Georgios Makrydimas

Assessment of sperm chromatin condensation and ploidy status using flow cytometry correlates to fertilization, embryo quality and pregnancy following in vitro fertilization

PurposeSperm flow cytometry (SFC) was used to evaluate the association of sperm chromatin condensation and ploidy with fertilization, embryo development, pregnancy and abortion rates following IVF.MethodsConventional semen analysis was performed in one hundred fifty men, as well as SFC analysis, after acridine orange and propidium iodide staining, for the evaluation of sperm maturity and ploidy respectively. Conventional IVF was performed in all couples.ResultsCouples with low percentages of mature spermatozoa presented with lower fertilization rates (p < 0.005), lower rates of grade A embryos (p < 0.003) and lower pregnancy rates (p < 0.006), compared to couples with high percentages of mature spermatozoa. Couples with low total aneuploidy rates presented with higher fertilization rates (p < 0.007), higher rates of grade A embryos (p < 0.004) and higher pregnancy rates (p < 0.003), compared to couples with high total aneuploidy rates.ConclusionsSperm chromatin condensation and ploidy constitute critical parameters for the evaluation of semen samples before IVF and for the identification of cases in need of ICSI application.

Semen quality is influenced by androgen receptor and aromatase gene synergism

STUDY QUESTION Does synergism between AR(CAG)(n) and CYP19(TTTA)(n) polymorphisms influence the quality of sperm? SUMMARY ANSWER AR(CAG)(n) and CYP19(TTTA)(n) polymorphisms had a synergistic effect on sperm concentration and motility. WHAT IS KNOWN ALREADY Androgens exert their action in the testicular tissue by binding to androgen receptor (AR), while their action is mediated by the aromatase P450 enzyme (CYP19). AR(CAG)(n) alleles are associated with sperm motility and CYP19(TTTA)(n) allelic variants have implications for sperm concentration and motility. STUDY DESIGN, SIZE, DURATION Two hundred oligozoospermic and 250 normozoospermic men who presented for infertility investigation were examined during a period of 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS Conventional semen analysis was performed. DNA was extracted from spermatozoa and both polymorphisms were genotyped by polymerase chain reaction. Serum hormone levels were determined. MAIN RESULTS AND THE ROLE OF CHANCE Six combined genotypes were identified between the 18 AR(CAG)(n) alleles with 12-32 repeats and the 6 CYP19(TTTA)(n) alleles with 7-12 repeats. A gradual reduction in the sperm concentration (10(6)/ml) and motility (%) from long AR allele-non-CYP19(TTTA)(7) allele carriers to long AR allele-CYP19(TTTA)(7) homozygotes and from short AR allele-non-CYP19(TTTA)(7) carriers to short AR allele-CYP19(TTTA)(7) homozygotes was observed in normozoospermic men (means ± SD; concentration: 93 ± 53.1 versus 65 ± 48.6 and 85 ± 60.1 versus 37 ± 17.2l, P < 0.002; motility: 63 ± 10.3 versus 55 ± 14.5 and 52 ± 19.6 versus 41 ± 13.7, P < 0.001, respectively). Similar associations were observed in oligozoospermic men (concentration: 10 ± 4.2 versus 9 ± 5.9 and 10 ± 6.3 versus 6 ± 3.1, P < 0.03; motility: 47 ± 17.1 versus 39 ± 6.2 and 39 ± 22 versus 27 ± 18.3, P < 0.003, respectively). The above associations of the combined genotypes with sperm concentration and motility were confirmed in the total study population (P < 0.006 and P < 0.001, respectively). LIMITATIONS, REASONS FOR CAUTION Our study population was limited to Greek Caucasian adult males, residents of Northwest Greece. WIDER IMPLICATIONS OF THE FINDINGS The confirmation of our findings in other populations would verify the significance of AR and CYP19 genes for spermatogenesis. STUDY FUNDING/COMPETING INTERESTS This study did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. The authors declare no conflicts of interest.

Association of paraoxonase gene polymorphisms with sperm parameters.

Paraoxonase (PON) is a high-density lipoprotein-associated enzyme that prevents low-density lipoprotein oxidation. PON proteins, localized in the seminiferous tubules and in spermatozoa, have been implicated in the pathogenesis of male infertility. In the present study, we sought to explore the contribution of the PON gene variants to sperm parameters. One hundred twenty oligospermic and 170 normozoospermic men were examined during infertility investigation. DNA was extracted from spermatozoa, and the PON1(L/M) 55, PON1(Q/R) 192, and PON2(S/C) 311 polymorphisms were genotyped by polymerase chain reaction and digestion with restriction enzymes. The analysis revealed that oligospermic men presented PON1 55L/L, PON1 192Q/Q, and PON2 311S/S genotypes less frequently than normozoospermic men (P < .01, P < .01, and P < .001, respectively), whereas the PON1 55M, PON1 192R, and PON2 311C alleles were significantly increased in oligospermic men (P < .004, P < .008, and P < .008, respectively). The presence of PON1 55L allele was associated with higher sperm motility in oligospermic men (P < .001), in normozoospermic men (P < .01), and in the total study population (P < .01), and the PON1 192Q allele was associated with higher sperm motility in oligospermic men (P < .01), in normozoospermic men (P < .04) and in the total study population (P < .03). On the other hand, the PON2 311S was associated with higher sperm concentration in oligospermic men (P < .03), in normozoospermic men (P < .008), and in the total study population (P < .001). In our series, the PON1 55M and PON1 192R alleles were associated with decreased sperm motility whereas the PON2 311C allele was associated with decreased concentration, supporting the significance of PON genes in semen quality.

Using semen flow cytometry to evaluate association of ploidy status and chromatin condensation of spermatozoa with conventional semen parameters: Clinical application in intrauterine insemination

OBJECTIVE To evaluate the association between the ploidy status and the nuclear chromatin condensation of spermatozoa with conventional semen parameters by using semen flow cytometry (SFC). The prognostic value of SFC on the successful outcome of intrauterine insemination (IUI) was examined. DESIGN Prospective study. SETTING Patients referred to the IVF Unit of Ioannina University School of Medicine. PATIENT(S) Ninety-two men with sperm count between 15×10(6) and 40×10(6) spermatozoa/mL and one hundred normozoospermic men were analyzed. INTERVENTION(S) Conventional semen analysis and SFC analysis after acridine orange and propidium iodide staining. IUI performed in 92 couples. MAIN OUTCOME MEASURE(S) Evaluation of sperm maturity and ploidy. Correlation with conventional semen parameters. RESULT(S) An association of the spermatozoa ploidy status with sperm morphology and motility was revealed. The highest aneuploidy rates were observed when <15% of spermatozoa had normal morphology. An inverse relation was found between sperm morphology and maturity. The pregnancy rates were significantly lower when semen with <15% normal forms (9% vs. 25%), low percentage of mature spermatozoa, and increased total aneuploidy rate were used for IUI. CONCLUSION(S) Ploidy status and sperm maturity are critical parameters for evaluation of the fertilizing capacity of spermatozoa. SFC could be used to evaluate semen samples before IUI and potentially prognose the outcome.

CYP19 gene variants affect the assisted reproduction outcome of women with polycystic ovary syndrome

Abstract Objective: Cytochrome P450 aromatase catalyzes the irreversible transformation of androgens into estrogens. The association of CYP19(TTTA)n polymorphism with the hormonal profile and the assisted reproduction outcome of women with polycystic ovary syndrome (PCOS) was explored. Methods: One hundred and thirty-two women with PCOS and 200 with male-factor infertility, as controls, participated in the current study. The CYP19(TTTA)n polymorphism was genotyped, while the hormonal profile was determined at the third day of the menstrual cycle. During oocyte retrieval, the follicular size, the follicle and oocyte numbers were recorded. Results: Genotype analysis revealed 6 CYP19(TTTA)n alleles with 7–12 repeats. In PCOS women, the CYP19(TTTA)7 allele presence was associated with lower serum E2 levels at the third day of the menstrual cycle (p < 0.009), lower large follicle (p < 0.02) and total oocyte numbers (p = 0.006), but with significantly higher pregnancy rates after assisted reproduction (p < 0.004). Conclusions: Potential associations of the CYP19(TTTA)7 allele with ovarian response to standard gonadotrophin stimulation and with assisted reproduction outcome were found in PCOS women, probably due to androgen/estrogen ratio alterations.

Sperm flow cytometric parameters are associated with ICSI outcome

The association of sperm nuclear chromatin condensation and ploidy with embryo development and outcome after intracytoplasmic sperm injection (ICSI) was explored. The study population consisted of 16 couples referred to Ioannina University Medical School In vitro Fertilization Unit with male factor infertility and serious impairments in sperm nuclear chromatin condensation and ploidy, according to sperm flow cytometry. Additionally, 20 couples with male factor infertility and relatively high sperm flow cytometry parameters participated as controls. The 35 cycles of the study population were characterized by a lower fertilization rate (P<0.001) as well as decreased grade A embryo rate (P=0.004) and increased grade C embryo rate (P=0.028), compared with the 29 cycles of the control group. Additionally, a significantly elevated arrested embryo rate (P<0.001) and a decreased clinical pregnancy rate (P<0.020) were observed in the couples of the study population. Consequently, high levels of sperm nuclear chromatin condensation abnormalities and sperm aneuploidies are probably associated with lower fertilization rates, impaired embryo quality, elevated arrested embryo rates and decreased pregnancy rates. These preliminary results strongly support the use of sperm flow cytometry as a potential prognostic tool of ICSI outcome.

Phosphatidylethanolamine N-methyltransferase and choline dehydrogenase gene polymorphisms are associated with human sperm concentration

Choline is a crucial factor in the regulation of sperm membrane structure and fluidity, and this nutrient plays an important role in the maturation and fertilizing capacity of spermatozoa. Transcripts of phosphatidylethanolamine N-methyltransferase (PEMT) and choline dehydrogenase (CHDH), two basic enzymes of choline metabolism, have been observed in the human testis, demonstrating their gene expression in this tissue. In the present study, we explored the contribution of the PEMT and CHDH gene variants to sperm parameters. Two hundred oligospermic and 250 normozoospermic men were recruited. DNA was extracted from the spermatozoa, and the PEMT -774G>C and CHDH +432G>T polymorphisms were genotyped. The genotype distribution of the PEMT -774G>C polymorphism did not differ between oligospermic and normozoospermic men. In contrast, in the case of the CHDH +432G>T polymorphism, oligospermic men presented the CHDH 432G/G genotype more frequently than normozoospermic men (62% vs. 42%, P<0.001). The PEMT 774G/G genotype was associated with a higher sperm concentration compared to the PEMT 774G/C and 774C/C genotypes in oligospermic men (12.5 ± 5.6 × 10(6) spermatozoa ml(-1) vs. 8.3 ± 5.2 × 10(6) spermatozoa ml(-1), P<0.002) and normozoospermic men (81.5 ± 55.6 × 10(6) vs. 68.1 ± 44.5 × 10(6) spermatozoa ml(-1), P<0.006). In addition, the CHDH 432G/G genotype was associated with higher sperm concentration compared to CHDH 432G/T and 432T/T genotypes in oligospermic (11.8 ± 5.1 × 10(6) vs. 7.8 ± 5.3 × 10(6) spermatozoa ml(-1), P<0.003) and normozoospermic men (98.6 ± 62.2 × 10(6) vs. 58.8 ± 33.6 × 10(6) spermatozoa ml(-1), P<0.001). In our series, the PEMT -774G>C and CHDH +432G>T polymorphisms were associated with sperm concentration. This finding suggests a possible influence of these genes on sperm quality.

Coelomic cells show apoptosis via Fas/FasL system: a comparative study between healthy human pregnancies and missed miscarriages

BACKGROUND The Fas/Fas ligand (FasL) system represents one of the main apoptotic pathways controlling placental apoptosis throughout gestation. In the current study, we have examined the Fas/FasL protein expression and the apoptotic incidents of coelomic cells, amniotic cells and trophoblastic tissue in first trimester human pregnancies and missed miscarriages (MM). METHODS Protein expression was determined by immunofluoresence, western blotting analysis, immunohistochemistry and indirectly by RT-PCR, whereas apoptotic cell death was assessed by in situ DNA fragmentation analysis. RESULTS Coelomic cells express Fas/FasL proteins, can undergo apoptosis and were the only cells in which apoptosis, Fas protein expression and FasL protein expression were accordingly increased along with gestational age (P = 0.001, P = 0.008; P = 0.012, respectively). In contrast, amniotic cells and trophoblast showed a consistency in the expression levels of Fas/FasL proteins in healthy pregnancies. MM were accompanied by increased Fas/FasL protein expression in all examined samples (P < 0.001). The increase of Fas/FasL protein expression was accompanied by proportional increase of apoptotic incidents among the coelomic cell population (P = 0.023, P = 0.009, respectively), whereas amniotic cells and trophoblast appeared to be resistant to Fas-induced apoptosis. The lowest expression of Fas/FasL proteins and the minimum occurrence of apoptotic incidents were detected in the trophoblast. CONCLUSIONS These data suggest that there is a different regulation and function of the Fas/FasL system in early human pregnancies. Aberration of the Fas-mediated apoptosis may represent one of the execution-step necessary for pregnancy loss in MM cases.

Aromatase (CYP19) gene variants influence ovarian response to standard gonadotrophin stimulation.

PurposeThe association of cytochrome P450 aromatase gene CYP19(TTTA)n polymorphism with ovarian response to FSH stimulation was explored.MethodsThree hundred women undergoing medically assisted reproduction and 300 women with at least one spontaneous pregnancy participated in the study. CYP19(TTTA)n polymorphism was genotyped, while serum hormones were determined. During oocyte retrieval, the follicular size, the follicle and oocyte numbers were recorded.ResultsSix CYP19(TTTA)n alleles with 7 to 12 repeats were revealed. Women homozygous for long CYP19(TTTA)n alleles presented with lower serum FSH levels at the third day of the menstrual cycle (p < 0.001) and higher large follicle numbers (p < 0.01), compared to women homozygous for short CYP19(TTTA)n alleles. The CYP19(TTTA)7 allele was associated with higher serum FSH levels (p < 0.003), with lower total follicle (p < 0.02) and large follicle numbers (p < 0.03), while CYP19(TTTA)7 allele-carriers presented more frequently with small follicles than CYP19(TTTA)7 allele-non carriers (p < 0.01).ConclusionsCYP19 genetic variants were associated with ovarian reserve and response to standard gonadotrophin stimulation of women undergoing in vitro fertilization.