials & Methods Subjects This prospective study was carried out in 519 males from infertile couples who attended the fertility clinic at Renji Hospital of Jiaotong University (Shanghai, China) in 2008. Semen analysis was processed according to World Health Organization (WHO) recommendations[19] and Kruger strict criteria[20]. The analysis objectives were chosen under following conditions: female age <35 years, only with oviduct obstruction. Other factors, such as hydrosalpinx, chromosome abnormality, which contribute to the female infertility were not included in this 42
study. Male with normal sperm parameter: concentration ≥ 15 × 106 /ml, a+b ≥ 10%, normal morphology ≥4%, undergoing in vitro fertilization (IVF). This research was proved by Ethics Committee of Renji Hospital. All subjects signed informed consent for participation in this study. Apparatus and reagents Flow cytometry (FACSCalibur flow type) was producted by BD Company of America), caspase-3 assay kit (30029) was purchased from the U.S. Biotium. Mitochondrial membrane potential (MMP) detection kit (M34152) was purchased from Invitrogen of Molecular Probes Company. Methods Semen samples treatment Semen samples were obtained via masturbation into sterile containers and were equilibrated at 37℃ for 20 min to allow liquefaction. The liquefied semen was loaded onto the 40% and 80% discontinuous gradient and centrifugated at 350 × g for 15 min. In a second step, the pellet was washed with 1.5 ml HTF-HEPES (Quinn’s Advantage HTFHEPES, USA), and centrifugated at 300 × g for 10 min, then the third step, the pellet was washed with HTF-fertilization medium and centrifugated at 300 ×g for 10 min. On the top of resulting pellet, 1 ml HTF-fertilization medium was loaded to allow swim-up for 60 min in the incubator with 37℃ and 5% CO2. The sperm cell suspension recovered from the upper 0.3 ml was objected to assisted reproduction process, and the remains as well as partly ejaculated spermatozoa were tested for the activation of CP3 and d-MMP, respectively. Flow cytometry was used to detect the activated CP3 and d-MMP in spermatozoa. CP3 and d-MMP detection A fluorescein lablled inhibitor of CP3 (Carboxyfluorescein FLICA, Immunochemistry Technologies, Bloomington, MN, USA) was used to test the activated CP3. The samples were washed twice with PBS (500×g, 5 min each time), and the suspensions were adjusted with PBS to the terminal concentration 0.5-1.0 × 106/ml. About 5 µl 0.2 mmol/L inhibitor solution was added to 0.2 ml suspension and incubated in room temperature for 15-30 min, then other 0.3 ml PBS was added to test. A lipophilic cationic dye (5,5', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl carbocyanine chloride) was used to detect d-MMP in spermatozoa (ApoAlert Mitosensor kitTM, Clontech, CA, USA). The samples were washed twice with PBS (500 × g, 5 min each time), and the suspensions were adjusted with PBS to the terminal concentration 0.51.0 ×106/ml. And the aliquots were incubated at 37℃, away from light, for 20 min in 1 µg lipophilic cation diluted in PBS. Spermatozoa apoptosis All fluorescence signals of labelled spermatozoa were analysed using the flow cytometer 43
FACS (Becton Dickinson, San Jose, CA, USA). A minimum of 10 000 spermatozoa were examined for each assay. The sperm population was gated using 90-degree and forwardangle light scatter to exclude debris and aggregates. The excitation wavelength was 488 nm supplied by an argon laser at 15 mW. The percentage of positive cells and the mean fluorescence were calculated using the flow cytometer software Expo32 ADC (Coulter, Krefeld, Germany). All of the preparation of spermatozoa and FACS analysis were carried out by the fixed technicist respectively. IVF-ET Ovarian stimulation was accomplished by exogenous gonadotropin administration following a desensitization protocol with long-acting GnRH analoques. Human chorionic gonadotropin (hCG : 10 000 IU) was given intramuscularly on the evening of the day when the mean diameter of the dominant follicle reached 18 mm. Oocyte retrieval was performed 36 h after injection of hCG. The pronucleus was observed 16 h later after fertilization, and the cleavage rate was calculated at 48 h. The morphological quality of the embryos on day 3 was assessed according to the number of blastomeres and the percentage of fragments. Embryos with no fragments were classified as class 1 embryo, those with less than 20% of the volume of the embryo consisting of fragments were classified as class 2. Those with 20%-50% fragmentation were classified as class 3 and those with more than 50% fragmentation as class 4. 2 pronuclei (2PN) rate=number of 2PN/(number of retrieved oocyte-number of immatured oocyte ) × 100%, cleavage rate=number of cleavage/number of 2PN × 100%, good-quality embryo rate=number of class 1 and 2/number of cleavage× 100%. At 42-44 h after insemination, up to 3 embryos with the best morphology were transferred to the uterus of the female partners. All the patients were contacted by phone calling, and the miscarriages result from TORCH affection or accidents were excluded. According to the clinical outcomes with IVF, they were divided into 3 groups: pregnancy and birth (liveborn group), pregnancy but abortion (miscarriage group), failure to pregnancy (failure group). Statistical analysis Data were expressed by mean ± SD (x- ± s) and statistical analysis was performed using the SPSS10.0 software. Single-factor analysis of variance was used to compare the data, ROC curve was performed to analyze mitochondrial membrane potential (MMP) and caspase-3 predictability of abortion. P<0.05 was considered to be siginificantly different.
Results The comparison of basic information is presented in Table 1. There was no significant difference in subjects’ age, the number of oocytes retrieved, cleavage rate, among 3 groups. 44
Table 1 Comparison of basic information of 3 groups according to the clinical outcomes (x- ± s) Item
Liveborn group
n
173
Miscarriage group
Failure group 317
29
Female age (years)
31.1 ± 3.5
31.4 ± 3.3
30.7 ± 3.0
Male age (years)
33.4 ± 5.1
33.6 ± 5.3
33.4 ± 4.9
Oocytes retrieved
11.7 ± 6.8
9.8 ± 6.0
10.4 ± 7.0
Fertilization rate (%)
83.9 ± 16.3
Cleavage rate (%)
81.7 ± 17.7
79.5 ± 21.3
57.4 ± 24.6 *: P<0.05, compared with miscarriage group
49.0 ± 25.4
*
Good-quality embryo rate (%)
82.4 ± 28.0*
78.3 ± 20.5
80.8 ± 28.6 #
50.6 ± 28.4#
#: P<0.05, compared with liveborn group
However, low fertilization rate was observed in miscarriage group (P<0.05), there was no significant difference between liveborn group and failure group (P>0.05). As for good-quality embryo rate, liveborn group was the highest (P<0.05), and the other 2 groups were not different significantly (P>0.05). The comparison of spermatozoa d-MMP/CP3 from ejaculated / processed semen among 3 groups is shown in Table 2. For ejaculated semen, miscarriage group had the highest rate of d-MMP and CP-3 activation (P<0.05), followed by failure group (P<0.05) and then the liveborn group (P<0.05); for processed semen, miscarriage group still showed the highest apoptosis rate (P<0.05), and no significant different was observed between liveborn group and failure group (P>0.05). Table 2 Comparison of spermatozoa d-MMP and CP3 from ejaculated or processed semen among 3 groups (x- ± s) Item n
Liveborn group
Miscarriage group
Failure group
173
29
317
Ejaculated d-MMP
24.8 ± 13.5*
40.2 ± 12.3
30.5 ± 11.8*#
CP3
31.9 ± 5.9
*
54.8 ± 7.5
36.7 ± 6.4*#
16.5 ± 9.0*
25.1 ± 7.7
17.9 ± 9.5*
27.4 ± 3.7
22.4 ± 4.0*
Processed d-MMP CP3
20.0 ± 3.7 *:P<0.05, compared with miscarriage group
*
#:P<0.05, compared with liveborn group
Figure 1 shows that there was a significant decrease of the percentage of d-MMP and CP3 after density gradient centrifugation (DGC) and swim-up. The results of receiver operating characteristic (ROC) analysis are shown in Table 3, the predictive value of apoptosis seemed to be more powerful for miscarriage than for 45
60
*: P <0.05, compared with ejaculated semen
Percentage (%)
50 Ejaculated semem
40 *
30
*
Processed semen
20 10 0 CP3
d-M M P
Figure 1 Comparison of spermatozoa d-MMP / CP3 between ejaculated and processed semen
Table 3 Az value of spermatozoa d-MMP / CP3 from ejaculated / processed for the clinical outcomes Item
Liveborn group
Miscarriage group
Failure group
173
29
317
d-MMP
0.629
0.828
0.307
CP3
0.349
0.914
0.550
d-MMP
0.506
0.725
0.444
CP3
0.449
0.839
0.473
n Ejaculated
Processed
liveborn and failure. For miscarriage, CP3 Az value (the area under the ROC curves) was higher than d-MMP Az value either in ejaculated or in processed (0.914, 0.828; 0.839, 0.725, respectively), and CP3 in ejaculate spermatozoa was the most effective one with 0.914 of Az value. Furthermore, the cutoff value of CP3 in ejaculated spermatozoa in this study was 42.0%, with sensitivity: 0.931, specificity: 0.804 (Figures 2, 3).
Discussion Apoptosis was one of the important reasons for male infertility[1]. Many studies showed that the spermatozoa apoptosis affected semen parameters significantly such as concentration, motility, and morphology[2-5]. Some spermatozoa function studies also proved that high level apoptosis would damage the spermatozoa function of penetration[7] and decondensation[8]. However, few studies were carried out to elucidate the impact of spermatozoa apoptosis on the clinical outcomes. At the present study, disruption of mitochondrial membrane potential (d-MMP) and caspase-3 (CP3) typical of apoptosis were detected according to the clinical outcomes, 46
Figure 2 The ROC curve of d-MMP / CP3 from ejaculated and processed for miscarriage
Figure 3 Scatter plot showing clinical outcomes and the cutoff value of CP3
including pregnancy and birth (liveborn group), pregnancy but abortion (miscarriage group), fail to pregnancy (failure group). There were no significant differences in the distribution of age, number of oocytes retrieved, cleavage rate. However, miscarriage group had the lowest fertilization rate and good-quality embryo rate among 3 groups, at the same time, miscarriage group had the highest apoptosis level. According to some previous studies, we believed that apoptosis with high level damages the fertilization by decreasing spermatozoa penetration. As far as the good-quality embryo rate was considered, we supposed it connected with the DNA damage, that we would discuss below. It is well known that ejaculated human spermatozoa may present various degrees of DNA damage. Although there were different theories to explain the DNA damage’s origins, apoptosis was the most important one[21]. ROS releasing from mitochondrial typical of event 47
in apoptosis was the main causes to DAN damage[12]. Furthermore, because of the special physical architecture of the spermatozoa (mitochondria and nucleus are in different cellular compartments), there was a risk of a sperm carrying damaged genome enter into the oocyte, resulting in poor embryo development, miscarriage, or birth defects[23]. In this study these were proved that miscarriage possessed the highest apoptosis level and lowest good-quality embryo rate. Density gradient centrifugation (DGC) followed by swim-up can effectively separate motile spermatozoa that are capable of fertilizing an oocyte[24]. The method is widely used for sperm preparation prior to assisted reproductive techniques (ART). In this study the apoptosis level was significantly decreased through DGC and swim-up preparation. The result was consisted with previous result (Figure 1). There was a significant difference in apoptosis rate from the ejaculated semen between liveborn group and failure group. However, there was no significant difference between the 2 groups after processing. The different elimination rate of apoptosis spermatozoa through DGC between the 2 groups was probably the explanation for the changes. A small sample size (n=20, totally) study about the relationship between CP3, processed through DGC + swim-up, and clinical outcomes showed that liveborn group had the lowest CP3 (liveborn: 14.3 ± 2.8%; miscarriage: 23.2 ± 9.5%; failure: 26.6 ± 13.6%)[25]. In this study the lowest CP3 of processed spermatozoa was observed in liveborn group (20.0 ± 3.7%), however, the highest one was in miscarriage group (27.4 ± 3.7%), and the middle one was in failure group (22.4 ± 4.0%), a little difference existed compared with the previous one. Small sample size was possibly the main reason for the different parts of the two study results. Although the direct link between miscarriage and CP3 has not been proved, DNA damage is possible to be the key event through which CP3 influenced the clinical outcomes. And the association between DNA damage and activation of CP3 was proved to be weak in fertile donors[5], but more pronounced in infertility patients[27]. The positive correlation between d-MMP and CP3 in human spermatozoa from fertile donors and infertility patients was proved by several studies[26,28]. At the present study, the similar result was obtained either in ejaculated (r =0.7, P<0.01) or in processed (r =0.6, P<0.05). Besides, CP3 directly participated the d-MMP and on the other side CP3 independent way also be present[29]. It was the possible reason, in this study, that the level of d-MMP was significantly lower than CP3 either in ejaculated or in processed (22.2% vs 35.7%; 16.6% vs 20.4%, respectively, P<0.05).
Conclusion The predictive value of CP3 was more powerful than d-MMP’s for miscarriage, 48
furthermore, ejaculated CP3 was better than processed’s, and for ejaculated CP3 the cutoff value was 42.0% with sensitivity: 0.931, specificity: 0.804 (Figure 2, 3). The establishment of the cutoff value may provide additional information for semen analysis.
Acknowledgement This project was supported by research funds with the approval of the Ethical Committee of the Faculty of Medicine, Jiaotong University, Shanghai, China.
References 1. Moustafa MH, Sharma RK, Thornton J, et al. Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility. Hum Reprod, 2004, 19(1):129-38. 2. Chen Z, Hauser R, Trbovich AM, et al. The relationship between human semen characteristics and sperm apoptosis:a pilot study. J Androl, 2006, 27(1):112-20. 3. Almeida C, Cardoso MF, Sousa M, et al. Quantitative study of caspase-3 activity in semen and after swimup preparation in relation to sperm quality. Hum Reprod, 2005, 20(5):1307-13. 4.
Taylor SL, Weng SL, Fox P, et al. Somatic cell apoptosis markers and pathways in human ejaculated sperm: Potential utility as indicators of sperm quality. Mol Hum Reprod, 2004, 10(11):825-34.
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Said T, Agarwal A, Grunewald S, et al. Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: An in vitro model. Biol Reprod, 2006, 74(3):530-7.
6. Paasch U, Sharma RK, Gupta AK, et al. Cryopreservation and thawing is associated with varying extend of activation of apoptotic machinery in subsets of ejaculated human spermatozoa. Biol Reprod, 2004, 71 (6):1828-37. 7.
Grunewald S, Said TM, Paasch U, et al. Relationship between sperm apoptosis signalling and oocyte penetration capacity. Int J Androl, 2008, 31(3):325-30.
8. Grunewald S, Reinhardt M, Blumenauer V, et al. Increased sperm chromatin decondensation in selected nonapoptotic spermatozoa of patients with male infertility. Fertil Steril, 2009, 92(2):572-7. 9. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod, 2000, 15(6):1338-44. 10. Paasch U, Grunewald S, Agarwal A, et al. Activation pattern of caspases in human spermatozoa. Fertil Steril, 2004, 81(Suppl 1):802-9. 11. Grunewald S, Sharma R, Paasch U, et al. Impact of caspase activation in human spermatozoa. Microsc Res Tech, 2009, 72(11):878-88. 12. de Iuliis GN, Thomson LK, Mitchell LA, et al. DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-29-deoxyguanosine, a marker of oxidative stress. Biol Reprod, 2009, 81(3):517-24. 13. Koppers AJ, de Iuliis GN, Finnie JM, et al. Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. J Clin Endocrinol Metab, 2008, 93(8):3199-207. 14. de Iuliis GN, Newey RJ, King BV, et al. Mobile phone radiation induces reactive oxygen species production and DNA damage in human spermatozoa in vitro. PLoS One, 2009, 4(7):e6446. 15. Aitken RJ, de Iuliis GN. On the possible origins of DNA damage in human spermatozoa. Mol Hum Reprod, 2010, 16(1):3-13. 49
16. Aitken RJ, de Iuliis GN, McLachlan RI. Biological and clinical significance of DNA damage in the male germ line. Int J Androl, 2009, 32(1):46-56. 17. Aitken RJ, Koopman P, Lewis SE. Seeds of concern. Nature, 2004, 432(1):48-52. 18 Aitken RJ, Gordon E, Harkiss D, et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod, 1998, 59(5):1037-46. 19 World Health Organization. WHO Laboratory Manual for the Examination of Human Semen-Cervical Mucus Interaction. 4th ed. Cambridge: Cambridge University Press, 1999. 20. Kruger TF, Coetzee K. The role of sperm morphology in assisted reproduction. Hum Reprod, 1999, 5 (2):172-8. 21. Aitken RJ, De Iuliis GN. On the possible origins of DNA damage in human spermatozoa. Mole Hum Reprod, 2010, 16(1):3-13. 23. Aitken RJ, De Iuliis GN. Value of DNA integrity assays for fertility evaluation. Soc Reprod Fertil Suppl, 2007, 65:81-92. 24. Chen SU, Ho HN, Chen HF, et al. Combination of directs swim-up technique and discontinuous Percoll gradient centrifugation for sperm preparation of oligoasthenozoo-spermic samples. Arch Androl, 1996, 37 (2):103-9. 25. Grunewald S, Reinhardt, Blumenauer, et al. Effects of post-density gradient swim-up on apoptosis signalling in human spermatozoa. J Androl, 2010, 42(2):127-31. 27. Marchetti C, Gallego MA, Defossez A, et al. Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: Correlation with apoptosis and sperm parameters. Hum Reprod, 2004, 19(5):1127-34. 28. Grunewald S, Paasch U, Said TM, et al. Caspase activation in human spermatozoa in response to physiological and pathological stimuli. Fertil Steril, 2005, 83(Suppl 1):1106-12. 29. Espinoza JA, Paasch U, Villegas JV. Mitochondrial membrane potential disruption pattern in human sperm. Hum Reprod, 2009, 24(9):2079-85. (Received on January 12, 2012)
50
http://www.RandC.cn [email protected]
Effect of Spermatozoa Apoptosis on the Clinical Outcomes with Human in vitro Fertilization Yu-bao WANG1, Lei-wen ZHAO2, Zu-qiong XIANG2, Xiu-zhuang ZHOU3 , You-lun GUI1, Chang-hai HE1 1. Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China 2. Shanghai Renji Hospital, Shanghai 200001, China 3. Zoneking Software Co., Ltd., Beijing 100190, China
Objective To analyze the effect of spermatozoa apoptosis on the clinical outcomes during in vitro Fertilization (IVF-ET). Methods A prospective analysis was carried out, 519 infertile couples were divided into 3 groups according to the clinical outcomes with IVF, including pregnancy and birth (liveborn group), pregnancy but abortion (miscarriage group), failure to pregnancy (failure group). Spermatozoa collected from 519 male partners were processed through density gradient centrifugation (DGC) and swim-up and the apoptosis of the spermatozoa, in ejaculated / processed semen, were evaluated using flow cytometry by determining the levels of disruption of mitochondrial membrane potential (d-MMP) and activation of caspase-3 (CP3). Results For ejaculated semen, apoptosis was significantly different among 3 groups individually, the result was as follows: liveborn group < failure group < miscarriage group (P<0.05). For processed semen, miscarriage had the highest apoptosis level (P<0.05), and there was no significant difference between liveborn group and failure group (P>0.05). Compared with the d-MMP, the activation of CP3, either in ejaculated or processed semen, showed more powerful predictive value for miscarriage other than liveborn and failure. The cutoff value of CP3 in ejaculated semen was 42.0%, with sensitivity: 0.931, specificity: 0.804. Conclusion The level of apoptosis of spermatozoa played a strongly impact on the clinical outcomes with IVF and the activation of CP3 in ejaculated semen possessed a high predictive value for miscarriage.
This work was supported by a grant from STCSM (Subject No: 074207002) Corresponding author: Yu-bao WANG; Tel: +86-21-64049215; E-mail: [email protected] 41
Key words: sperm apoptosis; caspase-3; mitochondrial membrane potential; miscarriage
It is well known that spermatozoa apoptosis plays an important role in spermiogenesis[1] and correlated with motility loss and abnormal morphology[2-5], causes DNA fragment[6], damages penetration capacity[7] and chromosomal decondensation after fertilized[8]. Reactive oxygen species (ROS) released from mitochondria and activation of caspase-3 (CP3) were considered as typical of apoptosis in ejaculated human spermatozoa[9-11]. And ROS contributed strongly to spermatozoa DNA fragmentation which frequently encountered in subfertile males [12-14]. The mechanism of ROS leading to the DNA damage was still unsolved but it was partly correlated with the disruption of mitochondrial membrane potential (d-MMP). On the other hand, the negative effects of DAN damage on the clinical outcomes, such as high rates of miscarriage and defected birth, were proved by series of studies[15-17]. Due to the special physical architecture of the spermatozoa (mitochondria and nucleus are in different cellular compartments), apoptosis can not precipitate immediate DNA cleavage typical of performance in somatic cells. As a consequence of this impediment to complete apoptosis, the spermatozoa with imcomplete apoptosis would not loss the ability to fertilize the oocyte[18]. So it is very possible for apoptosis spermatozoa, possessing high levels of DNA damage, to finish the fertilization. Because of the association between apoptosis and DNA damage, it is necessary to evaluate the impact of apoptosis on the clinical outcomes with IVF, and we also hypothesis that the levels of spermatozoa apoptosis may be one of the effective indicators for clinical outcomes. In this study, d-MMP and CP3 were calculated as the indicator of levels of spermatozoa apoptosis. The aim of this study was to compare the difference of apoptosis levels among 3 groups divided according to the clinical outcomes, including pregnancy and birth (liveborn group), pregnancy but abortion (miscarriage group) and failure to pregnancy (failure group), and evaluated the predictive values of spermatozoa apoptosis for clinical outcomes.
Materials & Methods Subjects This prospective study was carried out in 519 males from infertile couples who attended the fertility clinic at Renji Hospital of Jiaotong University (Shanghai, China) in 2008. Semen analysis was processed according to World Health Organization (WHO) recommendations[19] and Kruger strict criteria[20]. The analysis objectives were chosen under following conditions: female age <35 years, only with oviduct obstruction. Other factors, such as hydrosalpinx, chromosome abnormality, which contribute to the female infertility were not included in this 42
study. Male with normal sperm parameter: concentration ≥ 15 × 106 /ml, a+b ≥ 10%, normal morphology ≥4%, undergoing in vitro fertilization (IVF). This research was proved by Ethics Committee of Renji Hospital. All subjects signed informed consent for participation in this study. Apparatus and reagents Flow cytometry (FACSCalibur flow type) was producted by BD Company of America), caspase-3 assay kit (30029) was purchased from the U.S. Biotium. Mitochondrial membrane potential (MMP) detection kit (M34152) was purchased from Invitrogen of Molecular Probes Company. Methods Semen samples treatment Semen samples were obtained via masturbation into sterile containers and were equilibrated at 37℃ for 20 min to allow liquefaction. The liquefied semen was loaded onto the 40% and 80% discontinuous gradient and centrifugated at 350 × g for 15 min. In a second step, the pellet was washed with 1.5 ml HTF-HEPES (Quinn’s Advantage HTFHEPES, USA), and centrifugated at 300 × g for 10 min, then the third step, the pellet was washed with HTF-fertilization medium and centrifugated at 300 ×g for 10 min. On the top of resulting pellet, 1 ml HTF-fertilization medium was loaded to allow swim-up for 60 min in the incubator with 37℃ and 5% CO2. The sperm cell suspension recovered from the upper 0.3 ml was objected to assisted reproduction process, and the remains as well as partly ejaculated spermatozoa were tested for the activation of CP3 and d-MMP, respectively. Flow cytometry was used to detect the activated CP3 and d-MMP in spermatozoa. CP3 and d-MMP detection A fluorescein lablled inhibitor of CP3 (Carboxyfluorescein FLICA, Immunochemistry Technologies, Bloomington, MN, USA) was used to test the activated CP3. The samples were washed twice with PBS (500×g, 5 min each time), and the suspensions were adjusted with PBS to the terminal concentration 0.5-1.0 × 106/ml. About 5 µl 0.2 mmol/L inhibitor solution was added to 0.2 ml suspension and incubated in room temperature for 15-30 min, then other 0.3 ml PBS was added to test. A lipophilic cationic dye (5,5', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl carbocyanine chloride) was used to detect d-MMP in spermatozoa (ApoAlert Mitosensor kitTM, Clontech, CA, USA). The samples were washed twice with PBS (500 × g, 5 min each time), and the suspensions were adjusted with PBS to the terminal concentration 0.51.0 ×106/ml. And the aliquots were incubated at 37℃, away from light, for 20 min in 1 µg lipophilic cation diluted in PBS. Spermatozoa apoptosis All fluorescence signals of labelled spermatozoa were analysed using the flow cytometer 43
FACS (Becton Dickinson, San Jose, CA, USA). A minimum of 10 000 spermatozoa were examined for each assay. The sperm population was gated using 90-degree and forwardangle light scatter to exclude debris and aggregates. The excitation wavelength was 488 nm supplied by an argon laser at 15 mW. The percentage of positive cells and the mean fluorescence were calculated using the flow cytometer software Expo32 ADC (Coulter, Krefeld, Germany). All of the preparation of spermatozoa and FACS analysis were carried out by the fixed technicist respectively. IVF-ET Ovarian stimulation was accomplished by exogenous gonadotropin administration following a desensitization protocol with long-acting GnRH analoques. Human chorionic gonadotropin (hCG : 10 000 IU) was given intramuscularly on the evening of the day when the mean diameter of the dominant follicle reached 18 mm. Oocyte retrieval was performed 36 h after injection of hCG. The pronucleus was observed 16 h later after fertilization, and the cleavage rate was calculated at 48 h. The morphological quality of the embryos on day 3 was assessed according to the number of blastomeres and the percentage of fragments. Embryos with no fragments were classified as class 1 embryo, those with less than 20% of the volume of the embryo consisting of fragments were classified as class 2. Those with 20%-50% fragmentation were classified as class 3 and those with more than 50% fragmentation as class 4. 2 pronuclei (2PN) rate=number of 2PN/(number of retrieved oocyte-number of immatured oocyte ) × 100%, cleavage rate=number of cleavage/number of 2PN × 100%, good-quality embryo rate=number of class 1 and 2/number of cleavage× 100%. At 42-44 h after insemination, up to 3 embryos with the best morphology were transferred to the uterus of the female partners. All the patients were contacted by phone calling, and the miscarriages result from TORCH affection or accidents were excluded. According to the clinical outcomes with IVF, they were divided into 3 groups: pregnancy and birth (liveborn group), pregnancy but abortion (miscarriage group), failure to pregnancy (failure group). Statistical analysis Data were expressed by mean ± SD (x- ± s) and statistical analysis was performed using the SPSS10.0 software. Single-factor analysis of variance was used to compare the data, ROC curve was performed to analyze mitochondrial membrane potential (MMP) and caspase-3 predictability of abortion. P<0.05 was considered to be siginificantly different.
Results The comparison of basic information is presented in Table 1. There was no significant difference in subjects’ age, the number of oocytes retrieved, cleavage rate, among 3 groups. 44
Table 1 Comparison of basic information of 3 groups according to the clinical outcomes (x- ± s) Item
Liveborn group
n
173
Miscarriage group
Failure group 317
29
Female age (years)
31.1 ± 3.5
31.4 ± 3.3
30.7 ± 3.0
Male age (years)
33.4 ± 5.1
33.6 ± 5.3
33.4 ± 4.9
Oocytes retrieved
11.7 ± 6.8
9.8 ± 6.0
10.4 ± 7.0
Fertilization rate (%)
83.9 ± 16.3
Cleavage rate (%)
81.7 ± 17.7
79.5 ± 21.3
57.4 ± 24.6 *: P<0.05, compared with miscarriage group
49.0 ± 25.4
*
Good-quality embryo rate (%)
82.4 ± 28.0*
78.3 ± 20.5
80.8 ± 28.6 #
50.6 ± 28.4#
#: P<0.05, compared with liveborn group
However, low fertilization rate was observed in miscarriage group (P<0.05), there was no significant difference between liveborn group and failure group (P>0.05). As for good-quality embryo rate, liveborn group was the highest (P<0.05), and the other 2 groups were not different significantly (P>0.05). The comparison of spermatozoa d-MMP/CP3 from ejaculated / processed semen among 3 groups is shown in Table 2. For ejaculated semen, miscarriage group had the highest rate of d-MMP and CP-3 activation (P<0.05), followed by failure group (P<0.05) and then the liveborn group (P<0.05); for processed semen, miscarriage group still showed the highest apoptosis rate (P<0.05), and no significant different was observed between liveborn group and failure group (P>0.05). Table 2 Comparison of spermatozoa d-MMP and CP3 from ejaculated or processed semen among 3 groups (x- ± s) Item n
Liveborn group
Miscarriage group
Failure group
173
29
317
Ejaculated d-MMP
24.8 ± 13.5*
40.2 ± 12.3
30.5 ± 11.8*#
CP3
31.9 ± 5.9
*
54.8 ± 7.5
36.7 ± 6.4*#
16.5 ± 9.0*
25.1 ± 7.7
17.9 ± 9.5*
27.4 ± 3.7
22.4 ± 4.0*
Processed d-MMP CP3
20.0 ± 3.7 *:P<0.05, compared with miscarriage group
*
#:P<0.05, compared with liveborn group
Figure 1 shows that there was a significant decrease of the percentage of d-MMP and CP3 after density gradient centrifugation (DGC) and swim-up. The results of receiver operating characteristic (ROC) analysis are shown in Table 3, the predictive value of apoptosis seemed to be more powerful for miscarriage than for 45
60
*: P <0.05, compared with ejaculated semen
Percentage (%)
50 Ejaculated semem
40 *
30
*
Processed semen
20 10 0 CP3
d-M M P
Figure 1 Comparison of spermatozoa d-MMP / CP3 between ejaculated and processed semen
Table 3 Az value of spermatozoa d-MMP / CP3 from ejaculated / processed for the clinical outcomes Item
Liveborn group
Miscarriage group
Failure group
173
29
317
d-MMP
0.629
0.828
0.307
CP3
0.349
0.914
0.550
d-MMP
0.506
0.725
0.444
CP3
0.449
0.839
0.473
n Ejaculated
Processed
liveborn and failure. For miscarriage, CP3 Az value (the area under the ROC curves) was higher than d-MMP Az value either in ejaculated or in processed (0.914, 0.828; 0.839, 0.725, respectively), and CP3 in ejaculate spermatozoa was the most effective one with 0.914 of Az value. Furthermore, the cutoff value of CP3 in ejaculated spermatozoa in this study was 42.0%, with sensitivity: 0.931, specificity: 0.804 (Figures 2, 3).
Discussion Apoptosis was one of the important reasons for male infertility[1]. Many studies showed that the spermatozoa apoptosis affected semen parameters significantly such as concentration, motility, and morphology[2-5]. Some spermatozoa function studies also proved that high level apoptosis would damage the spermatozoa function of penetration[7] and decondensation[8]. However, few studies were carried out to elucidate the impact of spermatozoa apoptosis on the clinical outcomes. At the present study, disruption of mitochondrial membrane potential (d-MMP) and caspase-3 (CP3) typical of apoptosis were detected according to the clinical outcomes, 46
Figure 2 The ROC curve of d-MMP / CP3 from ejaculated and processed for miscarriage
Figure 3 Scatter plot showing clinical outcomes and the cutoff value of CP3
including pregnancy and birth (liveborn group), pregnancy but abortion (miscarriage group), fail to pregnancy (failure group). There were no significant differences in the distribution of age, number of oocytes retrieved, cleavage rate. However, miscarriage group had the lowest fertilization rate and good-quality embryo rate among 3 groups, at the same time, miscarriage group had the highest apoptosis level. According to some previous studies, we believed that apoptosis with high level damages the fertilization by decreasing spermatozoa penetration. As far as the good-quality embryo rate was considered, we supposed it connected with the DNA damage, that we would discuss below. It is well known that ejaculated human spermatozoa may present various degrees of DNA damage. Although there were different theories to explain the DNA damage’s origins, apoptosis was the most important one[21]. ROS releasing from mitochondrial typical of event 47
in apoptosis was the main causes to DAN damage[12]. Furthermore, because of the special physical architecture of the spermatozoa (mitochondria and nucleus are in different cellular compartments), there was a risk of a sperm carrying damaged genome enter into the oocyte, resulting in poor embryo development, miscarriage, or birth defects[23]. In this study these were proved that miscarriage possessed the highest apoptosis level and lowest good-quality embryo rate. Density gradient centrifugation (DGC) followed by swim-up can effectively separate motile spermatozoa that are capable of fertilizing an oocyte[24]. The method is widely used for sperm preparation prior to assisted reproductive techniques (ART). In this study the apoptosis level was significantly decreased through DGC and swim-up preparation. The result was consisted with previous result (Figure 1). There was a significant difference in apoptosis rate from the ejaculated semen between liveborn group and failure group. However, there was no significant difference between the 2 groups after processing. The different elimination rate of apoptosis spermatozoa through DGC between the 2 groups was probably the explanation for the changes. A small sample size (n=20, totally) study about the relationship between CP3, processed through DGC + swim-up, and clinical outcomes showed that liveborn group had the lowest CP3 (liveborn: 14.3 ± 2.8%; miscarriage: 23.2 ± 9.5%; failure: 26.6 ± 13.6%)[25]. In this study the lowest CP3 of processed spermatozoa was observed in liveborn group (20.0 ± 3.7%), however, the highest one was in miscarriage group (27.4 ± 3.7%), and the middle one was in failure group (22.4 ± 4.0%), a little difference existed compared with the previous one. Small sample size was possibly the main reason for the different parts of the two study results. Although the direct link between miscarriage and CP3 has not been proved, DNA damage is possible to be the key event through which CP3 influenced the clinical outcomes. And the association between DNA damage and activation of CP3 was proved to be weak in fertile donors[5], but more pronounced in infertility patients[27]. The positive correlation between d-MMP and CP3 in human spermatozoa from fertile donors and infertility patients was proved by several studies[26,28]. At the present study, the similar result was obtained either in ejaculated (r =0.7, P<0.01) or in processed (r =0.6, P<0.05). Besides, CP3 directly participated the d-MMP and on the other side CP3 independent way also be present[29]. It was the possible reason, in this study, that the level of d-MMP was significantly lower than CP3 either in ejaculated or in processed (22.2% vs 35.7%; 16.6% vs 20.4%, respectively, P<0.05).
Conclusion The predictive value of CP3 was more powerful than d-MMP’s for miscarriage, 48
furthermore, ejaculated CP3 was better than processed’s, and for ejaculated CP3 the cutoff value was 42.0% with sensitivity: 0.931, specificity: 0.804 (Figure 2, 3). The establishment of the cutoff value may provide additional information for semen analysis.
Acknowledgement This project was supported by research funds with the approval of the Ethical Committee of the Faculty of Medicine, Jiaotong University, Shanghai, China.
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