| Abstract | Cilj istraživanja: Glavni ciljevi istraživanja bili su: procjena plodnosti muških partnera u
parova uključenih u postupke pomognute oplodnje, utvrđivanje učestalosti kromosomskih
aberacija i mikrodelecija Y kromosoma u muškaraca s poremećajem plodnosti, utvrđivanje
utjecaja pojedinih staničnih, mikrobioloških i biokemijskih nalaza u sjemenu, kao i razine
hormona u krvi na smanjenje plodnosti i neplodnost, te da li i u kojoj mjeri oni mogu utjecati
na uspješnost metoda pomognute oplodnje.
Ispitanici i metode: Ispitivanu skupinu čini 305 pacijenata Odjela za humanu reprodukciju
Klinike za ginekologiju i porodništva KBC-a Rijeka.
Pacijenti su, prema nalazu sjemena po kriterijima SZO, podijeljeni u 6 skupina:
normozoospermija, oligozoospermija, azoospermija, astenozoospermija,
oligoastenozoospermija i teška oligoastenozoospermija.
U Zavodu za biologiju i medicinsku genetiku Medicinskog fakulteta Sveučilišta u Rijeci, za
svakog od 106 ispitanika je analizirano najmanje 20 mitoza citogenetičkim metodama
kratkotrajane kulture limfocita periferne krvi, bojenjem kromosoma Giemsom i G-pruganjem.
Molekularne analize mikrodelecija Y kromosoma su analizirane PCR metodom s uzetim
uzorkom DNK, dobivenim iz pune krvi ispitanika.
Ispitivanje je vršeno s dvjema multiplim lančanim reakcijama polimeraze pri čemu su rabljene
odgovarajuće DNK početnice (engl. primer). Multipla reakcija A obuhvatila je DNK početnice
za STS biljege (engl. sequence-tagged sites) sY14, sY84, sY127, sY254 i gen ZFX/ZFY.
Multipla reakcija B učinjena je za STS biljege: sY86, sY134, s Y255, s Y14 i gene ZFX/ZFY. U
dva muškarca s mikrodelecijama učinjene su dopunske reakcije s STS biljezima: sY87, sY88,
sY135, sY114, sY143, sY152, sY157, sY160 i genom RBMY1.
Mikrobiološko ispitivanje sjemena urađeno je standardnim bakteriološkim kulturama.
Određivana je i razina izoenzima LDHx u sjemenu, te razine osnovnih hormona hipofize i
testisa u serumu: prolaktin (PRL), folikulostimulirajući hormon (FSH), luteinizirajući hormon
(LH) i testosteron (T).
Rezultati: Usporedbom godina starosti i trajanja neplodnosti nije nađena statistički značajna
razlika između skupina ispitanika uključenih u metode pomognute oplodnje.
Usporedbom razine FSH između grupa ispitanika, nađena je statistički značajna razlika
između ispitanika s azoospermijom koji su imali značajno višu srednju vrijednost FSH od onih
s normozoospermijom, oligozoospermijom, astenozoospermijom, oligoastenozoospermijom i
teškom oligoastenozoospermijom.
Srednja vrijednost razine LH bila je također statistički začajno povišena (p<0.026) u grupi
ispitanika s azoospermijom u odnosu na grupu muškaraca s normalnim brojem spermija i na
grupu s oligoastenozoospermijom (p<0,016), ali su sve te srednje vrijednosti bile u granicama
normale. Izoenzim LDHX (laktat dehidrogenaza) bio je statistički značajno povišen u ispitanika s
oligoastenozoospermijom, teškom oligoastenozoospermijom i azoospermijom u odnosu na
one s normozoospermijom.
Bakterijske infekcije sjemena nađene u 31% ispitanika (41/132).
Major kromosomske aberacije bile su prisutne u 14,15% ispitanika (15/106); najčešće, u
34,78%, nađene su u grupi ispitanika s azoospermijom (8/23).
Najčešća aberacija muškog kariotipa u osoba uključenih u metode pomognute oplodnje je
Klinefelterov sindrom.
Od svih 106 testiranih muškaraca, dvojica su imala mikrodeleciju AZFc i AZFb+AZFc regije
(1,8%), a kako su svi su bili u grupi od 23 ispitanika s azoospermijom, učestalost
mikrodelecija u toj grupi bila je 8,7%, odnosno gotovo peterostruko viša nego u ukupnom
broju testiranih ispitanika sa smetnjama plodnosti (2/23).
Zaključak: Citogenetička obrada i gensko testiranje na mikrodelecije Y kromosoma trebale bi
biti obavezne za muškarce s azoospermijom, oligozoospermijom i teškom
oligoastenozoospermijom prije njihova uključenja u primjenu metoda pomognute oplodnje, jer
se u tim skupinama nalazi najveći broj aberacija i mikrodelecija koje mogu direktno utjecati na
uspješnost metoda pomognute oplodnje, a mogu biti i prenesene na potomstvo. |
| Abstract (english) | Objectives: The aim of this study is to investigate the male fertility in couples seeking IVF orIVF+ICSI and to evaluate the contribution of chromosome anomalies and Y chromosome
microdeletions. The objectives are also to evaluate the cellular and biochemical parameters of
the semen and the levels of hormones in the serum and their interference on the success of
assisted reproduction methods.
Patients and Methods: 305 male patients of the Department of Human Reproduction,
Obstetric and Gynecology Clinic, Clinical Hospital Center Rijeka were divided in 6 groups
according to the WHO criteria on sperm count: normozoospermia, oligozoospermia,
azoospermia, asthenozoospermia, oligoasthenozoospermia, and severe
oligoasthenozoospermia.
At the Department of Biology and Medical Genetics, University of Rijeka, School of Medicine
cytogenetic analyses were carried out in 106 male patients by the short peripheral blood
culture and the G-binding method. PCR method was used for the molecular analyses of the Y
chromosome microdeletions. Human genomic DNA was extracted by standard methods.
Multiplex reaction A was carried out using a combination of the oligonucleotide primers for the
Y specific sequence tagged sites (STSs): sY84, sY127, sY254, sY14 and for the gene
ZFX/ZFY. Multiplex reaction B was carried out with primers for STSs: sY86, sY134, sY255,
sY14 and for the gene ZFX/ZFY. In two men with chromosome Y microdeletions, additional
screening was performed for the next STS markers: sY87, sY88, sY135, sY114, sY143,
sY152, sY157, sY160 and for the gene RBMY1.
Content and the type of bacteria were analyzed in the semen as well as level of the LDHx
isoenzyme.
The level of the prolactin, FSH, LH and testosterone were analyzed for each patient
separately.
Results: There is no statistically significant difference in the age or the duration of infertility
between tested groups of males included in the methods of assisted reproduction (MAR).
Azoospermic males have statistically significantly higher level of FSH as compared to
normozoospermic, oligozoospermic, asthenozospermic, oligoasthenozoospermic and severe
oligoasthenozoospermic males.
The LH level is also significantly higher in the group of azoospermic male as compared to
normozoospermic and oligoasthenozoospermic ones.
Isoenzyme LDHx is significantly elevated in the oligoasthenozoospermic, severe
oligoasthenozoospermic and azoospermic groups of patients as compared to
normozoospermic group.
In 31% of the ejaculates (41/132) bacterial infections were found.
Major chromosomal aberrations were found in 14,15% (15/106) of patients; mostly, 34,78%,
in the azoospermic group (8/23). The Klinefelter syndrome was the most frequent aberration
of male karyotype in the analysed group of patients. The Y chromosome microdeletion of AZFc and AZFb+AZFc region was detected in two out of
106 patients analyzed (1.8%); both patients were from the azoospermic group (2/23) what
means that in this specific group with 8.7% microdeletions, the frequency of this anomaly is
fivefold higher than in the whole group analyzed.
Conclusion: The highest frequency of the major chromosomal aberrations and Y
chromosome microdeletion are found in the group of azoospermic and oligozoospermic
groups of patients. Cytogenetic analyses and testing for the Y chromosome deletion should
be mandatory for those groups of male patients seeking the methods of assisted
reproduction, since these anomalies may highly interfere with success of the methods and
they can be transmitted to offsprings. |
