Hum._Reprod.-2000-Zeyneloglu-853-6

8/10/2019 Hum._Reprod.-2000-Zeyneloglu-853-6

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Human Reproduction vol.15 no.4 pp.853856, 2000

Detection of chromosomal abnormalities by fluorescent

in-situ hybridization in immotile viable spermatozoa determined by hypo-osmotic sperm swelling test* Hulusi Bulent Zeyneloglu1! "ol#an Baltaci$

immature forms of sperm cells from the testis may be obtained %evinc &ge1 'li Haberal1 and %(Batioglu1 ith varyin! success.

"oor or absent fertili#ation rates are obtained in cases of 1$epartment of %bstetrics&'ynaecolo!y, and 2$epartment of

()*( usin! immotile e+aculated spermato#oa of unnon

-edical 'enetics, asent /niversity, nara, urey

viability a!y et al. , 15. otal immotility may be caused 3o hom correspondence should be addressed at -esa 7oru

by necro#oospermia, ultrastructural defects or metabolic imma

*itesi -anolya lo 24, )ayyolu, 0630, urey

turity of the spermato#oa.)f randomly selected immotile spermatozoa are used for (n cases of total necro#oospermia in the e+aculate, motile intracytoplasmic sperm inection +),%) pregnancy rates spermato#oa can sometimes be recovered from a testicular

are significantly decreased( .he hypo-osmotic swelling test biopsy ournaye et al. , 16. 9oever, hen selectin!

+H/%. is the only method available to detect the viablespermato#oa to in+ect into an oocyte, it is alays preferable

but immotile spermatozoa for ),%)( However evidence is to use the one that has shon some si!n of motility and ith

still lac#ing for the chromosomal abnormalities for the best morpholo!y available.

normal-loo#ing but immotile spermatozoa positive for

he hypoosmotic sellin! test 9%* or cell stainin!

H/%.( %perm samples from $0 infertile men with normal methods are often used to identify sperm viability. he mechan

chromosomal constitution were obtained( 'fter ercoll isms of 9%* and eosin: stainin! are mainly related to the

separation morphologically normal but immotile sperma- functional inte!rity of sperm membrane and normal functional

tozoa were transported individually into H/%. solution

activity of human spermato#oa ;eyendran et al. , 184. *ince for 1 min using micropipettes( ,ells that showed tail curling there is no available cellstainin! techni


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#oa in an astheno#oospermic population. *uccessful use of

colour fluorescence in-situ hybridization +3)%H for chro-

immotile spermato#oa has been reported in ()*( )asper

mosomes 4 5 and 16( .he same 3)%H procedure was

et al. , 16. lthou!h such cells have been successfully used applied for the motile spermatozoa from the same cohortin ()*(, controversies e=ist over hether sperm phenotype

which formed the control group( .he average aneuploidy abnormalities are indicative of !enotype abnormalities >n!el

rates were 1(70 and 1(89: in 1000 H/%. positive immotile et al. , 16. he multicolour fluorescence insitu hybridi#ation

and motile spermatozoa respectively detected by 3)%H for ?(*9 may be one approach to !ive an insi!ht into phenotype

each patient( /ur results indicate that morphologically

!enotype interaction of sperm cells, althou!h not the most

normal immotile but viable spermatozoa have an aneuplo- suitable techni


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held con+ointly ith the 54th nnual -eetin! of the merican *ociety

immotile spermato#oa ere transported individually into 9%*

for Geproduction, %ctober 4, 18, *an ?rancisco, ), /*.solution 9ypo10, *candinavian *cientific, 'othenbur!, *eden

H >uropean *ociety of 9uman Geproduction and >mbryolo!y

853

H(B(Zeyneloglu et al.

3igure $( "hoto!raphs of immotile spermato#oa after ?(*9 analysis

ith centromeric probes for the chromosomes 18 blue, I !reen

3igure 1( he spermato#oa incubated in hypoosmotic solution, the and : oran!e in a threecolour hybridi#ation protocol. ll probes

9%* C spermato#oa demonstrate curlin! and sellin! of the ere directly labelled ith fluorochromes and obtained

tails. *cale bar C 5 Em.

commercially. *cale bar C 5 Em.

usin! micropipettes 9unter *cientific, *affron Aalden, >sse= /7 of a microin+ection device ion, arishi!e, ;apanJ if the final


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.able )( )omparison of chromosomal status of motile and immotile sperm

ashout did not produce sufficient immotile spermato#oa, then the

cells from 20 infertile men after ?(*9 analysis spermato#oa from the interface beteen the !radients of "ercoll ere

(mmotile 9%*

-otile used. he spermato#oa ere incubated in hypoosmotic solution for

positive cells B

sperm cells B 10 min. he cells hich shoed tail curlin! ith sellin! in 9%*

viable cells ?i!ure 1 ere then transferred bac into 9? solution

I chromosome aneuploidy

0.46 0.43

to allo reversal of sellin!. hen the cells ere transferred to a

: chromosome aneuploidy

0.33 0.25

!lass microscope slide ith a small amount of 9? solution. he 18 chromosome aneuploidy

0.2D

0.23 cells ere alloed to dry in the drop of 9? solution on the slide

ny combinationn

0.81

0.D2 vera!e incidence of

1.D0 C 4.48

1.54 C 4.13 ith constant viein! under the inverted microscope. fter complete

spermato#oa ith abnormal

0.2.6 ran!e 0.82.5 ran!e

dryin! of the 9? solution, 1 drop 5 El of the )arnoyKs fi=ative 1

chromosomal status

ml acetic acid, 3 ml methanol as added to the spermato#oa. he cells ere constantly observed under the inverted microscope and

he to types of sperm cell ere not si!nificantly different.

another drop of fi=ative as added before complete dryin! and this step repeated 5 times.

$econdensations of the spermato#oa ere performed in 25 nmol&l

Results dithiothreitol $ in 0.1B trypsin iochrom 7', $1000 erlin,

he mean sperm concentration of the patients as D.3 C

'ermany. fter incubation ith $ for 15 min, slides ere rinsed

2.DC106&ml and median as D.5C106&ml ran!e 2.312. -ean


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tice in 2C**) sodium chloride and sodium citrate and dehydrated

percenta!e total motile type C type C type ) spermato

throu!h ethanol series D0, 85, 0B and air dried )oonen et al. ,#oa as 18.8 C D.6B median 1BJ ran!e D35. he mean

11. $ehydrated samples ere then denatured at D3L) in D0B

percenta!e normal morpholo!y accordin! to y!erber! strict formamide&2C**) and hybridi#ed overni!ht at 3DL), usin! probes

criteria as 1.8 C 1.0B median 2.0J ran!e 03. vera!e

for the chromosomes 18, I and : in a threecolour hybridi#ation protocol ischoff et al. , 14J lanco et al. , 16. ll probes ere I chromosome, :

chromosome and 18 chromosome anomaly

directly labelled ith fluorophores and obtained commercially @ysis

rates individually ere 0.46, 0.33 and 0.2DB respectively.(nc., $onerKs 'rove, (M, /*. he posthybridi#ation ash as

%verall, the avera!e incidence of spermato#oa ith abnormal

performed in 0.4C**)&0.3B "40 at D3L) for 2 min. he same

chromosomal status as 1.D0 C 4.48B 0.2.6B in immotile fi=ation and ?(*9 procedure ere applied for the motile sperm from

9%* positive sperm cells detected by ?(*9 for each patient.the same cohort, hich served as the control !roup. *lides ere

hese data are summari#ed in able (.

e=amined usin! a $"(&?()&e=as Ged tripleband pass filterbloc %n the other hand, ?(*9 analysis of motile spermato#oa

ion, ;apan alloin! the simultaneous visuali#ation of oran!e,

yielded the C chromosome, : chromosome and 18 chromo

!reen and blue fluorophores, and photo!raphed ith a camera ion some anomaly rates as 0.43, 0.25 and 0.23B respectively

?601 N$ ?i!ure 2. 9ybridi#ation efficiency as 5B in both

able (. %verall, the avera!e incidence of spermato#oa ith motile and immotile sperm !roups. n autosomal probe chromosome

abnormal chromosomal status in motile 9%* positive sperm

18 probe served as internal control. %nly intact cells and cells hich did not overlap ere scored. *lides ere scored blindly by to

cells detected by ?(*9 for each patient as 1.54 C 4.13B

independent investi!ators. he statistical analysis included O2ana

0.82.5B. he statistical analysis shoed that the difference lysis, and ttest. he mean values ere !iven ith their standard

in aneuploidy rate beteen the motile and immotile spermato

deviations C*$ in addition to median values and the ran!e.#oa as not si!nificant P C 0.23.

854

,hromosomal abnormalities in immotile spermatozoa Discussion -c(nnes et al. investi!ated sperm samples from infertile men

here is no described test ith noninvasive properties to

usin! ?(*9 ith probes for chromosomes 13 and 21 and found


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select a live spermato#oon for microin+ection purposes, since

a hi!hly si!nificant increase in the fre


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$espite these abnormalities, pre!nancies ere reported after

1D. hey found that the solution composed of 50B culture

intracytoplasmic in+ections of immotile spermato#oa from medium and 50B ater as the most appropriate for selection

e+aculate or testicular e=tract of men ith immotile cilia

of viable immotile spermato#oa for ()*(. Ae used a commercial syndrome 7ahraman et al. , 1DJ %lmedo et al. , 1D.

9%* solution in this study hich is mainly based on

9oever, antisperm antibodies, infection of the !enital tract ;eyendran solution and e did not detect any to=icity, since a

and prolon!ed periods of ane+aculation are most common

considerable number of the spermato#oa lived up to 24 h after

causes of total immotility @andervorst et al. , 1D. (n reversal of the hypoosmotic sellin! conditions.

addition, the lo ener!y production in the spermato#oa or

)hromosomal abnormalities are detected in P15B of

e=posure to radical o=y!en species may cause inadeven mitochondria of the midpiece. -en treated ith oral )oN10

in subfertile patients ith normo#oospermia, an increased rate

did better in the ne=t ()*( cycle after a failed cycle Mein

of chromosomal aberrations as detected -atsuda et al. ,and Mavon, 1D.

18. *e= and autosomal chromosomes ere found to be

(t is a common procedure to use morpholo!ically normal abnormal in 4.2 and 1.5B of infertile men respectively.

spermato#oa for ()*(, if available, and it has recently been

Ahile se= chromosome anomalies ere predominant in non confirmed in an >*9G> abstract that decreased fertili#ation

obstructive a#oospermic patients, autosomal chromosomal

rates result ith the in+ection of spermato#oa ith abnormal

anomalies ere more common in patients ith obstructive morpholo!y $e @os et al. , 1. (n addition, our collaborators

a#oospermia @an ssche et al. , 16. he most common

have already shon a relationship beteen diminished sperm abnormality involvin! se= chromosomes is II: and the most

maturity, increased cytoplasmic retention&head si#e and

common molecular defect is microdeletions in the Q? re!ion increased rate of chromosomal abnormalities as detected by

of the lon! arm of the : chromosome Gei+o et al. , 15.

multicolour ?(*9 -oretti et al. , 1D. case report has

ernardini investi!ated the cyto!enetic constitution of infertile


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recently been presented of a male ith :16 chromosome

and normal men and found hi!her rates of sperm aneuploidy

translocation and ?(*9 analysis of morpholo!ically normal for autosomes 1 and 1D ith decreasin! semen ., 'er!ely, ., Qeynelo!lu, 9.. et al. 1D Gelationship amon! studies to

further the evaluation of the men ith peripheral head si#e, morpholo!y and chromosome structure in human spermato#oa.

(n merican *ociety for Geproduction -edicine 53 nnual -eetin! 18

chromosomal abnormality. %ur results sho that the normal 22 %ctober, 1D, )incinnati, %9.Fertil. Steril. , =6, *158.

aryotype of the immotile but morpholo!ically normal sperma

a!y, Q."., Miu, ;., ;oris, 9. et al. 15 he result of intracytoplasmic sperm to#oa is

liely to decrease the ris of !enetic transmission


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in+ection is not related to any of the three basic sperm parameters.Hum.

Reprod. , 10, 1123112.

of the chromosomal abnormalities. (n conclusion, immotile eu!ebauer, $.)., euin!er, ;., ;ocenhovel, ?. et al. 10 R C 0K

spermato#oa from men ith normal aryotype in the absence

a=oneme in spermato#oa and some nasal cilia of a patient ith totally of microdeletions of the : chromosome may be safely used

immotile spermato#oa associated ith thicened sheath and short midpiece.

Hum. Reprod. , 8, 8186.for ()*( purposes.

%lmedo, *.., odar, ?., )hilli, ). et al. 1D *uccessful intracytoplasmic sperm

in+ection ith spermato#oa from a patient ith dysplasia of the

fibrous sheath and chronic respiratory disease.Hum. Reprod. , 1$, 14D14."alermo, '., ;oris, 9., $evroey, ". et al. 12 "re!nancies after

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spermato#oa for intracytoplasmic in+ection by the hypoosmotic sellin!

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Hum._Reprod.-2000-Zeyneloglu-853-6

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