Document Type : Original Article
Authors
1 Life Memorial Hospital, Bucharest, Romania;Department of Genetics, Faculty of Biology, University of Bucharest, Bucharest, Romania
2 Life Memorial Hospital, Bucharest, Romania
3 Department of Genetics, Faculty of Biology, University of Bucharest, Bucharest, Romania
Abstract
Keywords
Male infertility is a common and severe health
problem affecting 7% of populations (
Most men presenting with infertility are
found to have idiopathic oligo-astheno-terato-
zoospermia (OAT) (
Of the 7% of men suffering from infertility, 40%
have idiopathic infertility. The cause of infertility
in these men seems to be due to underlying genetic
abnormalities (
In infertile males, abnormal karyotype is more
frequent than in the general population (
Male factor problems commonly manifest through an alteration of one or more semen parameters and the following terminology are used to describe these changes:
At least 15 gene families, that are involved in
spermatogenesis, are found on the long arm of
chromosome Y (Yq) (
From 2007 to April 2011, 850 infertile men
(including 350 patients with oligo-asthenoteratozoospermia, 150 patients with severe oligozoospermia, 100 patients with
Patients had been referred for chromosomal analysis, fluorescence in situ hybridization (FISH) for detection of Yp chromosome microdeletions and polymerase chain reaction (PCR) for 15 sites of AZF region on Y chromosome. In this prospective study we investigated infertile men prior to intracytoplasmic sperm injection (ICSI) treatment. Informed consent was obtained from the patients and controls prior to collection of heparinized blood samples.
Karyotype analysis was performed on peripheral
blood lymphocytes. After 72 hours culture, the
cells were harvested, hipotonised and fixed using
3:1 methanol: acetic acid. The metaphases were
spread on slides. At least 10 metaphases were analyzed for each case and chromosomal abnormalities were reported according to the recommendations of the International System for Chromosome
Nomenclature (ISCN 2009) (
FISH was performed for 629 cases (using commercially available Vysis (Abbott) FISH probes
which are complementary to the region of interest on a particular chromosome) (
PCR was performed to screen the microdeletions in the AZF region of the Y chromosome.
Genomic DNA was extracted according to standard procedure from peripheral blood samples (
This original study was approved by The Ethics Committee of Life Memorial Hospital Statistical Analysis: The results of the two groups were compared by a two-tailed Fisher’s exact test and calculated online using Graph Pad (http://www.graphpad.com/quickcalcs/contingency1.cfm8).
Primer sequences of the sequence-tagged-sites (STSs) used in the detection of AZF loci (AZFa, AZFb and AZFc) and SRY
STS | Sequence 5' - 3' | Locus | Size (bp) |
---|---|---|---|
GTG ACA CAC AGA CTA TGC TTC | AZFa | 320 | |
ACA CAC AGA GGG ACA ACC CT | |||
GGC TCA CAA ACG AAA AGA AA | AZFb | 274 | |
CTG CAG GCA GTA ATA AGG GA | |||
GGG TGT TAC CAG AAG GCA AA | AZFc | 400 | |
GAA CCG TAT CTA CCA AAG CAG C | |||
GAA TAT TCC CGC TCT CCG GA | SRY | 495 | |
GCT GGT GCT CCA TTC TTG AG | |||
AGA AGG GTC TGA AAG CAG GT | AZFa | 326 | |
GCC TAC TAC CTG GAG GCT TC | |||
GTC TGC CTC ACC ATA AAA CG | AZFb | 301 | |
ACC ACT GCC AAA ACT TTC AA | |||
GTT ACA GGA TTC GGC GTG AT | AZFc | 126 | |
CTC GTC ATG TGC AGC CAC | |||
GAA TAT TCC CGC TCT CCG GA | SRY | 495 | |
GCT GGT GCT CCA TTC TTG AG | |||
Karyotyping was carried out in 850 infertile men
with impaired spermatogenesis. As shown in table 2,
a total of 108 patients (12.70%) had chromosomal abnormalities: 6 azoospermic patients with Klinefelter’s
syndrome (47,XXY), 2 patients with 47,XYY syndrome (confirmed by FISH) (
Chromosomal abnormalities in infertile men
Normal karyotype | Infertile maleNo./% | Fertile maleNo./% |
---|---|---|
108(12.70% ) | 4(8.16%) | |
8(7.41%) | - | |
100(92.59%) | 4(8.16%) | |
Isochomosom Y. Specific pattern of signals: an X chromosome specific signal (CEPX) and two signals characteristic of Y chromosome (SRY LSI). Result: 47,XYY. ish(CEPXx1) (SRYx2).
Screening of AZF microdeletions was carried
out in the 67 patients including 46 infertile patients
with
Chromosomal abnormality in studied groups
Patients | Autosomal abnormalities | Sex abnormalities | Polymorphic variants | Total abnormalities (autosomal + sex chromosome) | Total |
---|---|---|---|---|---|
20(2.35%) | 11(1,29%) | 77(9.06%) | 31(3.65) | 108(12.71%) | |
5(1,43%) | 1(0.29%) | 10(2.86%) | 6 (1.71%) | 16(4.57%) | |
8(8%) | 7(7%) | 15(15%) | 15 (15%) | 30(30%) | |
2(1.33%) | 1(0.67%) | 5(3.33%) | 3 (2%) | 8(5.33%) | |
1(1%) | - | 11(11%) | 1 (1%) | 12(12 %) | |
4(2.67%) | 2(1.33%) | 36(24%) | 6 (4%) | 42(28%) | |
- | - | 4(8.16%) | - | 4(8.16%) | |
Numerical and structural abnormalities in infertile men
Chromosomal aberrations | Karyotype | No. of cases | Frequency % | |
---|---|---|---|---|
Inversion | 46,XY,inv(9)(p11q13) | 22 | 2.59% | |
46,XY,inv(9)(p11q12) | 3 | 0.35% | ||
46,XY,inv(3)(p11q11.2)inv(9)(p11q13) | 1 | 0.12% | ||
46,XY,inv(1)(q23p13) | 1 | 0.12% | ||
46,XY,inv(1)(q13p31) | 1 | 0.12% | ||
46,XY,inv(10)(p11.2q21) | 1 | 0.12% | ||
46,XY,inv(5)(pterq13) | 1 | 0.12% | ||
Deletion | 46,X,delY(q11.2) | 1 | 0.12% | |
46,X,delY(q12) | 1 | 0.12% | ||
46,XY,t(1;19)(p13;13.3) | 1 | 0.12% | ||
46,XY,t(3;13)(p21;p11.2) | 1 | 0.12% | ||
46,XY,t(1;9)(q11;p13) | 2 | 0.24% | ||
45,XY,t(13;14)(q10q10) | 5 | 0.59% | ||
45,XY,t(14;15)(q10q10) | 1 | 0.12% | ||
46,XY,t(9;10)(q12q26) | 1 | 0.12% | ||
46,XY,t(9;3)(q32q28) | 1 | 0.12% | ||
46,XY,t(1;4)(q43q13) | 1 | 0.12% | ||
46,XY,t(7;8)(q31.1q24) | 1 | 0.12% | ||
46,XY,t(3;6)(q28;q13) | 1 | 0.12% | ||
Klinefelter SyndromSyndrom XX | 47,XXY | 6 | 0.71% | |
47,XYY | 2 | 0.24% | ||
46,XX | 1 | 0.12% | ||
Chromosomal polymorphisms in infertile men
Chromosomal polymorphic variations | Karyotype | No. of cases | Frequency % |
---|---|---|---|
46,XY,1qh+ | 10 | 9.26% | |
46,XY, 9qh+ | 9 | 8.33% | |
46,XY,16qh+ | 2 | 1.85% | |
46,X Yqh+ | 3 | 2.78% | |
46,XY, fra(17) | 7 | 6.48% | |
46,XY, fra(16) | 4 | 3.70% | |
46,XY, 14ps+ | 5 | 4.63% | |
46,XY, 15ps+ | 2 | 1.85% | |
46,XY, 21ps+ | 2 | 1.85% | |
46,XY, 22ps+ | 8 | 7.41% | |
This study was designed to explore the implication of chromosomal abnormalities (CA) in male infertility. In our study constitutional chromosomal abnormalities were identified in 12.70% (71.3% polymorphic variants) of infertile patients and 8.16% (100% polymorphic variants) in the control group. Structural chromosomal abnormalities were present in a high proportion of men with infertility problems. In fertile controls, structural abnormality was detected in 4 cases only and no deletion in AZF region was found.
We identified 6 cases of Klinefelter’ syndrome,
which is reported to be the most frequent chromosomal aberration causing
The incidence of chromosomal abnormalities in
patients with
We identified a very high prevalence of polymorphic variants in infertile men with normal spermogram (24%). This prevalence is statistically higher than for any other group (24% vs. at most 15%, p=0.001). This might suggest a role for polymorphic variants in human fertility.
The most common reported clinical diagnosis
among patients with inversion of chromosome
9 is
The exact mechanism by which chromosomal abnormalities induce infertility is still not completely
understood. Some authors suggest that presence of
abnormal chromatin interferes with meiotic division and affects sperm production (
Infertile men have a higher risk of constitutional
chromosomal rearrangements which may be the
cause of infertility (
However, because of the limited size of the control group in the present study, the actual frequency of chromosome abnormalities needs to be investigated in a further study with a larger control group.
The incidence of chromosome abnormalities in
patients with
On the other hand, normospermic men carried the most frequent aberration (polymorphic variants) which was 1.6 times more frequent than in azoospermic men and 3 times more frequent than in fertile men.
The high rate of chromosomal abnormalities among infertile men strongly suggests the need for cytogenetic analysis and detection of Y chromosome microdeletions prior to the application of assisted reproduction techniques.