Absence of sperm meiotic segregation error of chromosomes 1, 9, 12, 13, 16, 18, 21, X and Y in a case of 100 % necrozoospermia

Asian J Androl 2003 Jun; 5: 163-166             

Keywords: oligospermia; necrozoospermia; fluorescent in situ hybridization

Abstract

Varying degrees of necrozoospermia are common findings in cases of male sub-fertility; however, it is rare to find persistent and 100 % necrozoospermia. A case of persistent 100 % necrozoospermia was presented in this paper, where aneuploidy analysis was carried out on sperm. No known associations like thyrotoxicosis, genital infection, spinal injury and diabetes were found. Sperm fluorescent in situ hybridization (FISH) was carried out to evaluate sperm aneuploidy for chromosome 1, 9, 12, 13, 16, 18, 21, X and Y and did not show any excess of aneuploidy over controls. To the best of our knowledge, this is the first attempt on meiotic segregation analysis on 100 % necrozoospermic patient.

1 Introduction

Necrozoospermia is a rare condition and poorly documented cause of male infertility. Its incidence in infertile male was reported to be from 0.2 % to 0.5 % [1]. However, the precise incidence of true necrozoospermia is difficult to be determined from the literature, because many patients with immotile cilia syndrome (axonemal defects), protein carboxyl methylase deficiency, etc. were originally included in the necrozoospermia group. Moreover, the precise cellular defects leading to necrozoo-spermia are still to be defined.

Recent literature [2] has shown a correlation between poor semen quality and sperm aneuploidy. As patient of necrozoospermia requires assisted reproduction for fertility, it is important to understand the sperm aneuploidy rate before going to the procedure. Necrospermia of 100 % magnitude is very rare and literature search through MEDLINE did not show any reports analyzing the sperm chromosome aneuploidy in 100 % necrozoo-spermic patients. In the present study, we attempted to investigate the sperm aneuploidy by fluorescent in situ hybridization (FISH) in a case of persistent 100 % necrozoospermia.

2 Patients

The case presented here was a 43-year-old male and referred to our center from a private assisted reproduction center with the diagnosis of necrozoospermia for reproductive counseling. He had been married for 18 years without any issue. Clinical examination revealed no abnormality (general, local and systemic). There was no paraparesis, varicocele or hydrocele. The volume of both testes was normal. There was no history suggestive of chronic persistent genital infection or exposure to gonadotoxins, e.g., cigarette smoke, alcohol, alkylating agent, gossypol and pesticides. There was no history of recurrent chest infections (Kartagener Syndrome) or anosmia (Kallmann Syndrome).

The patient was investigated initially for routine semen examination, semen culture and sensitivity, gonadotropins, thyroid function, anti-sperm antibody, peripheral blood chromosomes and later for sperm aneuploidy. The semen samples were obtained by masturbation directly into a wide mouthed sterile centrifuge tube (50 mL) after abstinence (5 days) as well as after frequent ejaculations (10 times~15 times over a period of 5 days~7 days). Later, sperm were obtained from open testicular biopsy for hypoosmotic swelling (HOS) test.

The results of semen analysis at our center (repeated several times over one year) and in the past at outside laboratories (several times over ten years) were similar. These showed essentially normal according to World Health Organization [3] criteria except the presence of dead sperm and higher normal total sperm count [(80~160)10⁶/mL]. Initially, approximately 5 % sperm were sluggishly motile, however, since last five years all sperm were dead. Sperm examination after frequent ejaculation was performed to deplete stored sperm in vas deferens and caudal epididymis. No improvement in sperm viability was seen in these samples. Later, necro-zoospermia was confirmed by eosin Y, eosin-nigrosin and cresyl blue staining and HOS test. Repeated semen culture and sensitivity test showed no evidence of any infection (aerobic and anaerobic). The observations on gonadotropins (FSH and LH), thyroid function, blood sugar, autoantibodies and antisperm antibodies (re-peatedly; mixed anti-globulin test) were also normal. The peripheral blood chromosomes showed normal male karyotype (46, XY). To assess the potential of intracytoplasmic sperm injection (ICSI), HOS test was carried out on sperm obtained from testicular biopsy. The test was positive i.e., swelling of 10 %~15 % sperm nuclei and tail curling, indicating some live sperm and hence chance for successful ICSI.

FISH was carried out on nondecondensed sperm nuclei as per Halder and Tutscheck [4]. Semen sample was washed three times in PBS solution (pH 7.4) and sperm were fixed using 3 washes in the fixative (3:1 methanol:acetic acid). Finally, sperm were re-suspended in 100 mL~200 mL fresh fixative depending upon the volume. Approximately 10 mL were applied onto un-coated glass slides. The slides were then treated with 70 % acetic acid for 1 min and dehydrated in alcohol series (70 %, 90 % and 100 %, 3 min each). The fixed sperm on slide were treated in pepsin solution (10 mg/L, pH 1.5~2.0) for 20 minutes at 37 , washed in water, fixed in 1 % paraformaldehyde in 4 oC for ten minutes and finally dehydrated as before. Then probes preparation, hybridization, post hybridization washing and visualization were routinely performed. Probes and sperm DNA was denatured together for 5 minutes at 75 oC in an oven and incubated at 37 oC in moist chamber for 12 h~18 h (overnight). During the study, lymphocyte metaphase spread was used to check hybridization specificity. The slides were screened using a fluorescent microscope (Nikon Optiphot, Japan) and images were captured using a digital imaging system (Applied Imaging, UK).

3 Discussion

The use of FISH in assessment of sperm aneuploidy of infertile male appears to be most desirable. Because sperm karyotype through hamster egg and human sperm fertilization potentially bias the results. Here, only spermatozoa that are able to fertilize hamster oocytes are karyotyped (natural selection) and this may exclude spermatozoa that have a poor fertilization potential. Further-more, in the present case, sperm karyotype was not possible due to necrozoospermia. Sperm FISH did not show any increase in aneuploidy rate with chromosomes 1, 9, 12, 13, 16, 18, 21, X and Y (Table 1) when compared with healthy fertile men.

Table 1. Results of sperm FISH.

Note: Sperms without FISH signal were excluded from analysis.

In this study, we have carried out aneuploidy analysis on necrozoospermic sperm to find out any association of chromosomal aneuploidy and hence to aid reproductive counseling before assisted reproduction. As chromosomal aneuploidy is commonly associated with failure of gametes, male infertility, etc., it is justifiable to investigate the aneuploidy with a case of idiopathic 100 % necrozoospermia.

Although the precise mechanism leading to necrozoo-spermia was not clear, it was generally assumed that spermatozoa loose their viability during a prolonged stay in the male genital tract in conditions like spinal cord injury, genital tract infection or distal obstructions (sperm autoantibody related). However, in our case these conditions were unlikely, because we did not find any viable sperm even after repeated ejaculation with repeated sterile culture and negative anti-sperm antibody test. In addition, a high total sperm count also argued against the possibility of infection, toxin exposure, autoantibody, etc. Furthermore, testicular biopsy showed a few viable sperm (10 %~15%) instead of plenty. In this case, spermatogenesis/spermiogenesis appeared to be normal and it seemed that spermatozoa died soon after their production. Hence, it was justifiable to suspect chromosomal or genetic causes, including accelerated apoptosis. Apoptosis is an important mechanism for normal spermatogenesis and its disorders may lead to poor sperm quality [4]. Apoptotic sperm can be quantified with different staining (annexin V, Hoechst 33258, DAPI, propidium iodide, etc.) and flow cytometry as well as by terminal deoxy-nucleotidyl transferase-mediated dUTP [deoxy-uridine triphosphate] nick end labeling (TUNEL) assay [5-7]. In the literature, the percentage of apoptotic sperm varied from 0.1 % (normal fertile semen samples) to 50 % (seminoma carriers) [8]. We were unable to evaluate the apoptotic rate for technical reasons. In conclusion, we believe that the necrozoospermia in the present case was not due to the above-mentioned nine (1, 9, 12, 13, 16, 18, 21, X and Y) chromosome aneuploidy.

Acknowledgements

Authors acknowledge the Intramural Research Committee of Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India for funding the project entitled Meiotic segregation analysis in infertile men with seminal abnormality.

References

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[3] World Health Organization. WHO Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th edition, Cambridge:Cambridge University Press; 1999. p 60-1.
[4] Halder A, Tutscheck B. Analysis of meiotic segregation in human nondecondensed interphase spermatozoa by triple color rapid direct fluorescent in situ hybridization. Indian J Med Res 1998; 107: 94-7.
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[6] Oosterhuis GJ, Mulder AB, Kalsbeek-Batenburg E, Lambalk CB, Schoemaker J, Vermes I. Measuring apoptosis in human spermatozoa: a biological assay for semen quality? Fertil Steril 2000; 74: 245-50.
[7] Ricci G, Perticarari S, Fragonas E, Giolo E, Canova S, Pozzobon C, et al. Apoptosis in human sperm: its correlation with semen quality and the presence of leukocytes. Hum Reprod 2002; 17: 2665-72.
[8] Baccetti B, Collodel G, Piomboni P. Apoptosis in human ejaculated sperm cells(notulae seminologicae 9). J Submicrosc Cytol Pathol 1996; 28: 587-96.

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Correspondence to: Dr Ashutosh Halder, Assistant Professor, Dept. of Reproductive Biology, All India Institute of Medical Sciences, New Delhi-110 029, India.
Tel: +91-11-2659 3304 ext. 4211, Fax: +91-11-26588663
E-mail: ashutoshhalder@yahoo.co.in
Received 2002-08-30   Accepted 2003-02-20

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