Role of Sperm Function Tests in Unexplained Infertility
                     f oJObst. t;,. Cyn. of lndra Vol. 51, No.5: September/October 2001 Pg 146-149 
Role of Sperm Function Tests in Unexplained Infertility 
Suvarna S. Khadilkar, Parathe, S.M. Tayde 
Oeparllnent of Obstetrics and Gynaecology, Grant Medical College and Cam a & A/bless Hospital , Mumbai, Malwrnslztra , India. 
Summary 
Unexplained infertility is a challenging problem for treating doctor. The apparent normal semen analysis 
does not rule out the functional abnormalities of sperms. The role of sperm function by H.O.S. test 
[H ypoosmotic swelling test] in Lmexplained infertility has been studied and analyzed here. 35 patients of 
unexplained infertility were screened for sperm function test by H.O.S. We observed that 20% of patients 
had abrtormal H.O.S. test and the results correlated well with morphological abnormalities. Abnorma 1 
HOS indicates poor membrane integrity. Thus it may adversely affect the results of various ARTS. 
J 
Introduction all practical purposes, detailed history , c lini ca l 
examination and routine semen anal ysis ts ~uffictcnl 
Unexplained infertility is a challenging problem for most of the patients. But this spectrum of patients 
for the treating doctor but is a frustrating experience for with unexplained infertility needs further evaluation 111 
the infertile coup le. The couple goes from one hospital to the form of sperm function tests. H.O.S. or Hypoosmotic 
another, changing protocols after protocols for months swelling test is one important sperm function test which 
together, just to realize that nothing seems to be working is studied in this paper. The aims & objectives of this 
inspite of every thing being apparently normal. Many paper were to assess sperm function by H.O.S. test & 
things need to be looked at in such patients. Nidation perform detailed analysis of sperm morphology and 
failure, luteinized unruptured follicles (L.U.F.) and LPD other routine parameters on patients with unexplained 
or luteal phase deficiency are few of the factors being infertility. We studied whether any of the routine 
studied all over the world. However "assessment of parameters of semen analysis correlated well with HOS 
sperm functions in unexplained infertility" is an test results. 
important factor that needs to be studied. 
Materials and Methods 
The role of sperm function by H.O.S. test 
(hypoosmotic swelling testl in unexplained infertility 35 patients diagnosed to have unexplained 
has been studied and analyzed here. The routine semen infertility, attending infertility clinic in Cama Hospital , 
analysis repeatedly comes apparently normal, but one were selected for the study. All the patients had j ' 
does not know whether the longevity & fertilizing undergone all routi.ne investigations, spectfic infertility 
property of sperms is normal or not. There is definitely investigations like D & C, laparoscopy I HSC and 
something beyond "normal routine semen analysis". For routine semen analysis. None of the inves tigation s 
146 
Unexplained infertilitlJ 
showed any obvious abnormality. The c.::ouples had Technique: HOS solution was prepared in our lUI 
regular coi tus for period of one year or more which did laboratory at Cama Hospital and was stored at 4°C temp . 
not result in any concep tions. 
Composition of H.O.S. Solution: 
The semen samples of these patients were Fructose:- 1.351 gm 
cma lyzed in different labs and had always shown normal Sodium Citrate:- 0.735gm 
routine anc1lys1s as per W.H.O. recommendation for the Distilled Water:-100 ml. 
same. (W. H.O. 1992) [Table I]. All the patients selected 1 ml of HOS solution was taken in a tube and warmed at 
had counts above 20 millions/ml, with motility of more 37°C for 10 minutes. 0.1 ml of semen sample was then 
than 50"'n with at least 25% R.L.Ps [rapid linear added to H.O.S. solution, mixed well and incubated at 
progrc.:;sive]. This was not always specified in all the 37°C for 30 minutes. 10 ul (Micro litre) of incubated 
reports. All had more than 30% morphologically normal mixture was taken on a labeled clean glass s lide covered 
sperms. All the patients were asked to collect semen with a cover slip & examin.ed under microscope at 400X 
samp les by masturbation in wide-mouthed sterile glass magnification. 
beakers after the period of 3-4 days of abstinence. After Percentage of sperms having coiled tails was calculated 
liquefaction of samples a drop was taken on a slide for in two fields and the mean was taken. 
the initial examination which included initial coiling 
and initial count. Thereafter the samples were processed Interpretation 
in two parts. Routine analysis was done on the first part HOS Positive % = Total mean percentage post HOS 
with grea t care and Sperm morphology was done on coiling- Initial coiling percentage. 
stained smears under higher magnification [1000X] Normal H.O.S. = > 60% sperms with coiled tails. [HOS 
applying 'st ricter criteria'. A separate count was kept of positive%] 
head, mid piece and the tail abnormalities. Inference=Good prognosis. Longevity, Viability and 
fertilization potential is good & normal. 
Table I Abnormal HOS=< 60% sperms with coiled tails. 
WHO Criteria for Normal Semen Analysis [1992] Inference= poor prognosis. Indica tes d egenerati Vl' 
Seminal Parameter Normal values changes in sperm membrane secondary to either 
Volume 2ML Infection or some inherited disorder. 
Motility >50'Yo Forward progressive, H.O.S, test results and other seminal parameters were 
>25% R.L.P.s [rapid linear compared and studied in details. 
progressive] 
Morphology >30% normal Table II 
Vitality > 75% alive Sperm Count and HOS Test. 
W.B.C. Cone. 70 5 dissolve protein (eg. Gelatin coated on the s lide) is 
seen under microscope as 'haloes ' o f dissolved 
148 
llllexplni11ed iHfertility 
gelattn around sperm heads, when treated with A consideration of the hypoosmotic swelling 
suitable reagents. test in combination with the sperm viability tn 
2. N.C.D. test. uclcar Chromatin Decondensation hypoosmotic solution will enhance the detection ol 
Test: [Copalkrishnan ct al-1991] differences in membrane properties on the sperm surface. 
This tests the ability of the sperms to form male Sperm specimens failing both the VHOS and HOS tests 
pronucleus by decondensation of nuclear chromatin. have been associated with poor fertilizing ability and 
3. SMA! test: Sperm Mitochondrial Activity Index test poor in vitro fertilization outcome. The tests will help 
[Gopalkrishnan et al-1990] identify the specimens requiring spcoal sperm 
This tests the ability of spermatic mitochondrial processing. Percoll gradient method should be avoided 
enzvmcs to bring about good motility of sperms. as it may further damage the wca k acrosoma I 
4. f\.0. test: Acridine Orange staining of spermatozoa. membranes. 
I Tejclda cl a!, 1984] 
Thi-, test detects the ability of sperm nuclear Conclusion 
chromatin to resist denaturation with the help of 
fluorescent stain. Our study emphasizes that in the work up of 
unexplained infertility, sperm function tests have a 
All the above tests arc useful however need definite place. Even though the tests do not have much 
special stains & reagents, they are more time consumirtg therapeutic value, they have a good prognostic value. 
& should be done in proper laboratory set up. H.O.S. test 20% abnormal sperm function assessed by H.O.S. test in 
gives overall membrane integrity, longevity, so gives a this study calls for doing this test and perhaps other 
fair idea about the prognosis. relevant tests as a step before selecting the ART protocol 
for the patients with unexplained infcrtility.ICSI may be 
Osmolarity of semen in fertile men ranges better choice for patients with abnormal sperm function 
between 360-380M osmol (velasquez et al, 1977). Normal tests. 
Osmolarity of semen is an important factor for successful 
fertilization. The osmolarity of the HOS solution is about Acknowledgements 
150M osmols. 
We would like to thank the Dean, Grant medical 
Recently at Loma Linda University in college, Head of the department of obstetrics and 
California, Chan et al, 1996 reported a new method gynaecology, Grant Medical College and the 
combinmg the supravital stain [Eosine Y] test with the Superintendent Cama and Alblcss hospitals, Mun1bai 
hypoosmotic sperm swelling test (VHOS) which reduced for allowing us to publish this data. 
the number of false positive results from HOS tests. We 
eliminated this possibility by doing the initial coiling References 
and subtracting it from the post HOS coiling. 1. Chan PJ, Corselli L Patton W, Jacobson J: The sperm 
viability in hypoosmotic solution test, Loma Linda 
Univeristy, CAUSA, (1996). Chan PJ, Tredway DR, 
Corselli L Pang SC, SUBC: Hum Rep rod; Sept 6(8): 
1115, 1991. 
2. Gopalkrishnan K, Hinduja IN, Anandkumar TC: 
Mol. Androl; 3: 243, 1990. 
3. Gopalkrishna K, Hinduja IN, and Anandkumar TC: 
Arch Androl; 27: 43, 1991. 
4. Gopalkrishna K: Current Science 68 (4): 353, 1995. 
5. I.C.M.R. Bulletin; Vol. 26 No. 10:97. Octo. 1996. 
6. Jeyendran, RS, Vander Ven HH, Perez Pelaez MM, 
Carabo BG and Zaneveld LCD, J. Reprod Fertil 70: 
219, 1984. 
7. Tejada RI, MitchellJC, Norman A MarikJJ, Friedman 
S: Fertil steril42: 87, 1984. 
8. Velazquez A Fedrovi N; Delgado H. Rosado A; 
Int. J. Fertil, 22-92,1977. 
9. World Health Organization (1992) Laboratory 
Manual for the examination of human semen & 
sperm- cervical mucus interaction. In WHO special 
programme for research development & Research 
Fig 1: Microphotograph showing post HOS coiled tails training for Human Reproduction, y