Combined Effect of Trolox and EDTA on Frozen-Thawed Sperm Quality.

The freezing and thawing process not only is associated with serious damage to sperm such as damage to the plasma membrane and the acrosomal membrane but also changes the membrane permeability to some ions including calcium. Also, the generation of oxygen free radicals is increased during the freezing-thawing process. The purpose of this study was to evaluate of the effects of Trolox as an antioxidant and edetic acid (EDTA) as a calcium chelator on frozen-thawed (FT) sperm and compare these effects with those on fresh sperm. This study was done on these men of 25 healthy men, who referred to Shiraz Infertility Centerbetween2012 and2013. Normal samples were transferred to the ReproductivePhysiology Laboratory, Department of Physiology, Shiraz University of Medical Sciences, Shiraz. The samples were divided into two groups randomly: fresh and FT sperm groups. Each group was divided into five subgroups: control group, the solvent group (0.1%dimethyl sulfoxide [DMSO]), Trolox group (200µM), EDTA group (1.1mM), and Trolox+EDTA group. The percentages of motility, viability, and acrosome-reacted sperm were tested. The percentages of motility and viability in the FT sperm were lower than those in the fresh sperm. The progressive motility of the FT sperm was improved nonsignificantly with Trolox+EDTA. However, the effect of Trolox+EDTA on the progressive motility of the FT sperm was much more than that on the fresh sperm. The fewest acrosome-reacted sperm were observed in the EDTA-containingFT sperm. Antioxidant supplementation or omission of extracellular calcium may partly improve motility and also reduce acrosomal damage in FT sperm.

Reproductive outcome following thawed embryo transfer in management of ovarian hyperstimulation syndrome.

BACKGROUND: The purpose of the study was to compare clinical pregnancy and delivery rates with fresh and frozen embryo transfer in patients admitted to Shiraz- Human Assisted Reproductive Center with ovarian hyperstimulation syndrome (OHSS). METHODS: OHSS patients randomly divided in two groups, group A (n=50) with fresh embryo transfer and group B (n=50) with frozen embryo transfer. We used vitrification method for freezing the embryos. Patient age, combination of female and male factors, total number of retrieved oocytes, number of cryopreserved embryo, number of transferred embryos, clinical pregnancy and delivery rates were recorded for all patients. All statistical calculations were done using SPSS software. Generalized linear model was used to adjust the confounding factors to compare the clinical pregnancy and delivery rates between two groups. The p<0.05 was considered statistically significant. RESULTS: Mean (±SD) ages of these patients were 26.78±3.5 and 28.42±4.2 yrs in fresh (A) and frozen (B) embryo transfer groups respectively. Combinations of male and female factors were 28.3% and 32.1% respectively. Average numbers of oocytes retrieved in two groups were 22.14±4.3 and 21.02±4.9, and after fertilization, embryos cryopreserved per patient yielded averages of 13.82±3.5 and 12.5±4.3. Thaw and ET were performed and the means for transferred embryos were 3.22±0.6 and 4.1±0.7. We didn't find any significant differences in implicit parameters between the two groups. The pregnancy and delivery rates in OHSS patients were significantly higher in frozen embryo transfer, 63.1% and 45.6%, compared with fresh embryo transfer, 55.1% and 35.4%, respectively. CONCLUSION: The pregnancy and delivery rates in OHSS cases, both fresh and subsequently with frozen embryo transfer, were exceptionally high. There was statistically significant difference of pregnancy and delivery rates between fresh and frozen embryo transfer. As a result, an elective embryo freezing policy to moderate the severity and duration of OHSS has compromising outcomes for women at risk of OHSS.

Value of sperm chromatin dispersion test in couples with unexplained recurrent abortion.

OBJECTIVE: To evaluate and compare standard sperm parameters and sperm chromatin integrity by sperm chromatin dispersion test (SCD) in ejaculates from men whose partners have a history of recurrent pregnancy loss and from control group of fertile men. METHODS: Thirty couples with unexplained recurrent abortion (case group) and 30 fertile couples (control group) referring to Shiraz infertility center were included. Sperm parameters were assessed in semen samples from two groups and then staining with SCD procedure. The results were analyzed by performing ANOVA and Tukey(,)s tests. RESULTS: In control group, nucleoids with big (65.93 ± 2.35), small (12.4 ± 0.60) and without halo (11.6 ± 0.50) showed significant difference with case group (41.40 ± 1.43), (21.16 ± 1.11) and (23.26 ± 1.10) respectively. In the RPL group spermatozoa with high percentage of abnormal parameters (morphology and motility) was observed (p ≤ 0.05). CONCLUSION: This study strengthens the current literature associating sperm quality with recurrent pregnancy loss, and emphasizes the important of evaluating male factor by tests such as SCD in addition to conventional sperm parameters.

Effect of combined density gradient centrifugation on X- and Y- sperm separation and chromatin integrity.

BACKGROUND: It has been claimed that by using different washing methods, the sperms can be separated according to size, motility, density, chromosomal content and surface markings and charge. These methods also reduce sperm chromatin deficiencies and screen the sperms before applying in assisted reproduction techniques. OBJECTIVE: This study compared simple density gradient methods and a combined method with albumin density gradient and PureSperm separation (alb/PureSperm) for sex preselection by double fluorescence in situ hybridization (FISH) versus chromomycin A3 staining to determine chromatin integrity. MATERIALS AND METHODS: 30 normal semen samples were prepared with PureSperm, albumin gradients and alb/PureSperm. All samples were then stained by FISH and chromomycin A3. The results were compared with SPSS 11.5 and the Kruskal-Wallis test. RESULTS: The proportion of X-bearing spermatozoa by PureSperm separation (47.58±5.67) and Y-bearing spermatozoa by albumin gradient (46.13±3.83) methods were slightly higher than in putative normal sperm samples (1:1), but there were no significant differences in the X- or Y- bearing spermatozoa counts among the three methods. Albumin gradient separation tended to underestimate abnormal spermatozoa compared to PureSperm and combined alb/PureSperm. CONCLUSION: Routine separation methods slightly enriched X- or Y- bearing spermatozoa, but the differences were not significant for clinical purposes. The combined alb/PureSperm method had no advantages for assessing sex ratio or chromatin integrity compared to simpler gradient methods.

The effects of laser assisted hatching on pregnancy rates.

BACKGROUND: For infertile women aged over 35 years, failure of the ZP (zona pellucida) to rupture is believed to be associated with a decreased implantation rate in in vitro fertilization (IVF) or intra cytoplasmic sperm injection (ICSI). OBJECTIVE: In this research, laser assisted hatching (LAH) was offered to patients with advanced maternal age to evaluate a possible benefit. MATERIALS AND METHODS: Nine hundred thirty two cycles of IVF/ICSI in females were analyzed. Women included in this study were allocated in 4 groups. In group I and II, embryos were cultured and transferred with and without LAH in women aged ≤35, whereas embryos of group III and IV were examined with and without LAH in women aged ≥ 35. Laser manipulations were performed using a suturn-Tm3 system using 2-3 pulses of 0.8 millisecond with 400 voltage duration. The size of the hole made in the zona was measured to be 5-10 µm, depending on the zona thickness of each individual embryo. RESULTS: The performance of LAH significantly increased clinical pregnancy rates in all patients. In group I and II, the chemical (50.99% and 31.61% respectively), clinical (50% and 30.69% respectively) and multiple pregnancies (22.27% and 5.94% respectively) significantly differ between these groups. In the patients with advanced female age ≥35 the performance of LAH significantly increased chemical (30.12%) and clinical pregnancy (27.71%) rates compared to whom without LAH (18.96% and 16.37% respectively). CONCLUSION: Our data demonstrate in the patients who were less than 35 years old, multiple pregnancy rates were significantly increased compared to other groups who aged over 35 years old. In addition benefit of LAH in improving pregnancy rates after IVF or ICSI in women of advanced age (≥35) was shown.

Evaluation of sperm's chromatin quality with acridine orange test, chromomycin A3 and aniline blue staining in couples with unexplained recurrent abortion.

OBJECTIVE: To evaluate the sperm's chromatin quality in couples with spontaneous recurrent abortion. METHODS: Thirty couples with spontaneous recurrent abortion (case group) and 30 fertile couples (control group) referring to Zeinabieh Gynecology clinic of Shiraz were included. Semen samples were collected for each participant and were used for standard semen analysis and sperm nuclear maturity tests including Chromomycin A3 (CMA3), Aniline Blue (AB) staining and Acridine Orange (AO) test (by light microscopy). RESULT: Patients in case group had significantly higher percentage of CMA3 (p < 0.001) and AB (p < 0.001) positive spermatozoa compared to controls. However AO results did not differ significantly between groups (p = 0.656). Sperm morphology and progressive motility were negatively correlated with CMA3 (p = 0.001 and p = 0.043) and AB (p = 0.015 and p = 0.031) respectively. CONCLUSION: Evaluation of the sperm's quality via CMA3 and AB staining could be considered as one of the complementary tests of semen analysis for assessment of male factor in couples with spontaneous recurrent abortion.

Short-term metformin treatment for clomiphene citrate-resistant women with polycystic ovary syndrome.

OBJECTIVE: To evaluate the effect of a short-course pretreatment with metformin on hyperandrogenism, insulin resistance, cervical scores, and pregnancy rates in women with clomiphene citrate (CC)-resistant polycystic ovary syndrome (PCOS). METHODS: Thirty-seven women with CC-resistant PCOS were randomly assigned to be pretreated with 500 mg of metformin or placebo 3 times per day for 2 cycles, and 100 mg of CC was given on days 5 through 9 of the second cycle in both groups. Luteinizing hormone (LH), follicle stimulating hormone (FSH), dehydroepiandrostendione sulfate (DHEAS), total testosterone (T), glucose, and insulin levels were measured at baseline and after the first cycle, as well as body mass index (BMI), cervical score, and pregnancy rate. RESULTS: After 1 cycle, BMI, total T level, and percentage of participants with insulin resistance were significantly decreased in the metformin group, without any significant decrease in LH, FSH, and DHEAS levels; and in the second cycle, CC treatment resulted in a higher ovulation rate and a thicker endometrium in the metformin group. The pregnancy rate and cervical scores were also higher in that group. CONCLUSION: The short-course pretreatment with metformin decreased hyperandrogenism and insulin resistance and improved cervical sores, ovulation rate, and pregnancy rate among women with CC-resistant PCOS.