Male infertility affects 20% to 25% of couples and in the half cases, conception failures etiology remains idiopathic [26]. It is estimated that approximately 15% of spermatogenic failure and/or sperm dysfunction is the result of gene mutations and chromosomal aberrations [26]. Some genetic disturbances can lead to androgen-estrogen imbalances, resulting in disrupted sperm function and conception problems. Studies on the polymorphic variations of estrogen receptors are becoming more common, but they are insufficient in addressing the genetic changes in estrogens and their receptors concerning the physiology and pathophysiology of the male reproductive system.
Impaired sperm production results from a multitude of factors: oxidative stress, environmental factors, hormonal imbalance, congenital diseases, genetic causes, and a host of other issues. Still, in approximately 50% of male infertility cases, the etiology remains unknown [20, 31].
Estradiol is a key factor for germ cell survival [25], and when production is disturbed, impaired sperm production occurs. Two main estrogen receptor subtypes mediate estrogen-related actions and are present in different stages of human germ cells [7, 8]. In recent years, there has been increasing interest in estrogen signal transduction disorders that may result from genetic polymorphisms in genes encoding estrogen receptors. There have been few studies investigating the direct relationship of these polymorphisms with semen parameters and sperm fertilizing ability. These studies have produced conflicting results. The results may vary due to ethnic differences in the population surveyed, genetic background of the participants, population sample size, or environmental factors or habits [5, 28]. The studies focusing on the ethnic diversity of single nucleotide polymorphisms (SNPs) showed a dependence on the relationship between the genes encoding estrogen receptor SNPs and osteoporosis [22, 38].
Estrogens have come to the forefront for their role in the pathophysiology of male infertility. These steroid hormones are produced in the testis from testosterone through the actions of cytochrome P450, and they act via specific receptors known as estrogen receptor 1 (ESR1) and 2 (ESR2) [11, 18]. Separate genes encode these receptors: the
The development and increasing availability of
This study aims to analyze the relationship between
One hundred sixteen infertile patients were enrolled in the study. All couples underwent IVF treatment. The indication for this treatment consisted of the following: tubal factors, male factor and idiopathic infertility. Exclusion criteria for females referred to age over 39 years, FSH in the 3rd to 5th day of the cycle over 12 mIU/mL, grade III or IV endometriosis according to American Society of Reproductive Medicine (ASRM), and polycystic ovarian syndrome. For males, the exclusion criteria consisted of azoospermia and hypogonadotropic hypogonadism. The sperm parameters assessed on the day of ovum pickup were used to determine eligibility for either classical IVF or intracytoplasmic sperm injection (ICSI). Only couples with at least two mature oocytes retrieved per patient were qualified for the study. Finally, forty couples underwent conventional IVF treatment, whereas 76 couples underwent ICSI.
All the cohorts of Caucasian descent came from Central European population (Poland). Male patients with chromosomal abnormalities or erectile disorders who could not provide the sperm samples in the process of masturbation were also excluded from the study.
The major division of patients included the method of fertilization (IVI and ICSI). Forty couples underwent conventional in vitro fertilization and 76 intracytoplasmic sperm injection. The criteria for the fertilization method choice concerned mainly sperm quality. Patients with normozoospermia (accordingly 2010 WHO manual [36]) were qualified for classical IVF, whereas in both conditions: 1) worse sperm quality, or 2) after unsuccessful IVF attempt, in patients with normozoospermia ICSI fertilization was performed. The concentration, motility, morphology, and viability of spermatozoa were significantly better in subgroups which underwent conventional IVF. Subsequently, we analyzed the polymorphisms prevalence with sperm parameters (concentration, motility, morphology, HOS [as the indicator of sperm vitality] and patients’ classification to the normozoospermia group (defined using the 2010 WHO manual [36]). The polymorphisms occurring in ESR1 and
For every seminal parameter, we divided the patients into two groups – parameters either below or within the normal range as determined by the 2010 WHO manual [36]. Additionally, we analyzed the genotype distribution in the patient subgroups with and without normozoospermia, as confirmed by concentration, motility with progressive movement, and sperm morphology. This was performed to assess whether the analyzed genotypes are dominant in the above-mentioned subgroups.
On the day of ovarian puncture, semen samples were obtained from patients following three days of sexual abstinence. All samples underwent standard evaluation of concentration, motility, morphology, and viability according to the 2010 WHO manual. Hypoosmotic test (HOS) was used as a viability marker. Samples with leukocyte concentration >106/mL were excluded from the study.
Conventional IVF and ICSI were performed according to the standards of the Department of Infertility and Reproductive Endocrinology [10]. For ovarian stimulation, a protocol utilizing a GnRH agonist has been described previously [34]. Briefly, when the dominant follicles had a diameter > 17 mm and estradiol concentrations were 150–200 pg/mL/follicle, 10,000 IU of human chorionic gonadotropin (HCG; Pregnyl, Organon) was injected intramuscularly to facilitate final oocyte maturation. After 36 hours, the ovaries were punctured under transvaginal ultrasonography control and the follicular fluid was aspirated to obtain the oocytes. The retrieved oocytes were evaluated microscopically and only mature cells in MII were qualified for further IVF processing.
After liquefaction, the sperm samples were prepared by centrifugation using a SpermGrad (Vitrolife, Sweden). Spermatozoa were then dissolved in G-IVF Plus medium (Vitrolife, Sweden) and incubated in 37°C for two hours prior to the IVF procedure.
For conventional IVF, oocytes were placed in 5-well dishes with G-1 Plus medium (Vitrolife, Sweden), inseminated with 50.000–100.000 motile sperm, and incubated. For ICSI, MOPS Plus buffer and sperm were placed in ICSI dishes and covered with a layer of OVOIL oil (Vitrolife, Sweden). Single oocytes were placed in each well. Spermatozoa exhibiting the proper morphology and motility were immobilized and injected into the oocytes using a microinjection needle. Oocytes were then placed in G-1 Plus medium, covered with OVOIL oil, and incubated. Approximately 16 to 18 hours after insemination, fertilization was evaluated. The presence of two pronuclei in the oocyte was considered as completed fertilization process.
Sample collection. Blood samples were obtained from each male subject to isolate genomic DNA. Approximately 2.7 mL of peripheral blood was collected from all male patients in S-Monovette® EDTA tubes (SARSTEDT AG & Co., Numbrecht, Germany) for genetic analysis of
Genotyping. The genomic DNA (gDNA) from each male patient was isolated from peripheral blood with the Axy-Prep Blood Genomic DNA Miniprep Kit (Axygen Scientific, Inc. Union City, CA, USA) according to the manufacturer’s protocol. DNA concentration and purity were determined spectrophotometrically, and 50 to 200 ng of gDNA was used in each polymerase chain reaction (PCR) and restriction analysis. Ten percent of the randomly selected samples were purified using AxyPrepPCR Clean-up Kit according to the manufacturer’s protocol (Axygen). The samples were then sequenced to confirm identity in relation to the known sequences in the NCBI (National Center for Biotechnology Information) gene database.
For each
The remaining PCR products were purified according to the AxyPrepPCR Clean-up Kit manufacturer’s protocol (Axygen), and restriction fragments length polymorphism (RFLP) analysis was conducted. The purified PCR product was used in each restriction analysis for
Statistical analysis was performed using Statistica 13 (TIBCO Software, Tulusa, USA). Deviation from the HWE was examined using the Michael H. Court’s (2005–2008) online calculator Excel-based HWE Test (
The average age of male patients was 34 years, and their partners mean age was 32. The mean seminal parameters of the entire study group were as follows: sperm concentration of 2.5E7/mL, 18% sperm showing progressive motility, 4% spermatozoa with normal morphology and 53% live sperm. As defined using the 2010 WHO manual, normozoospermia, in all basic parameters (motility, concentration, and morphology), was observed in 28 patients (24% of the study group) [36]. Sperm parameters in the subgroups which underwent conventional IVF and ICSI are presented in Table 1. The concentration, motility, morphology, and viability of spermatozoa were significantly better in the subgroups which underwent conventional IVF. There was no difference in the average fertilization rate between groups (67% vs. 73%; p>0.05).
Average sperm parameters in patients who underwent conventional IVF and ICSI
Mean ± SD | Median | Mean ± SD | Median | p | |
---|---|---|---|---|---|
Concentration [mln/mL] | 39±16 | 40 | 18±18 | 13 | <0.0001a |
Progressive motility [%] | 25±9 | 21 | 14±10 | 12 | <0.0001a |
Correct morphology [%] | 5±2 | 5 | 3±2 | 3 | <0.0001a |
HOS [%] | 62±11 | 63 | 48±19 | 50 | <0.0001b |
IVF – patients treated with conventional in vitro fertilization, ICSI – patients treated with intracytoplasmic sperm injection; HOS – hypoosmotic test, SD – standard deviation; a – Mann-Whitney U test; b – Student’s t-test
Restriction analysis was fully conclusive for 102 patients in the case of
Using the chi-squared test and the Fisher exact test, we evaluated the association between sperm parameters and
Genotype | Sperm concentration | P | Sperm progressive motility | P | Sperm morphology | P | HOS | P | Normozoospermia | P | |||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
< 15 mln/mL | ≥ 15 mln/mL | < 32% | ≥ 32% | < 4% | ≥ 4% | < 58% | ≥ 58% | yes | no | ||||||
TT | 11 (31%) | 13 (19%) | 0.4a | 19 (25%) | 5 (20%) | 0.83a | 8 (24%) | 16 (23%) | 0.98b | 12 (22%) | 12 (25%) | 0.33a | 5 (22%) | 19 (24%) | 0.82a |
TC | 16 (46%) | 36 (54%) | 38 (49%) | 14 (56%) | 17 (52%) | 35 (51%) | 25 (46%) | 27 (56%) | 13 (56%) | 39 (49%) | |||||
CC | 8 (23%) | 18 (27%) | 20 (26%) | 6 (24%) | 8 (24%) | 18 (26%) | 17 (32%) | 9 (19%) | 5 (22%) | 21 (27%) | |||||
Powerd | 0.65 | 0.24 | 0.07 | 0.72 | 0.25 | ||||||||||
T | 38 (54%) | 62 (46%) | 0.28a | 76 (49%) | 24 (48%) | 0.87a | 33 (50%) | 67 (49%) | 0.85a | 49 (45%) | 51 (53%) | 0.27a | 23 (50%) | 77 (49%) | 0.85a |
C | 32 (46%) | 72 (54%) | 78 (51%) | 26 (52%) | 33 (50%) | 71 (51%) | 59 (55%) | 45 (47%) | 23 (50%) | 82 (51%) | |||||
Powerd | 0.63 | 0.06 | 0.06 | 0.63 | 0.06 | ||||||||||
AA | 7 (20%) | 11 (16%) | 0.81a | 15 (19%) | 3 (12%) | 046b | 7 (21%) | 11 (16%) | 0.51a | 11 (20%) | 7 (15%) | 0.75a | 2 (9%) | 16 (20%) | 0.22a |
AG | 14 (40%) | 31 (46%) | 35 (46%) | 10 (40%) | 16 (49%) | 29 (42%) | 23 (53%) | 22 (46%) | 9 (39%) | 36 (46%) | |||||
GG | 14 (40%) | 25 (38%) | 27 (35%) | 12 (48%) | 10 (30%) | 29 (42%) | 20 (27%) | 19 (39%) | 12 (52%) | 27 (34%) | |||||
Powerd | 0.21 | 0.74 | 0.66 | 0.70 | 0.99 | ||||||||||
A | 28 (40%) | 53 (40%) | 0.95a | 65 (42%) | 16 (32%) | 0.2a | 30 (46%) | 51 (37%) | 0 25a | 45 (35%) | 36 (38%) | 0.54a | 13 (28%) | 68 (43%) | 0.07a |
G | 42 (60%) | 81 (60%) | 89 (58%) | 34 (68%) | 36 (64%) | 87 (63%) | 63 (65%) | 60 (62%) | 33 (72%) | 90 (57%) | |||||
Powerd | 0.05 | 0.82 | 0.73 | 0.06 | 0.99 | ||||||||||
AA | 3 (10%) | 5 (8%) | 1b | 6 (8%) | 2 (10%) | 0.49b | 2 (6%) | 6 (10%) | 0.54b | 5 (10%) | 3 (6%) | 0.78b | 2 (11%) | 6 (8%) | 0.21b |
AG | 13 (42%) | 26 (43%) | 28 (40%) | 11 (52%) | 11 (36%) | 28 (46%) | 19 (39%) | 20 (47%) | 11 (58%) | 28 (38%) | |||||
GG | 15 (48%) | 30 (49%) | 37 (52%) | 8 (38%) | 18 (58%) | 27 (44%) | 25 (51%) | 20 (47%) | 6 (31%) | 39 (54%) | |||||
Powerd | 0.08 | 0.67 | 0.72 | 0.35 | 0.72 | ||||||||||
A | 19 (31%) | 36 (30%) | 0.87a | 40 (28%) | 15 (36%) | 0.35a | 15 (24%) | 40 (33%) | 0.23a | 29 (30%) | 26 (30%) | 0.92a | 15 (39%) | 40 (27%) | 0.15a |
G | 43 (69%) | 86 (70%) | 102 (72%) | 27 (64%) | 47 (76%) | 82 (67%) | 69 (70%) | 60 (70%) | 23 (61%) | 106 (73%) | |||||
Powerd | 0.06 | 0.68 | 0.82 | 0.05 | 0.92 | ||||||||||
GG | 26 (84%) | 53 (95%) | 0.13b | 60 (90%) | 19 (95%) | 0.68b | 23 (82%) | 56 (95%) | 0.11b | 40 (87%) | 39 (95%) | 0.27b | 17 (94%) | 62 (90%) | 1b |
GA | 5 (16%) | 3 (5%) | 7 (10%) | l (5%) | 5 (18 %) | 3 (5%) | 6 (13%) | 2 (5%) | 1 (6%) | 7 (10%) | |||||
Powerd | 0.8 | 0.34 | 0.88 | 0.6 | 0.34 | ||||||||||
G | 57 (92%) | 109 (97%) | 0.14b | 127 (95%) | 39 (98%) | 0.68b | 51 (91%) | 115 (97%) | 0.11b | 86 (93%) | 80 (98%) | 0.28b | 35 (97%) | 131 (95%) | 1b |
A | 5 (8%) | 3 (3%) | 7 (5%) | 1 (2%) | 5 (9%) | 3 (3%) | 6 (7%) | 2 (2%) | 1 (3%) | 7 (5%) | |||||
Powerd | 0.68 | 0.44 | 0.79 | 0.73 | 0.34 | ||||||||||
AA | 0 | 4 (7%) | 0.21b | 2 (3%) | 2 (9%) | 0.36b | 0 | 4 (6%) | 0.06c | 1 (2%) | 3 (7%) | 0.39b | 2 (10%) | 2 (3%) | 0.23b |
AT | 13 (36%) | 15 (25%) | 23 (31%) | 5 (23%) | 14 (42%) | 14 (22%) | 17 (32%) | 11 (26%) | 4 (20%) | 24 (31%) | |||||
TT | 23 (64%) | 41 (68%) | 49 (66%) | 15 (68%) | 19 (58%) | 45 (72%) | 35 (66%) | 29 (67%) | 14 (70%) | 50 (66%) | |||||
Powerd | 0.99 | 0.92 | 0.99 | 0.91 | 0.84 | ||||||||||
A | 13 (18%) | 23 (19%) | 0.84a | 25 (17%) | 7 (16%) | 0.94a | 14 (21%) | 22 (17%) | 0.53a | 19 (18%) | 17 (20%) | 0.75a | 8 (20%) | 28 (18%) | 0.82a |
T | 59 (82%) | 97 (81%) | 121 (83%) | 35 (84%) | 52 (79%) | 104 (83%) | 87 (82%) | 69 (80%) | 32 (80%) | 124 (82%) | |||||
Powerd | 0.07 | 0.07 | 0.27 | 0.11 | 0.11 |
SNP - single nucleotide polymorphism, HOS - hypoosmotic test;
- Pearson’s chi-squared p-value;
- Fisher’s exact test p-value;
-Fisher-Freeman-Halton exact test p-value;
Powerd - G*Power power post-hoc calculated
Similarly, we observed no differences in sperm parameters between the genotypes using the Kruskal-Wallis and Mann Whitney U tests (p>0.05 for all sperm parameters in every SNP) (Table 3). For
Comparison of median sperm parameters and fertilization rate of conventional IVF and ICSI between the genotypes for
Genotype | Sperm concentration [mln/mL] | Sperm motility [%] | Sperm morphology [%] | HOS [%] | Oocytes fertilized in conventional IVF [%] | Oocytes fertilized in ICSI [%] |
---|---|---|---|---|---|---|
PvuII | p = 0.36a | p = 0.6a | p = 0.9a | p = 0.54a | p = 0.16a | p = 0.75a |
TT | ||||||
TC | ||||||
CC | ||||||
XbaI | p = 0.31a | p = 0.71a | p = 0.33a | p = 0.78a | p = 0.73a | p = 0.97a |
AA | ||||||
AG | ||||||
GG | ||||||
AluI | p = 0.94a | p = 0.88a | p = 0.69a | p = 0.72a | p = 0.25a | p = 0.97a |
AA | ||||||
AG | ||||||
GG | ||||||
RsaI | p = 0.32b | p = 0.71b | p = 0.21b | p = 0.47b | p = 0.53b | p = 0.67b |
GG | ||||||
GA | ||||||
AlwNI | p = 0.1b | p = 0.12a | p = 0.31a | p = 0.4a | p = 0.79a | p = 0.86a |
CC | ||||||
CT | ||||||
TT |
SNP – single nucleotide polymorphism,
– Kruskal-Wallis test,
– Mann-Whitney U test
Furthermore, we analyzed the association between SNP genotypes and fertilization rates from two methods of
Genotype | Conventional IVF | Fertilization completed | No fertilization | p | ICSI | Fertilization completed | No fertilization | p |
---|---|---|---|---|---|---|---|---|
TT | 6 (17%) | 6 (21%) | 0 | 0.02c | 18 (27%) | 16 (26%) | 2 (40%) | 0.84a |
TC | 20 (55%) | 18 (62%) | 2 (29%) | 32 (48%) | 30 (50%) | 2 (40%) | ||
CC | 10 (28%) | 5 (17%) | 5 (71%) | 16 (24%) | 15 (24%) | 1 (20%) | ||
Powerd | 0.99 | 0.64 | ||||||
T | 32 (44%) | 30 (52%) | 2 (36%) | 0.02b | 68 (52%) | 62 (51%) | 6 (60%) | 0.75b |
C | 40 (56%) | 28 (48%) | 12 (64%) | 64 (48%) | 60 (49%) | 4 (40%) | ||
Powerd | 0.77 | 0.06 | ||||||
AA | 4 (12%) | 3 (10%) | 1 (14%) | 0.61a | 14 (21%) | 13 (21%) | 1 (20%) | 0.16a |
AG | 16 (44%) | 14 (49%) | 2 (29%) | 29 (44%) | 27 (45%) | 2 (40%) | ||
GG | 16 (44%) | 12 (41%) | 4 (57%) | 23 (35%) | 21 (34%) | 2 (40%) | ||
Powerd | 0.57 | 0.14 | ||||||
A | 24 (33%) | 20 (34%) | 4 (29%) | 0.76b | 57 (43%) | 53 (43%) | 4 (40%) | 1b |
G | 48 (67%) | 38 (66%) | 10 (71%) | 75 (57%) | 69 (57%) | 6 (60%) | ||
Powerd | 0.14 | 0.06 | ||||||
AA | 2 (6%) | 1 (4%) | 1 (14%) | 0.64a | 6 (10%) | 5 (9%) | 1 (20%) | 0.63a |
AG | 14 (45%) | 11 (46%) | 3 (43%) | 25 (41%) | 23 (41%) | 2 (40%) | ||
GG | 15 (49%) | 12 (50%) | 3 (43%) | 30 (49%) | 28 (50%) | 2 (40%) | ||
Powerd | 0.72 | 0.79 | ||||||
A | 18 (29%) | 13 (27%) | 5 (36%) | 0.52b | 37 (30%) | 33 (29%) | 4 (40%) | 0.48b |
G | 44 (71%) | 35 (73%) | 9 (64%) | 85 (70%) | 79 (71%) | 6 (60%) | ||
Powerd | 0.36 | 0.76 | ||||||
- | ||||||||
GG | 24 (80%) | 23 (79%) | 1 (100%) | - | 53 (93%) | 46 (92%) | 7 (100%) | |
GA | 6 (20%) | 6 (21%) | 0 | 4 (7%) | 4 (8%) | 0 | ||
Powerd | - | - | ||||||
G | 54 (90%) | 52 (90%) | 2 (100%) | - | 110 (96%) | 96 (96%) | 14 (100%) | - |
A | 6 (10%) | 6 (10%) | 0 | 4 (4%) | 4 (4%) | 0 | ||
Powerd | - | - | ||||||
CC | 2 (13%) | 2 (8%) | 0 | 1c | 2 (3%) | 2 (3%) | 0 | 0.28c |
CT | 5 (17%) | 4 (17%) | 1 (17%) | 23 (35%) | 23 (38%) | 0 | ||
TT | 23 (70%) | 18 (75%) | 5 (83%) | 41 (62%) | 36 (59%) | 5 (100%) | ||
Powerd | 0.29 | 0.99 | ||||||
C | 9 (15%) | 8 (17%) | 1 (8%) | 0.67b | 27 (20%) | 27 (22%) | 0 | - |
T | 51 (85%) | 40 (83%) | 11 (92%) | 105 (80%) | 95 (78%) | 10 (100%) | ||
Powerd | 0.46 | - |
SNP – single nucleotide polymorphism, IVF – in vitro fertilization, ICSI – intracytoplasmic sperm injection;
– Pearson’s chi-squared p-value;
– Fisher’s exact test p-value;
– Fisher-Freeman-Halton exact test p-value;
Powerd – G*Power power post-hoc calculated
For
To assess the relationship of sperm parameters with the
We found no statistically significant differences for the
In the analysis of sperm concentration as related to rs2234693 and rs9340799 polymorphisms (defined by
Solakidi et al. reported ESR1 localized to the equatorial segment of the sperm head. They suggest the possible involvement of this receptor in combining the cell membranes of the male and female gametes [32]. It seems, therefore, that individual polymorphisms of the gene encoding
In the
Significantly higher occurrences of the TC genotype of the
Because of the polygenic nature of spermatogenic disorders, additional loci could be involved in spermatozoa defects and conception ability. This may be related to estrogens, their receptors, or other core genes involved in estrogenic and estrogen-related pathways. In most cases, we did not discover a relationship between both estrogen receptors polymorphisms and sperm function, except forrs2234693 and fertilization rate. There was no association with