Could zinc prevent reproductive alterations caused by cigarette smoke in male rats?

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Zona thinning with a noncontact diode laser in ICSI embryos from women of advanced age

Purpose: the objective of this study was to determine if the zona thinning (ZT) technique improved the rates of implantation and clinical pregnancy for patients aged, greater than or equal to38 years submitted to an ICSI program.Methods: A total of 100 patients submitted to ICSI and aged, greater than or equal to38 years were divided in a prospective and randomized manner into two groups: Group I - patients submitted to ZT (n = 50); a laser diode with 1.48 mum wavelength (Fertilaser) was used for the ZT procedure with 1-2 irradiations of 10 ms applied to four different positions on the zona pellucida (ZP) of each embryo to thin 60-90% of the ZP (each point with a 15-20 mum length of ZT). Group II - patients with no ZT (n = 50). In both groups, embryo transfer was performed on the second or third day.Results: the age of Group I patients (39.8 +/- 1.3) did not differ (p = 0.67) from that of Group II patients (40 +/- 1.9). The number of oocytes retrieved at metaphase II from Group I (6.4 +/- 4.2) and Group II (6.8 +/- 5) was similar (p = 0.94). Normal fertilization rates and cleavage rates were similar (p = 0.78 and p = 0.63, respectively) for Group I (71.5 +/- 22% and 96.7 +/- 11%) and Group II (73.5 +/- 19.7% and 96 +/- 11%, respectively). The number of embryos transferred was similar (p = 0.53) for the two groups (Group I = 3.1 +/- 1.3; Group II = 2.9 +/- 1.1). The thickness of the ZP of Group I embryos (16.9 +/- 2.4 mum) did not differ (p = 0.97) from that of Group II embryos (16.9 +/- 2.3 mum). The rates of embryo implantation and clinical pregnancy per embryo transfer were similar (p = 0.67, p = 0.61) for Group I (7 and 16%, respectively) and for Group II (8.2 and 22%, respectively).Conclusions: These results suggest that ZT in the population aged, 38 years may have no impact on ICSI success rates. However, this conclusion is limited to a situation in which length of the laser ZT was less than or equal to 20 mum and the laser was applied to four different positions.

Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa

The aim of this investigation was to determine the presence of abnormal sperm chromatin packaging in spermatozoa with large nuclear vacuoles (LNV) selected via high magnification by analysing the pattern of chromomycin A3 (CMA3) staining. A prospective observational study was designed to analyse semen samples obtained from 66 men undergoing infertility diagnosis and treatment. The numbers of cells with normal (dull yellow staining of the sperm head/CMA3-negative) and abnormal (bright yellow fluorescence of the sperm head/CMA3-positive) chromatin packaging were determined on slides with normal and LNV spermatozoa. The presence of bright yellow fluorescence (CMA3-positive) was significantly higher (p < 0.0001) in spermatozoa with LNV than in normal spermatozoa (719/1351; 53.2% vs. 337/835; 40.3%, respectively), reflecting a higher percentage of abnormal chromatin packaging in spermatozoa with large LNV. Our data support the hypothesis that the presence of LNV reflects the presence of abnormal chromatin packaging, which may facilitate sperm DNA damage. As sperm nuclear vacuoles are evaluated more precisely at high magnifications using motile sperm organelle morphology examination (MSOME), the present results support the use of high-magnification sperm selection for intracytoplasmic sperm injection (ICSI).

GnRH agonist versus GnRH antagonist in IVF/ICSI cycles with recombinant LH supplementation: DNA fragmentation and apoptosis in granulosa cells

Objective: To compare the level of apoptosis and DNA fragmentation in the human granulosa cell (GC) layer exposed to an agonist or antagonist of GnRH in intracytoplasmic sperm injection (ICSI) cycles supplemented with recombinant LH (rLH).Study design: Patients without ovulatory dysfunction, aged <= 37 years and in their first ICSI cycle were prospectively randomised to receive either a long GnRH agonist protocol or a multi-dose antagonist protocol. In both groups, recombinant FSH supplemented with rLH was used for ovarian stimulation, and the GCs were collected during oocyte denudation. The GCs were then analysed for DNA fragmentation by TUNEL assay and for apoptosis using the annexin-V assay. The outcomes were given as the percentage of GCs with DNA fragmentation and apoptosis out of the total number of GCs analysed. Comparison of the agonist versus the antagonist group was performed using the Mann-Whitney test.Results: DNA fragmentation: 32 patients were included in either the GnRH agonist group (n = 16) or the antagonist group (n = 16). The percentage of GCs with positive DNA fragmentation did not differ significantly (P = 0.76) between the agonist group (15.5 +/- 9.4%) and the antagonist group (18.8 +/- 13.3%). Apoptosis: 28 patients were included in either the GnRH agonist group (n = 14) or the antagonist group (n = 14). The percentage of GCs positive for apoptosis did not differ significantly (P = 0.78) between the agonist group (34.6 +/- 14.7%) and the antagonist group (36.5 +/- 22%).Conclusions: The results suggest that therapy with either an agonist or antagonist of GnRH is associated with comparable levels of DNA fragmentation and apoptosis in granulosa cells in ICSI cycles supplemented with rLH. (C) 2012 Elsevier B.V. All rights reserved.

Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-stranded or normal double-stranded DNA in spermatozoa with large nuclear vacuoles (LNV) selected by high magnification. Fresh semen samples from 30 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and LNV were selected at x8400 magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation in spermatozoa with LNV (29.1%) was significantly higher (P < 0.001) than in spermatozoa with NN (15.9%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono- and oligonucleosomes), and single-strand breaks (nicks) in high molecular weight DNA occur more frequently in spermatozoa with LNV. Similarly, the percentage of denatured-stranded DNA in spermatozoa with LNV (67.9%) was significantly higher (P < 0.0001) than in spermatozoa with NN (33.1%). The high level of denatured DNA in spermatozoa with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibres. The results show an association between LNV and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high magnification for ICSI.

Significance of extruded nuclear chromatin (regional nuclear shape malformation) in human spermatozoa: implications for ICSI

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-strand or normal double-strand DNA in spermatozoa with extruded nuclear chromatin (ENC) selected by high magnification. Fresh semen samples from 55 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and ENC were selected at 8400x magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation was not significantly different (p = 0.86) between spermatozoa with ENC (19.6%) and those with NN (20%). However, the percentage of spermatozoa with detectable denatured-stranded DNA in the ENC spermatozoon group (59.1%) was significantly higher (p < 0.0001) than in the NN group (44.9%). The high level of denatured DNA in spermatozoa with ENC suggests premature decondensation and disaggregation of sperm chromatin fibres. The results show an association between ENC and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high-magnification intracytoplasmic sperm injection.

Seasonal variability in the fertilization rate of women undergoing assisted reproduction treatments

The objective of this study was to evaluate whether seasonality affects human-assisted reproduction treatment outcomes. For this, 1932 patients undergoing intracytoplasmic sperm injection (ICSI) were assigned to a season group according to the day of oocyte retrieval: winter (n = 435), spring (n = 444), summer (n = 469) or autumn (n = 584). Analysis of variance was used to compare the ICSI outcomes. The fertilization rate was increased during the spring (winter: 67.9%, spring: 73.5%, summer: 68.7% and autumn: 69.0%; p < 0.01). In fact, a nearly 50% increase in the fertilization rate during the spring was observed (odds ratio 1.45, confidence interval 1.20-1.75; p < 0.01). The oestradiol concentration per number of oocytes was significantly higher during the spring (winter: 235.8 pg/mL, spring: 282.1 pg/mL, summer: 226.1 pg/mL and autumn: 228.7 pg/mL; p = 0.030). This study demonstrates a seasonal variability in fertilization after ICSI, where fertilization is higher during the spring than at any other time.

Decreased fertility in poor responder women is not related to oocyte morphological status

Introduction: In women showing impaired fertility, a decreased response to ovarian stimulation is a major problem, limiting the number of oocytes to be used for assisted reproduction techniques (ART). Despite the several definitions of poor response, it is still a matter of debate whether young poor responder patients also show a decrease in oocyte quality. The objective in this study was to investigate whether poor ovarian response to the superstimulation protocol is accompanied by impaired oocyte quality.Material and methods: This study included 313 patients younger than 35 years old, undergoing intracytoplasmic sperm injection. Patients with four or fewer MII oocytes (poor-responder group, PR, n = 57) were age-matched with normoresponder patients (NR, n = 256).Results: A higher rate of oocyte retrieval and a trend towards an increase in MII oocyte rate were observed in the NR group when compared to the PR group (71.6 +/- 1.1% and 74.1 +/- 1.0% vs. 56.3 +/- 2.9% and 66.5 +/- 3.7%; p < 0.0001 and p = 0.056, respectively). A trend toward increased implantation rates was observed in the NR group when compared to the PR group (44 and 24.5 +/- 2.0% vs. 28.8 and 16.4 +/- 3.9%; p = 0.0305 and p = 0.0651, respectively).Conclusions: Low response to ovarian stimulation is apparently not related to impaired oocyte quality. However, embryos produced from poor responder oocytes show impaired capacity to implant and to carry a pregnancy to term.

Are poor responders patients at higher risk for producing aneuploid embryos in vitro?

Purpose To test the hypothesis that aged women with poor ovarian response express an increase on embryo chromosomal alterations when compared to aged women who presented normal response.Methods Couples undergoing intracytoplasmic sperm injection cycles with preimplantation genetic screening, were subdivided into two groups: Poor Responder group (n = 34), patients who produced a parts per thousand currency sign4 oocytes; and Normoresponder group (n = 50), patients who produced a parts per thousand yen5 oocytes. Groups were compared regarding cycles' outcomes and aneuploidy frequency.Results There were no significant differences between and groups regarding the fertilization rate (p = 0.6861), clinical pregnancy (p = 0.9208), implantation (p = 0.6863), miscarriage (p = 0.6788) and the percentage of aneuploid embryos (p = 0.270). Embryo transfer rate was significantly lower on poor responder group (p = 0.0128) and logistic regression confirmed the influence of poor response on the chance of embryo transfer (p = 0.016).Conclusions Aged females responding poorly to gonadotrophins are not at a higher risk for producing aneuploid embryos in vitro.

Inflammatory Response Patterns in ICSI Patients: A Comparative Study Between Chronic Anovulating and Normally Ovulating Women

The aim of this study was to evaluate inflammatory response in chronic anovulating infertility women undergoing intracytoplasmic sperm injection. Thirteen infertile women with chronic anovulation and 23 normally ovulating women were prospectively evaluated. N-acetylglucosaminidase (NAG), myeloperoxidase (MPO), monocyte chemoattractant protein 1 (MCP-1), and C-reactive protein (CRP) concentrations were evaluated in serum and follicular fluid. Women with chronic anovulation presented higher NAG and MPO activity in follicular fluid when compared with normally ovulating women. Serum MPO activity was higher in the control group compared to the chronic anovulation group. Both serum and follicular fluid CRP concentrations were higher in women with chronic anovulation in comparison with the control group. Higher MCP-1 follicular fluid concentrations and serum levels of CRP were associated with the occurrence of ovarian hyperstimulation syndrome. Patients with chronic anovulation exhibited significantly higher follicle macrophage/neutrophil activation as well as unspecific inflammatory response by comparison with normally ovulating women.

Relationship between visualization of meiotic spindle in human oocytes and ICSI outcomes: a meta-analysis

The objective of this meta-analysis was to investigate the influence of meiotic spindle visualization in human oocytes on intracytoplasmic sperm injection (ICSI) outcomes. Search strategies included on-line Surveys of databases (MEDLINE, em BASE, Science Citation Index, Cochrane Controlled Trials Register and Ovid). The fixed effect was used for odds ratio. Ten trials fulfilled the inclusion criteria comparing in-vitro and clinical ICSI outcomes with or without visualization of meiotic spindle in fresh and in-vivo matured oocytes. According to the meta-analysis, the results showed statistically significant higher fertilization rate (P < 0.0001) when the meiotic spindle was viewed than when it was not. Moreover, the percentage of pro-nuclear-stage embyros with good morphology (P = 0.003), cleavage rate (P < 0.0001), percentage of day-3 top-quality embryos (P = 0.003) and percentage of embryos that reached the blastocyst stage (P < 0.0001) were statistically significantly better among, embryos derived from oocytes in which meiotic spindle was viewed compared with those in which meiotic spindle was not observed. However, these differences were not observed in the clinical pregnancy or implantation rates. This observation has clinical relevance mainly in countries where there is a legal limit on the number of oocytes to be fertilized. However, additional controlled trials are needed to further confirm these results.

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

Background: The present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).Methods: A total of 16592 prepared spermatozoa were selected and classified into two groups: Group I, spermatozoa which presented their head attached to an HA substance (HA-bound sperm), and Group II, those spermatozoa that did not attach to the HA substance (HA-unbound sperm). HA-bound and HA-unbound spermatozoa were evaluated according to the following sperm forms: 1-Normal morphology: normal nucleus (smooth, symmetric and oval configuration, length: 4.75+/-2.8 mu m and width: 3.28+/-0.20 mu m, no extrusion or invagination and no vacuoles occupied more than 4% of the nuclear area) as well as acrosome, post-acrosomal lamina, neck, tail, besides not presenting a cytoplasmic droplet or cytoplasm around the head; 2-Abnormalities of nuclear form (a-Large/small; b-Wide/narrow; c-Regional disorder); 3-Abnormalities of nuclear chromatin content (a-Vacuoles: occupy >4% to 50% of the nuclear area and b-Large vacuoles: occupy >50% of the nuclear area) using a high magnification (8400x) microscopy system.Results: No significant differences were obtained with respect to sperm morphological forms and the groups HA-bound and HA-unbound. 1-Normal morphology: HA-bound 2.7% and HA-unbound 2.5% (P = 0.56). 2-Abnormalities of nuclear form: a-Large/small: HA-bound 1.6% vs. HA-unbound 1.6% (P = 0.63); b-Wide/narrow: HA-bound 3.1% vs. HA-unbound 2.7% (P = 0.13); c-Regional disorders: HA-bound 4.7% vs. HA-unbound 4.4% (P = 0.34). 3. Abnormalities of nuclear chromatin content: a-Vacuoles >4% to 50%: HA-bound 72.2% vs. HA-unbound 72.5% (P = 0.74); b-Large vacuoles: HA-bound 15.7% vs. HA-unbound 16.3% (P = 0.36).Conclusions: The findings suggest that HA binding assay has limited efficacy in selecting motile spermatozoa with normal morphology at high magnification.

IVF/ICSI outcomes after culture of human embryos at low oxygen tension: a meta-analysis

Background: Improved pregnancy, implantation, and birth rates have been reported after the use of reduced O2 concentration during embryo culture, mainly due to a reduction of the cumulative detrimental effects of reactive oxygen species. However, some studies have failed to report any positive effects. The objective of this meta-analysis was to evaluate the effect of a low-O2 environment on IVF/intracytoplasmic sperm injection (ICSI) outcomes.Methods: All available published and ongoing randomised trials that compared the effects of low (similar to 5%; OC similar to 5) and atmospheric (similar to 20%; OC similar to 20) oxygen concentrations on IVF/ICSI outcomes were included. Search strategies included online surveys of databases from 1980 to 2011. The outcomes measured were fertilisation rate, implantation rate and ongoing pregnancy rates. The fixed effects model was used to calculate the odds ratio.Results: Seven studies were included in this analysis. The pooled fertilisation rate did not differ significantly (P = 0.54) between the group of oocytes cultured at low O2 tension and the group at atmospheric O2 tension. Concerning all cycles, the implantation (P = 0.06) and ongoing pregnancy (P = 0.051) rates were not significantly different between the group receiving transferred sets containing only OC similar to 5 embryos and the group receiving transferred sets with only OC similar to 20 embryos. In a meta-analysis performed for only those trials in which embryos were transferred on day 2/3, implantation (P = 0.63) and ongoing pregnancy (P = 0.19) rates were not significantly different between the groups. In contrast, when a meta-analysis was performed using only trials in which embryos were transferred on days 5 and 6 (at the blastocyst stage), the group with transferred sets of only OC similar to 5 embryos showed a statistically significantly higher implantation rate (P = 0.006) than the group receiving transferred sets with only OC similar to 20 embryos, although the ongoing pregnancy (P = 0.19) rates were not significantly different between the groups.Conclusions: Despite some promising results, it seems too early to conclude that low O2 culture has an effect on IVF outcome. Additional randomised controlled trials are necessary before evidence-based recommendations can be provided. It should be emphasised that the present meta-analysis does not provide any evidence that low oxygen concentration is unnecessary.

GnRH agonist versus GnRH antagonist in assisted reproduction cycles: oocyte morphology

Background: The selection of developmentally competent human gametes may increase the efficiency of assisted reproduction. Spermatozoa and oocytes are usually assessed according to morphological criteria. Oocyte morphology can be affected by the age, genetic characteristics, and factors related to controlled ovarian stimulation. However, there is a lack of evidence in the literature concerning the effect of gonadotropin-releasing hormone (GnRH) analogues, either agonists or antagonists, on oocyte morphology. The aim of this randomized study was to investigate whether the prevalence of oocyte dysmorphism is influenced by the type of pituitary suppression used in ovarian stimulation.Methods: A total of 64 patients in the first intracytoplasmic sperm injection (ICSI) cycle were prospectively randomized to receive treatment with either a GnRH agonist with a long-term protocol (n: 32) or a GnRH antagonist with a multi-dose protocol (n: 32). Before being subjected to ICSI, the oocytes at metaphase II from both groups were morphologically analyzed under an inverted light microscope at 400x magnification. The oocytes were classified as follows: normal or with cytoplasmic dysmorphism, extracytoplasmic dysmorphism, or both. The number of dysmorphic oocytes per total number of oocytes was analyzed.Results: Out of a total of 681 oocytes, 189 (27.8 %) were morphologically normal, 220 (32.3 %) showed cytoplasmic dysmorphism, 124 (18.2%) showed extracytoplasmic alterations, and 148 (21.7%) exhibited both types of dysmorphism. No significant difference in oocyte dysmorphism was observed between the agonist- and antagonist- treated groups (P > 0.05). Analysis for each dysmorphism revealed that the most common conditions were alterations in polar body shape (31.3%) and the presence of diffuse cytoplasmic granulations (22.8%), refractile bodies (18.5%) and central cytoplasmic granulations (13.6%). There was no significant difference among individual oocyte dysmorphisms in the agonist- and antagonist-treated groups (P > 0.05).Conclusions: Our randomized data indicate that in terms of the quality of oocyte morphology, there is no difference between the antagonist multi-dose protocol and the long-term agonist protocol. If a GnRH analogue used for pituitary suppression in IVF cycles influences the prevalence of oocyte dysmorphisms, there does not appear to be a difference between the use of an agonist as opposed to an antagonist.

The influence of leukocytospermia on the outcomes of assisted reproductive technology

Background: It is not well established whether the increased number of leukocytes in the seminal fluid impairs the outcomes of assisted reproductive technology (ART). This investigation analysed the outcomes of the intracytoplasmic sperm injection (ICSI) and intracytoplasmic morphologically selected sperm injection (IMSI) cycles in couples in which the male partner exhibited leukocytospermia.Methods: A total of 100 cycles in 100 couples were included in this study. For the ICSI or IMSI procedures, the patients were divided into two groups according to the presence or absence of leukocytospermia and then matched by (female) age:- ICSI: Group I (n = 25): Leukocytospermia - semen samples with a leukocyte count of greater than or equal to 1 x 10(6)/mL; and Group II (n = 25): Non-leukocytospermia - semen samples with a leukocyte count < 1 x 10(6)/mL.- IMSI: Group I (n = 25): Leukocytospermia; and Group II (n = 25): Non-leukocytospermia.The endpoints included the rates of fertilisation, implantation, clinical pregnancy, miscarriage, ongoing pregnancy and live birth. Student's t-tests, Mann-Whitney tests and Chi-square tests were performed, and P < 0.05 was considered significant.Results: The data from the ICSI groups showed that leukocytospermia did not have a negative influence on the rates of fertilisation (Group I: 57.9+/-30.2%, Group II: 61.9+/-27.7%; P = 0.74), implantation (Group I: 12.3%; Group II: 13.5%; P = 0.93), clinical pregnancy (Group I: 24%; Group II: 24%; P = 1.0), miscarriage ( Group I: 0, Group II: 0), ongoing pregnancy (Group I: 24%; Group II: 24%; P = 1.0), or live births (Group I: 24%; Group II: 24%; P = 1.0). Similarly, the data from the IMSI groups also showed that the leukocytospermia did not have a negative influence on the rates of fertilisation (Group I: 67.6+/-24.6%, Group II: 59.5+/-28.1%; P = 0.36), implantation (Group I: 17.5%; Group II: 16.7%; P = 0.90), clinical pregnancy (Group I: 28%; Group II: 24%; P = 1.0), miscarriage (Group I: 14.3%; Group II: 0; P = 0.33), ongoing pregnancy (Group I: 24%; Group II: 24%; P = 1.0), or live births (Group I: 24%, 6/25; Group II: 24%, 6/25; P = 1.0).Conclusions: The results indicate that the leukocytospermia may not have a negative effect on the outcomes of ICSI or IMSI cycles. Nevertheless, it seems that it is necessary to more precisely determine the effects, if any, of seminal leukocytes on fertilisation and implantation processes. Such efforts will help to establish a more reliable leukocyte threshold, which could eventually demonstrate whether there is a negative influence on the ART procedures.