Prior studies suggested sperm with damaged DNA permits fertilization but may lead to failure of embryo implantation following blastocyst formation. Quantitative correlations between DNA damage and risk of implantation failure have, however, so far not been performed. The aim of this study was to investigate two FSHR gene polymorphisms G919A (Ala307Thr) and A2039G (Asn680Ser) in Eastern Ukrainian Caucasian men with abnormally low fertility. The molecular genetic analysis was performed by real-time PCR, with the level of DNA fragmentation measured by the sperm chromatin dispersion (SCD) method. The relationship between DNA fragmentation in sperm and these genetic polymorphisms was estimated. Compared to homozygotes, the risk of high-level DNA fragmentation (>20%) increased in men up to age 35 years 16-fold for heterozygotes GA of polymorphic variant G919A, 28-fold for homozygotes AA of polymorphic variant G919A; and 16-fold for heterozygotes GG of polymorphic variant A2039G. A statistically significant positive correlation between number of alternative alleles of the FSHR gene in genotype and degree of DNA fragmentation is proved (r s = 0.70, P < 0.01).
In developed countries, 10% - 15% of married couples encounter infertility. Up to 50% of all infertility occurs in the male, while approximately 20% of cases have male as well as female causes [
DNA fragmentation is defined as diminished or essential absence of chromatin compaction in nuclei of spermatozoa, and has for years been under investigation as a possible cause of reduced male fertility [
Follicle stimulating hormone (FSH), luteinizinghormone (LH) and testosterone are the major hormones influencing sperm formation/maturation. FSH does not induce the process of spermatogenesis, but is necessary for formation of mature spermatozoa, with the FSH beta-subunit, known as the follicle stimulating hormone gene beta polypeptide (FSHB, 11p13) determining reproductive function [
Almost 150 polymorphic variants of the FSHR gene are currently under investigation in men and women. Polymorphisms of the FSHR gene have been associated with increases of FSH, decreases of progesterone and with decreasing sperm concentrations in semen; effects of single nucleotide polymorphisms (SNP) have also been investigated [
The aim of this study was to investigate two FSHR gene polymorphisms G919A (Ala307Thr) and A2039G (Asn680Ser) in Eastern Ukrainian Caucasian men with abnormally low fertility.
This study was performed at the Center of Human Reproduction (“Clinic of Professor Feskov,” Kharkov, Ukraine). In the study, all included patients signed an informed consent. The Institutional Review Board of the center approved both the study protocol and informed consent. Seventy-one couples with diagnosis of male factor infertility participated in the study between 4th quarter 2013 and 2nd quarter 2014. Blood (10 ml) of the 71 men (ages 22 - 45 years) was obtained via routine draw for karyotyping and molecular genetic analysis. In 11 azoospermic patients, there was no semen evaluation possible.
Chromosomes samples for cytogenetic analyses were obtained from peripheral blood lymphocytes via Giemsa banding (G-banding, GTG), and results were presented following the International System of Human Cytogenetic Nomenclature [
Since distribution of frequencies in data sets for alternative alleles did not follow a Gaussian distribution, non-parametric methods were utilized for statistical analyses. Frequency differences in genotypes were compared by Fischer’s test. Relationships between variables were examined by correlation analyses. Verifications of associations of alleles/genotypes and of equality of distributions were accomplished by the Chi-square test (χ2 test) at significance levels of P = 0.05, P = 0.01 and P = 0.001, and odds ratios (OR) and confidence interval (CIs) were calculated [
As already noted, 11 of 71 males were azoospermic. Among those, three were heterozygous for the delF508 mutation in the CFTR gene, and three others demonstrated karyotype abnormalities {45, XY, rob (13;21) (q10;q10); 47, XXY [
Interestingly, all 60 remaining study patients, who produced sperm and did undergo DNA fragmentation assessments, demonstrated normal 46, XY karyotypes. Since age impacts the level of sperm DNA fragmentation, we restricted the remaining investigations to 51 of these 60 men, who were under the age of 35 years at time of study [
Based on allele frequencies, we calculated the theoretical number of genotypes for a randomly mating population: The actual distribution of genotypes of the polymorphic variant G919A did not statistically differ from the theoretically expected one at equilibrium: N (df = 2,
The actual distribution of genotypes of the polymorphic variant A2039G was also not significantly different from what was theoretically expected at equilibrium in the group of younger patients below age 35 (n = 51, df = 2,
The relationship between DNA fragmentation in sperm and these genetic polymorphisms was estimated. Among 51 younger probands with different genotypes, DNA fragmentation, however, those with alternatively homozygous or heterozygous alleles was significantly higher than is those with homozygotes alleles in that
Polymorphism | Alleles frequencies | |
---|---|---|
Total group, N = 71 | Younger group (<35 y.o.), n = 51 | |
G919A | РG = 0.697, qA = 0.303 | РG = 0.726, qA = 0.275 |
A2039G | РA = 0.739, qG = 0.261 | РA = 0.775, qG = 0.225 |
Statistics (G919A) | df = 2, | df = 2, |
Statistics (A2039G) | df = 2, | df = 2, |
SNP | Genotypes | Distribution | |||
---|---|---|---|---|---|
Total group, N, % | Younger group (<35 y.o.), n, % | ||||
Actual | Theoretical | Actual | Theoretical | ||
A2039G, N (%) | AA | 43 (60.6) | 39 (55.0) | 33 (64.7) | 30 (60.0) |
AG | 19 (26.8) | 27 (38.0) | 13 (25.5) | 18 (35.0) | |
GG | 9 (12.6) | 5 (7.0) | 5 (9.8) | 3 (5.0) | |
G919A, N (%) | GG | 38 (53.5) | 35 (49.3) | 29 (56.9) | 27 (52.7) |
GA | 23 (32.4) | 30 (42.2) | 16 (31.2) | 20 (39.9) | |
AA | 10 (14.1) | 6 (8.5) | 6 (11.9) | 4 (7.4) | |
Statistics (A2039G) | df = 2, | df = 2, | |||
Statistics (G919A) | df = 2, | df = 2, |
DNA fragmentation in the former group exceeded the threshold level of 20.0% more often P < 0.001 (
Average sperm DNA fragmentation in homozygotes with basic alleles GG (11.7% ± 5.7%) and in heterozygotes GA (26.2% ± 8.8%) with polymorphic variant G919A were also statistically different (Mann-Whitney U test: Ucritical = 149, Uactual = 68; P < 0.01;
These findings can be summarized as demonstrating that in comparison to homozygotes with basic alleles, the heterozygotes GA with polymorphic variant G919A show a 16-fold greater risk of high level DNA fragmenta- tion (OR = 15.83, 95% CI 3.50 - 71.54, P < 0.05), while the homozygotes AA show a 28-fold risk (OR = 27.76, 95% CI 3.33 - 231.70, P < 0.05). For heterozygotes AG and homozygotes GG with polymorphic variant A2039G, the risk of developing a higher level of DNA fragmentation was 16-fold greater (OR = 15.55, 95% CI 3.42 - 70.76, Р < 0.05; OR = 15.55, 95% CI 1.98 - 122.15, P < 0.05, respectively) when compared to homozygotes having only basic allele.
We tried to evaluate the levels of FSH and testosterone for patients in the study. 26 patients with basic GGAA alleles demonstrated FSH levels of 9.49 ± 2.61 mIU/mL and testosterone levels of 25.48 ± 5.49 nmol/L. FSH level was, thus, approximately 30% below what is considered the median 12.5 of normal range 5.0 - 20.0 mIU/ mL, while average testosterone of basic allele patients was practically equal to the median 25.2 within the normal
SNP | Genotypes | N (%) | Average age, x ± mx | DNA fragmentation level, % | Number of patients with DNA fragmentation level > 20% |
---|---|---|---|---|---|
G919A | GG | 29 (56.9) | 30.2 ± 2.5 | 11.7 ± 5.7 | 3 (10.3*) |
GA | 16 (31.2) | 30.6 ± 1.8 | 26.2 ± 8.8 | 11 (68.8*) | |
AA | 6 (11.9) | 31.8 ± 2.6 | 36.5 ± 16.2 | 5 (83.3) | |
A2039G | AA | 33 (64.7) | 30.1 ± 2.3 | 13.9 ± 6.9 | 5 (15.2**) |
AG | 13 (25.5) | 31.3 ± 2.2 | 25.6 ± 8.4 | 10 (76.9**) | |
GG | 5 (9.8) | 31.4 ± 2.7 | 39.3 ± 17.2 | 4 (80) | |
Statistics | *df = 2, | ||||
**df = 2, |
Genotypes | Quantity | Average age, x ± mx | DNA fragmentation level, % | Number of patients with DNA fragmentation level >20% |
---|---|---|---|---|
GGAA | 26 | 29.9 ± 2.5 | 10.7 ± 5.1 | 1 (3.9%) |
GGAG | 3 | 32.7 ± 1.8 | 20.3 ± 7.1 | 2 (66.7%) |
GAAA | 6 | 30.5 ± 1.2 | 24.5 ± 8.7 | 3 (50.0%) |
GAAG | 10 | 30.6 ± 2.2 | 27.6 ± 8.6 | 8 (80.0%) |
AAAA | 1 | 34.0 ± 0.0 | 22.5 ± 0.0 | 1 (100%) |
AAGG | 5 | 31.4 ± 2.7 | 39.3 ± 17.2 | 4 (80.0%) |
Statistics | (Spearman correlation) df = 3, rs = 0.70 ± 0.10, P < 0.01 |
range 12.1 - 38.3 nmol/L. Analysis revealed that our cohort of 51 patients is not large enough for statistically significant conclusions, but certain polymorphism-related deviations of hormonal level of polymorphic patients were observed in comparison with patients having basic allele of FSHR gene. For example, among 10 patients with GAAG polymorphism, the hormonal levels were FSH = 12.66 ± 8.55 and testosterone = 18.92 ± 3.50; i.e. their average FSH was 33.4% exceeding basic allele’s one (9.49), but close to normal median, while average testosterone was 34.7% below basic allele’s one.
After certain preparatory treatment, near dozen of all 51 couples entered the IVF cycles. At that, compare with patients having polymorphic abnormalities of FSHR gene, for the couples having basic FSHR gene allele, we observed not only the easy obtaining of good quality blastocysts, but also a better success rate of embryo implantation. However, a small size of patient group in the study does not allow making any statistically significant conclusions.
Among 51 couples with male factor infertility in the study, there were 26 male patients with basic allele of FSHR gene and 25 patients with polymorphic abnormality in said gene. This allows us to assume that near 50% (but not less than 10%) of infertile men might express a polymorphic variant of the FSHR gene. Another assumption is about obtaining of blastocyst of good quality in IVF cycle. Based on our limited observations in the small-size study, we may expect that in a large-cohort study all basic-allele patients will easy obtain good quality blastocysts in sufficient amount, while only part of polymorphic patients will also be capable to do so. Third important assumption includes the expectation that both quality of blastocyst and probability of success of embryo implantation depends on specific type of polymorphism.
For confirmation of our hypothesis with statistical significance, we may suggest a retrospective analysis of the human genomic samples that accumulated in specialized laboratories. For example, Affiliated Genetics Inc. (Salt Lake City, UT) holds a multi-thousand sample bank and collaborates with IVF clinics nationwide; this allows producing a statistically significant analysis of correlation of FSHR gene polymorphism (FGP) with outcome of IVF treatment for the cycles that have been already performed.
Ahead of completion of retrospective analysis, we may speculate that the applying of pre-IVF diagnostic on FGP-presence in male patient with further PGD-selection of the embryo, which has no such genetic abnormality as FGP, can not only lead to increase of implantation success rate, but also eliminate the risk of FGP-related infertility in genetic successor despite presence of FGP in the father’s genome.
Normal function of cells involved in spermatogenesis, including production of FSH by these cells, is dependent on the expression of the FSH receptor (FSHR). A better understanding of factors important for FSHR gene transcription is, therefore, potentially important for finding ways to either modulate endocrine regulation or cell- specific gene expression, thereby preventing and/or treating the risk of infertility. Some polymorphism can be assumed to affect the gene’s transcription and, therefore, effects. However, the impact of FSHR variants, including single nucleotide polymorphisms (SNPs) on male fertility is currently not yet well understood.
Reported results have been contradictory: Some studies did not observe significant differences of FSH level in serum of normal and infertile patients, reaching the conclusion that SNP distribution does not matter for male reproductive function [
Here we went beyond those investigations: Based on the criteria set by the European Association of Urologists, suggesting that to be normally fertile, no more than 20% of the ejaculate should demonstrate damaged DNA, we investigated the DNA damage’s associations with specific polymorphisms in the FSHR gene among male patients with reduced fertility. To avoid previously noted suspicions that ethnic backgrounds may affect these associations, we performed this study in a genetically homogenous population of Caucasian Ukrainians. Furthermore, in order to eliminate reported age-dependent associations, we concentrated on a younger patient subgroup under age 35 years.
In this, therefore, very homogenous study group we detected statistically significant difference in DNA fragmentation levels between homozygotes with basic alleles GG and heterozygotes GA of polymorphic variant G919A of FSHR gene, and between homozygotes with basic alleles АА and heterozygotes AG for polymorphic variant A2039G of FSHR gene. The observed associations suggest that FSHR gene expression might influence the process of chromatin compaction in sperm nuclei and, consequently, affect quality of sperm and, therefore, male fertility.
Our data, therefore, demonstrate that FSHR gene polymorphisms seem to have a direct influence on sper- matogenesis, and that certain polymorphisms, therefore, might contribute to multi-genetic male infertility. These observations, therefore, confirm previous qualitative hypotheses [
If confirmed by further studies, these results also suggest that here reported genetic associations might be used clinically to prospectively assess risk toward male infertility. We here, thus, for the first time demonstrated quantitatively that alternative alleles of polymorphic variants G919A and A2039G of the FSHR gene might represent increased risk for high-level DNA fragmentation (>20%) in sperm of young men up to age 35, who present with reduced fertility. These observations advance our understanding of potential genetic causes of selected cases of male infertility, which previously have not been understood in their etiologies. Once confirmed, these data suggest that DNA fragmentation in selected cases of presumed male infertility may be indicated in association with determinations of FSHR gene polymorphisms.
If future observations statistically significantly confirm our hypothesis regarding the influence of FSHR male polymorphism (through spermatogenesis and embryogenesis) both on blastocyst formation and embryo transfer success, we may predict that the prescreening of embryo’s FSHR gene code prior ET will increase pregnancy rate, and, therefore, such prescreening will be recommended in IVF for infertile men expressing FSHR polymorphism. Further research on influence of FSHR female polymorphism on embryogenesis is also suggested in this regard.
IevgeniiaZhylkova,OleksandrFeskov,OlenaFedota, (2016) FSHR Gene Polymorphisms Causes Male Infertility. Open Journal of Genetics,06,1-8. doi: 10.4236/ojgen.2016.61001