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Sperm function tests after vasovasostomy

Ren-Qian WEN, Mei-Yi LIE, Pei-ling TIAN, Ning YANG, Yan-Jia JIANG, Ai-Ping CHEN

Family Planning Research Institute of Guangdong, Guangzhou 510600, China

Asian J Androl  2000 Jun; 2: 111-114

Keywords: semen; sperm function test; vasectomy; vasovasostomy
Abstract
Aim: To evaluate the sperm function after vasovasostomy. Methods: Semen samples from 42 subjects after vasovasostomy (Group A: 1-6 months, Group B: 6-12 months; Group C: 12-18 months after vasectomy reversal) were investigated. Semen from 34 normal fertile men was used as controls. Sperm function tests, including hypo-osmotic swelling test (HOST), acridine orange (AO) fluorescence, acrosome reaction (triple-stain), cervical mucus penetration test (CMPT), etc were done. Results: After vasectomy reversal, the percentage of HOST was significantly lower than that of the normal fertile men. In regard to AO, there were no significant differences between the three vasovasostomy groups and between these 3 groups and the controls. With triple-stain, the percentage of normal acrosome reaction was significantly lower in Group A as compared with the controls, but not in Groups B and C. There were no significant differences in the results of CMPT between the vasovasostomy groups and the controls. However, the number of poor type was significantly higher in Groups A and C than in the controls; the percentage of negative type were higher in Groups A and B than in the controls. Conclusion: After vasovasostomy a lower level of HOST remained for one year and  gradually recovered after one year. Six months after vasectomy reversal, the percentage of acrosome reaction could be changed from lower level to normal range. The data of AO indicated that the genetic material (double-stranded DNA) in spermatozoa was not affected by vasovasostomy. To evaluate the result of CMPT after vasectomy reversal, not only the normal results but also the abnormal results (poor and negative types) should also be considered.

1 Introduction

Sperm function tests recommended by the World Health Organization[1,2] and relevant authors[3,4] to be routinely used in research and clinical laboratories generally include sperm hypo-osmotic swelling test (HOST), cervical mucus penetration test (CMPT), acridine orange (AO) fluorescence, and acrosome reaction (triple-stain). After vasectomy the microenvironment and functions of testis and epididymis may be changed[5,6]. Our previous report has shown that after vasectomy the spermatozoa stored in the proximal vas deferens and epididymis might be damaged with impaired function[7]. There were quite a few research articles indicating the presence of abnormal semen parameters, such as lower sperm counts, lower sperm motility, and higher malformation rates, after vasectomy reversal[8-11]. Sharlip[12] reported that the causes of failure to achieve pregnancy after vasovasostomy were epididymal dysfunction and sperm antibodies. However, these reasons may only partly explained the lower pregnancy rate after vasectomy reversal[13]. In about 50% of patients, the sperm concentration, motility and morphology improved over 12 months following reversal[14], but some of the sperm functions may still be abnormal, which may be the important causes of the low pregnancy rate.

Various sperm function tests are employed to evaluate different aspects of sperm function: the HOST is used to evaluate sperm membrane integrity, the CMPT, the ability to penetrate cervical mucus, the triple-stain, the acrosome reaction, and the AO, to distinguish normal and abnormal nuclear chromatin.

2 Materials and methods

Forty-two subjects after vasectomy reversal, aged 28-45 years and the duration of vasectomy, 1-5 years, were recruited. Thirty-four fertile (having a child) men, aged 23-42 years, served as the control. They had no chronic illnesses and their testicular size was all within the normal range (15-25 mL) measured by the Preder orchidometer.

After 3-7 days of abstinence, semen samples were collected by masturbation at 1-6 months (Group A), 6-12 months (Group B) and 12-18 months (Group C) after vasectomy reversal. After routine semen analysis, the HOST and the capillary tube CMPT were performed according to the methods recommended by WHO[1,2]. Two hundred of spermatozoa underwent HOST were counted and the spermatozoa with b to g tail swelling were reported. The score of cervical mucus used for the CMPT was greater than 10[1], obtained from fertile women. For AO the method of Tejada et al[4] was employed. Slides were read under a fluorescence microscope within 2 hours after staining. At least 200 spermatozoa were observed the red and green cells were recorded. Spermatozoa with their heads stained green (normal sperm) were reported. For triple-stain, the method recommended by Tablot et al[3] was used. Two hundred cells on a slide were counted and 4 staining patterns were recorded. The percentage of spermatozoa with a normal acrosome reaction (category d[3]) was reported.

Statistical analysis was performed using one-way analysis of variance (between Groups A, B and C), Student's t-test and Chi-Square test with SPSS (Statistical Packet for Social Science) program.

3 Results

The results (means) of sperm concentration, sperm motility and normal sperm morphology from 34 normal sebjects (controls) were (90.252.2)106/mL, (58.518.2)% and (76.216.8)%, respectively. They were all within the normal range. In the vasectomy reversal Groups A, B, and C, the mean sperm count was markedly lower, being 34.5 (Group A), 20.8 (Group B) and 32.7 (Group C) millions, as well as the mean sperm motility, being 43.0%, 53.9%, and 54.6% for Groups A, B and C, respectively. However, for these groups, the mean normal morphology rate appeared to be within the normal range, being 50.7%, 60.8%, and 59.4%, respectively.

Table listed the results of HOST, AO fluorescence, triple-stain, and CMPT. The percentage of HOST in Groups A, B, and C was significantly lower than that of the controls (P<0.05); HOST in Group C was significant higher than that in Group A (F=4.1660, P<0.05). With AO, there was no significant differences within the vasovasostomy groups (F=1.3930, P>0.05) and between these groups and the controls (P>0.05). The data of triple-stain seemed to be similar to those of spermatozoa without an acrosome[3]. According to one-way analysis of variance, there was a significant difference between Groups A and C (F=3.6388, P<0.05). Meanwhile, the percentage of triple-stain in Group A was significant lower than that of the normal controls (t=2.22, P<0.05). In CMPT there was no significant difference between the vasovasostomy groups. However,  the values of poor type in Group A (2= 8.191, P<0.01) and Group C (2=4.429, P<0.05) were significant higher than that of the normal controls. The percentage of negative type in Group A (2=3.895, P<0.05) and Group B (2=4.427, P<0.05) were higher than those of normal controls.

Table. Results (means) of sperm function tests. bP< 0.05, cP<0.01, compared with controls; eP<0.05 compared with Group A.

 

Fertile controls

Group A

Group B

Group C

HOST (%):

81.09.4
(n=11)

56.915.5c
(n=36)

56.715.3c
(n=18)

66.910.9ce
(n=24)

AO (%):

61.114.7
(n=21)

57.811.5
(n=26)

65.18.7
(n=11)

56.413
(n=18)

Triple-stain:

23.913.0
(n=11)

15.86.3b
(n=17)

20.78.2
(n=13)

21.86.4e
(n=15)

CMPT (%):

good:

50.0 (17/34)

29.7 (11/37)

25.0 (4/16)

26.1 (6/23)

fair:

41.2 (14/34)

21.6 (8/37)

50.0 (8/16)

34.8 (8/23)

poor:

8.8 (3/34)

37.8 (14/37)c

12.5 (2/16)

30.4 (7/23)b

negative:

0 (0/34)

10.8 (4/37)b

12.5 (2/16)b

8.7 (2/23)

4 Discussion

After vasectomy reversal the reasons of lower pregnancy rate are antisperm antibodies[8,12,15], abnormal sperm morphology[10], epididymal dysfunctions[12], etc. Although WHO does not recommend HOST as a sperm function test[2], HOST can give additional information on the integrity and compliance of the sperm tail membrane. In this study we indicated that after vasectomy reversal the percentage of HOST in the semen was significantly lower than that of the controls. The result is similar to those of spermatozoa in proximal vas deferens (mature spermatozoa before ejaculation) of vasectomized men[16]. It was suggested that after vasectomy, the cell membrane integrity from spermatozoa stored in epididymis and proximal vas deferens was decreased[7] and this lower level remained for one year after vasectomy reversal. It was gradually recovered one year after vasovasostomy. Sperm movement is dependent largely on the integrity of sperm tail membrane, which is very important for the natural fertilizing process.

The AO fluorescence has been recommended as a new functional test for male fertility. It was shown that the percentage of normal sperm with green fluorescence at the head was lower in the infertile than the fertile men[17]. In present study we did not find significant difference within the vasovasostomy groups and between these groups and the normal controls. The authors believe that the genetic material, which AO is said to evaluate, are not affected by vasectomy or vasovasostomy and it is apparently not the cause of lowered pregnancy rate after vasectomy reversal. Our results showed that only in Group A the percentage of normal acrosome reaction was significantly lower than that of the normal controls, whereas those in Group B and Group C were not, suggesting that within 6 months after vasovasostomy the acrosome reaction was lower and was recovered 6 months after vasectomy reversal. Pampiglions et al[18] indicated that an acrosomal response of <31.3% (using triple-stain technique after stimulation by the calcium ionophore A23187) was a predictive value for spermatozoa that failed to fertilize. In this study A23187 was not used and the result of the control men was only 23.9%. Both studies indicated that the triple-stain technique was a useful test for evaluating sperm fertilizing ability.

The CMPT capillary tube test was recommended by WHO[1] as one of the routine sperm function method. With CMPT, good and fair were considered as normal result, and  poor and negative, abnormal. In the present study no significant difference was found between vasovasostomy groups and the normal controls. It is suggested that after vasectomy and vasovasostomy the ability of sperm to penetrate cervical mucus was reserved. However, the percentage of poor in Groups A and C, and the percentage of negative in Groups A and B were significantly higher than that of the normal controls. Although after vasectomy reversal, the good and fair were within the normal level, there was definitely a decreasing tendency. It was thus indicated that in evaluating the result of CMPT after vasectomy reversal, not only the normal results (good and fair) but also the abnormal results (poor and negative), shuld be considered.

5 Acknowledgements

This work received financial support from the State Family Planning Committee (China). We thank WHO for the gift of assay reagents.

References

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Correspondence to Dr Ren-Qian Wen, Family Planning Research Institute of  Guangdong, 17 Mei Dong Road, Dongshan District, Guangzhou 510600, China 
Fax: +86-20-8777 7331 
E-mail wenrq@163.net
Received 2000-03-13   Accepted 2000-05-09