Association between leukaemia inhibitory factor gene polymorphism and pregnancy outcomes after assisted reproduction techniques.

Certain gene polymorphisms are associated with implantation failure and pregnancy loss. Studies of leukaemia inhibitory factor (LIF) gene polymorphisms are scarce. The LIF single nucleotide polymorphism (SNP) thymine (T)/guanine (G) (rs929271) was studied in women to determine whether an association existed with pregnancy outcomes after intracytoplasmic sperm injection (ICSI); 411 women who underwent ICSI were recruited. DNA was extracted from the peripheral blood, and the LIF gene SNP T/G (rs929271) was genotyped using real-time polymerase chain reaction. Participants were divided into three groups according to their LIF genotype: T/T (n = 168), T/G (n = 202) and G/G (n = 41). All IVF and ICSI procedures were carried out under the same clinical and laboratory conditions. The ICSI cumulative results (from fresh plus frozen cycles) of each genotype group were analysed. The G/G genotype in women was associated with a higher implantation rate (T/T: 15.9%, T/G: 16.2%, G/G: 27.0%; P < 0.05), ongoing pregnancy rate/patient (T/T: 31.5%, T/G: 36.1%, G/G: 53.7%; P < 0.05) and ongoing pregnancy rate/transfer (T/T: 18.5%, T/G: 20.2%, G/G: 36.7%; P < 0.05). LIF SNP T/G (rs929271) seems to be a susceptibility biomarker capable of predicting implantation efficiency and pregnancy outcomes.

The Relationship between Vascular Endothelial Growth Factor 1154G/A Polymorphism and Recurrent Implantation Failure.

OBJECTIVE: The aim of this study was to investigate the relationship between herpesvirus-associated ubiquitin-specific protease (HAUSP A/G, rs1529916), tumor protein p53 (TP53 Arg/Pro, rs1042522), leukemia inhibitory factor (LIF G/T, rs929271), glycoprotein 130 (gp130 A/T, rs1900173) and vascular endothelial growth factor (VEGF G/A, rs1570360) polymorphisms and recurrent implantation failure (RIF) in Brazilian women. SUBJECTS AND METHODS: A total of 120 women with RIF (i.e. those with ≥5 cleaved embryos transferred and a minimum of 2 failed in vitro fertilization/intracytoplasmic sperm injection attempts) were included. The control group involved 89 women who had experienced at least 1 live birth (without any infertility treatment). DNA was extracted from the peripheral blood of all participants, and the abovementioned single-nucleotide polymorphisms (SNPs) were genotyped by real-time polymerase chain reaction. The data were evaluated using Fisher's test. RESULTS: A significant difference between the RIF and control groups was found in the VEGF gene where the GG genotype showed a 2.1-fold increased chance of not being included in the RIF group, while the presence of an A allele increased this risk 1.6-fold. No significant differences were found for the other polymorphisms. CONCLUSION: This study showed an association between the VEGF -1154G/A polymorphism and RIF in Brazilian women.

A new ovarian response prediction index (ORPI): implications for individualised controlled ovarian stimulation.

BACKGROUND: The objective was to present a new ovarian response prediction index (ORPI), which was based on anti-Müllerian hormone (AMH) levels, antral follicle count (AFC) and age, and to verify whether it could be a reliable predictor of the ovarian stimulation response. METHODS: A total of 101 patients enrolled in the ICSI programme were included. The ORPI values were calculated by multiplying the AMH level (ng/ml) by the number of antral follicles (2-9 mm), and the result was divided by the age (years) of the patient (ORPI=(AMH x AFC)/Patient age). RESULTS: The regression analysis demonstrated significant (P<0.0001) positive correlations between the ORPI and the total number of oocytes and of MII oocytes collected. The logistic regression revealed that the ORPI values were significantly associated with the likelihood of pregnancy (odds ratio (OR): 1.86; P=0.006) and collecting greater than or equal to 4 oocytes (OR: 49.25; P<0.0001), greater than or equal to 4 MII oocytes (OR: 6.26; P<0.0001) and greater than or equal to 15 oocytes (OR: 6.10; P<0.0001). Regarding the probability of collecting greater than or equal to 4 oocytes according to the ORPI value, the ROC curve showed an area under the curve (AUC) of 0.91 and an efficacy of 88% at a cut-off of 0.2. In relation to the probability of collecting greater than or equal to 4 MII oocytes according to the ORPI value, the ROC curve had an AUC of 0.84 and an efficacy of 81% at a cut-off of 0.3. The ROC curve for the probability of collecting greater than or equal to 15 oocytes resulted in an AUC of 0.89 and an efficacy of 82% at a cut-off of 0.9. Finally, regarding the probability of pregnancy occurrence according to the ORPI value, the ROC curve showed an AUC of 0.74 and an efficacy of 62% at a cut-off of 0.3. CONCLUSIONS: The ORPI exhibited an excellent ability to predict a low ovarian response and a good ability to predict a collection of greater than or equal to 4 MII oocytes, an excessive ovarian response and the occurrence of pregnancy in infertile women. The ORPI might be used to improve the cost-benefit ratio of ovarian stimulation regimens by guiding the selection of medications and by modulating the doses and regimens according to the actual needs of the patients.

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 × 10(6)/mL; and Group II (n = 25): Non-leukocytospermia - semen samples with a leukocyte count < 1 × 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.

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 effects of male age on sperm analysis by motile sperm organelle morphology examination (MSOME).

BACKGROUND: This study aimed to investigate the influence of age on sperm quality, as analysed by motile sperm organelle morphology examination (MSOME). METHODS: Semen samples were collected from 975 men undergoing evaluation or treatment for infertility. Sperm cells were evaluated at 8400× magnification using an inverted microscope equipped with Nomarski (differential interference contrast) optics. Two forms of spermatozoa were considered: normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV, defined as vacuoles occupying > 50% of the sperm nuclear area). At least 200 spermatozoa per sample were evaluated, and the percentages of normal and LNV spermatozoa were determined. The subjects were divided into three groups according to age: Group I, less than or equal to 35 years; Group II, 36-40 years; and Group III, greater than or equal to 41 years. RESULTS: There was no difference in the percentages of normal sperm between the two younger (I and II) groups (P >0.05). The percentage of normal sperm in the older group (III) was significantly lower than that in the younger (I and II) groups (P < 0.05). There was no difference in the percentage of LNV spermatozoa between the younger (I and II) groups (P >0.05). The percentage of LNV spermatozoa was significantly higher in the older group (III) than in the younger (I and II) groups (P < 0.05). Regression analysis demonstrated a significant decrease in the incidence of normal sperm with increasing age (P < 0.05; r = -0.10). However, there was a significant positive correlation between the percentage of spermatozoa with LNV and male age (P < 0.05, r = 0.10). CONCLUSION: The results demonstrated a consistent decline in semen quality, as reflected by morphological evaluation by MSOME, with increased age. Considering the relationship between nuclear vacuoles and DNA damage, these age-related changes predict that increased paternal age should be associated with unsuccessful or abnormal pregnancy as a consequence of fertilisation with damaged spermatozoa. Given that sperm nuclear vacuoles can be evaluated more precisely at high magnification, these results support the routine use of MSOME for ICSI as a criterion for semen analysis.

Pregnancy outcomes in women with repeated implantation failures after intracytoplasmic morphologically selected sperm injection (IMSI).

BACKGROUND: The purpose of this study was to compare laboratory and clinical outcomes of intracytoplasmic morphologically selected sperm injection (IMSI) and conventional intracytoplasmic sperm injection (ICSI) in couples with repeated implantation failures. METHODS: A total of 200 couples with at least two prior unsuccessful ICSI cycles were enrolled: 100 couples were submitted to IMSI and 100 were submitted to routine ICSI. For IMSI, spermatozoa were selected at 8400× magnification using an inverted microscope equipped with Nomarski (differential interference contrast) optics. For conventional ICSI, spermatozoa were selected at 400× magnification. Clinical outcomes were evaluated between the two groups. RESULTS: Study patients were comparable in age, number of treatment failures, aetiology of infertility, percentage of normal form assessed by MSOME (motile sperm organelle morphology examination), semen parameters, total number of oocytes collected, number of mature oocytes collected, total number of embryos transferred and number of high-quality embryos transferred. No statistically significant differences between the two groups were observed with regard to rates of fertilisation, implantation and pregnancy/cycle. Although not statistically significant, rates of miscarriage (IMSI:15.3% vs ICSI:31.7%), ongoing pregnancy (IMSI:22% vs ICSI:13%) and live births (IMSI:21% vs ICSI:12%) showed a trend towards better outcomes in the IMSI group. In addition, analysis of subpopulations with or without male factor showed similar results. CONCLUSIONS: Our results suggest that IMSI does not provide a significant improvement in clinical outcome compared to ICSI, at least in couples with repeated implantation failures after conventional ICSI. However, it should be noted that there were clear trends for lower miscarriage rates (≈50% reduced) and higher rates of ongoing pregnancy and live births (both nearly doubled) within the IMSI group. Further confirmation as well as randomized large-scale trials are needed to confirm the beneficial effects of IMSI in couples with poor reproductive prognoses.

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 (~5%; OC~5) and atmospheric (~20%; OC~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~5 embryos and the group receiving transferred sets with only OC~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~5 embryos showed a statistically significantly higher implantation rate (P=0.006) than the group receiving transferred sets with only OC~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.

Efficacy of the motile sperm organelle morphology examination (MSOME) in predicting pregnancy after intrauterine insemination.

BACKGROUND: Although the motile sperm organelle morphology examination (MSOME) was developed merely as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in the evaluation of semen quality. The aim of this study was to determine the prognostic value of normal sperm morphology using MSOME with regard to clinical pregnancy (CP) after intrauterine insemination (IUI). METHODS: A total of 156 IUI cycles that were performed in 111 couples were prospectively analysed. Each subject received 75 IU of recombinant FSH every second day from the third day of the cycle. Beginning on the 10th day of the cycle, follicular development was monitored by vaginal ultrasound. When one or two follicles measuring at least 17 mm were observed, recombinant hCG was administered, and IUI was performed 12-14 h and 36-40 h after hCG treatment. Prior to the IUI procedure, sperm samples were analysed by MSOME at 8400× magnification using an inverted microscope that was equipped with DIC/Nomarski differential interference contrast optics. A minimum of 200 motile spermatozoa per semen sample were evaluated, and the percentage of normal spermatozoa in each sample was determined. RESULTS: Pregnancy occurred in 34 IUI cycles (CP rate per cycle: 21.8%, per patient: 30.6%). Based on the MSOME criteria, a significantly higher percentage of normal spermatozoa was found in the group of men in which the IUI cycles resulted in pregnancy (2.6+/-3.1%) compared to the group that did not achieve pregnancy (1.2+/-1.7%; P = 0.019). Logistic regression showed that the percentage of normal cells in the MSOME was a determining factor for the likelihood of clinical pregnancy (OR: 1.28; 95% CI: 1.08 to 1.51; P = 0.003). The ROC curve revealed an area under the curve of 0.63 and an optimum cut-off point of 2% of normal sperm morphology. At this cut-off threshold, using the percentage of normal sperm morphology by MSOME to predict pregnancy was 50% sensitive with a 40% positive predictive value and 79% specificity with an 85% negative predictive value. The efficacy of using the percentage of normal sperm morphology by MSOME in predicting pregnancy was 65%. CONCLUSIONS: The present findings support the use of high-magnification microscopy both for selecting spermatozoa and as a routine method for analysing semen before performing IUI.

Evaluation of zona pellucida birefringence intensity during in vitro maturation of oocytes from stimulated cycles.

BACKGROUND: This study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF) intensity and the nuclear (NM) and cytoplasmic (CM) in vitro maturation of human oocytes from stimulated cycles. RESULTS: The ZP-BF was evaluated under an inverted microscope with a polarizing optical system and was scored as high/positive (when the ZP image presented a uniform and intense birefringence) or low/negative (when the image presented moderate and heterogeneous birefringence). CM was analyzed by evaluating the distribution of cortical granules (CGs) throughout the ooplasm by immunofluorescence staining. CM was classified as: complete, when CG was localized in the periphery; incomplete, when oocytes presented a cluster of CGs in the center; or in transition, when oocytes had both in clusters throughout cytoplasm and distributed in a layer in the cytoplasm periphery Nuclear maturation: From a total of 83 germinal vesicle (GV) stage oocytes, 58 of oocytes (69.9%) reached NM at the metaphase II stage. From these 58 oocytes matured in vitro, the high/positively scoring ZP-BF was presented in 82.7% of oocytes at the GV stage, in 75.8% of oocytes when at the metaphase I, and in 82.7% when oocytes reached MII. No relationship was observed between NM and ZP-BF positive/negative scores (P = 0.55). These variables had a low Pearson's correlation coefficient (r = 0.081). Cytoplasmic maturation: A total of 85 in vitro-matured MII oocytes were fixed for CM evaluation. Forty-nine oocytes of them (57.6%) showed the complete CM, 30 (61.2%) presented a high/positively scoring ZP-BF and 19 (38.8%) had a low/negatively scoring ZP-BF. From 36 oocytes (42.3%) with incomplete CM, 18 (50%) presented a high/positively scoring ZPBF and 18 (50%) had a low/negatively scoring ZP-BF. No relationship was observed between CM and ZP-BF positive/negative scores (P = 0.42). These variables had a low Pearson's correlation coefficient (r = 0.11). CONCLUSIONS: The current study demonstrated an absence of relationship between ZP-BF high/positive or low/negative score and nuclear and cytoplasmic in vitro maturation of oocytes from stimulation cycles.

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 µm and width: 3.28+/-0.20 µm, 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.

Administration of single-dose GnRH agonist in the luteal phase in ICSI cycles: a meta-analysis.

BACKGROUND: The effects of gonadotrophin-releasing hormone agonist (GnRH-a) administered in the luteal phase remains controversial. This meta-analysis aimed to evaluate the effect of the administration of a single-dose of GnRH-a in the luteal phase on ICSI clinical outcomes. METHODS: The research strategy included the online search of databases. Only randomized studies were included. The outcomes analyzed were implantation rate, clinical pregnancy rate (CPR) per transfer and ongoing pregnancy rate. The fixed effects model was used for odds ratio. In all trials, a single dose of GnRH-a was administered at day 5/6 after ICSI procedures. RESULTS: All cycles presented statistically significantly higher rates of implantation (P<0.0001), CPR per transfer (P=0.006) and ongoing pregnancy (P=0.02) in the group that received luteal-phase GnRH-a administration than in the control group (without luteal-phase-GnRH-a administration). When meta-analysis was carried out only in trials that had used long GnRH-a ovarian stimulation protocol, CPR per transfer (P=0.06) and ongoing pregnancy (P=0.23) rates were not significantly different between the groups, but implantation rate was significant higher (P=0.02) in the group that received luteal-phase-GnRH-a administration. On the other hand, the results from trials that had used GnRH antagonist multi-dose ovarian stimulation protocol showed statistically significantly higher implantation (P=0.0002), CPR per transfer (P=0.04) and ongoing pregnancy rate (P=0.04) in the luteal-phase-GnRH-a administration group. The majority of the results presented heterogeneity. CONCLUSIONS: These findings demonstrate that the luteal-phase single-dose GnRH-a administration can increase implantation rate in all cycles and CPR per transfer and ongoing pregnancy rate in cycles with GnRH antagonist ovarian stimulation protocol. Nevertheless, by considering the heterogeneity between the trials, it seems premature to recommend the use of GnRH-a in the luteal phase. Additional randomized controlled trials are necessary before evidence-based recommendations can be provided.

Motile sperm organelle morphology examination (MSOME): intervariation study of normal sperm and sperm with large nuclear vacuoles.

BACKGROUND: Although the motile sperm organelle morphology examination (MSOME) was developed only as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in evaluation of semen quality, with potential clinical repercussions. The present study aimed to evaluate individual variations in the motile sperm organelle morphology examination (MSOME) analysis after a time interval. METHODS: Two semen samples were obtained from 240 men from an unselected group of couples undergoing infertility investigation and treatment. Mean time interval between the two semen evaluations was 119+/-102 days. No clinical or surgical treatment was realized between the two observations. Spermatozoa were analyzed at greater than or equal to 8400x magnification by inverted microscope equipped with DIC/Nomarski differential interference contrast optics. At least 200 motile spermatozoa per semen sample were evaluated and percentages of normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV/one or more vacuoles occupying>50% of the sperm nuclear area) were determined. A spermatozoon was classified as morphologically normal when it exhibited a normal nucleus (smooth, symmetric and oval nucleus, width 3.28+/-0.20 microm, length 4.75+/-0.20 microm/absence of vacuoles occupying>4% of nuclear area) as well as acrosome, post-acrosomal lamina, neck and tail, besides not presenting cytoplasm around the head. One examiner, blinded to subject identity, performed the entire study. RESULTS: Mean percentages of morphologically normal and LNV spermatozoa were identical in the two MSOME analyses (1.6+/-2.2% vs. 1.6+/-2.1% P=0.83 and 25.2+/-19.2% vs. 26.1+/-19.0% P=0.31, respectively). Regression analysis between the two samples revealed significant positive correlation for morphologically normal and for LNV spermatozoa (r=0.57 95% CI:0.47-0.65 P<0.0001 and r=0.50 95% CI:0.38-0.58 P<0.0001, respectively). CONCLUSIONS: The significant positive correlation and absence of differences between two sperm samples evaluated after a time interval with respect to normal morphology and LNV spermatozoa indicated that MSOME seems reliable (at least for these two specific sperm forms) for analyzing semen. The present result supports the future use of MSOME as a routine method for semen analysis.

Single-embryo transfer reduces clinical pregnancy rates and live births in fresh IVF and Intracytoplasmic Sperm Injection (ICSI) cycles: a meta-analysis.

BACKGROUND: It has become an accepted procedure to transfer more than one embryo to the patient to achieve acceptable ongoing pregnancy rates. However, transfers of more than a single embryo increase the probability of establishing a multiple gestation. Single-embryo transfer can minimize twin pregnancies but may also lower live birth rates. This meta-analysis aimed to compare current data on single-embryo versus double-embryo transfer in fresh IVF/ICSI cycles with respect to implantation, ongoing pregnancy and live birth rates. METHODS: Search strategies included on-line surveys of databases from 1995 to 2008. Data management and analysis were conducted using the Stats Direct statistical software. The fixed-effect model was used for odds ratio (OR). Fixed-effect effectiveness was evaluated by the Mantel Haenszel method. Seven trials fulfilled the inclusion criteria. RESULTS: When pooling results under the fixed-effect model, the implantation rate was not significantly different between double-embryo transfer (34.5%) and single-embryo transfer group (34.7%) (P = 0.96; OR = 0.99, 95% CI 0.78, 1.25). On the other hand, double-embryo transfer produced a statistically significantly higher ongoing clinical pregnancy rate (44.5%) than single-embryo transfer (28.3%) (P < 0.0001; OR:2.06, 95% CI = 1.64,2.60). At the same time, pooling results presented a significantly higher live birth rate when double-embryo transfer (42.5%) (P < 0.001; OR: 1.87, 95% CI = 1.44,2.42) was compared with single-embryo transfer (28.4%). CONCLUSION: Meta-analysis with 95% confidence showed that, despite similar implantation rates, fresh double-embryo transfer had a 1.64 to 2.60 times greater ongoing pregnancy rate and 1.44 to 2.42 times greater live birth rate than single-embryo transfer in a population suitable for ART treatment.

Effects of recombinant LH supplementation to recombinant FSH during induced ovarian stimulation in the GnRH-agonist protocol: a matched case-control study.

BACKGROUND: Some studies have suggested that the suppression of endogenous LH secretion does not seem to affect the majority of patients who are undergoing assisted reproduction and stimulation with recombinant FSH (r-FSH). Other studies have indicated that a group of normogonadotrophic women down-regulated and stimulated with pure FSH preparations may experience low LH concentrations that compromise the IVF parameters. The present study aimed to compare the efficacy of recombinant LH (r-LH) supplementation for controlled ovarian stimulation in r-FSH and GnRH-agonist (GnRH-a) protocol in ICSI cycles. METHODS: A total of 244 patients without ovulatory dysfunction, aged <40 years and at the first ICSI cycle were divided into two groups matched by age according to an ovarian stimulation scheme: Group I (n = 122): Down-regulation with GnRH-a + r-FSH and Group II (n = 122): Down-regulation with GnRH-a + r-FSH and r-LH (beginning simultaneously). RESULT(S): The number of oocytes collected, the number of oocytes in metaphase II and fertilization rate were significantly lower in the Group I than in Group II (P = 0.036, P = 0.0014 and P = 0.017, respectively). In addition, the mean number of embryos produced per cycle and the mean number of frozen embryos per cycle were statistically lower (P = 0.0092 and P = 0.0008, respectively) in Group I than in Group II. Finally the cumulative implantation rate (fresh+thaw ed embryos) was significantly lower (P = 0.04) in Group I than in Group II. The other clinical and laboratory results analyzed did not show difference between groups. CONCLUSION: These data support r-LH supplementation in ovarian stimulation protocols with r-FSH and GnRH-a for assisted reproduction treatment.

New therapeutic protocol for improvement of endometrial receptivity (PRIMER) for patients with recurrent implantation failure (RIF) - A pilot study.

OBJECTIVE: To evaluate whether or not one should use a new Protocol for Endometrial Receptivity Improvement (PRIMER) based on platelet-rich plasma (PRP) and granulocyte colony-stimulation factor (G-CSF) to enhance ongoing pregnancy rates in patients with recurrent implantation failure (RIF). METHODS: Women undergoing IVF/ICSI were prospectively divided into two groups: - PRIMER/RIF group (n:33): patients with RIF (defined as ≥2embryo transfers (ETs) and at least 5 morphologically good embryos transferred) in which intrauterine PRP injection and subcutaneous G-CSF-injection were performed. - Control group (n:33): patients in their first IVF/ICSI attempt/cycle (without PRP or G-CSF injection). The PRP was prepared using autologous fresh-whole blood processed to increase platelet-concentration in 2 to 4 fold. All patients undergoing the PRP-treatment received 0.7ml of it through intrauterine-injection 48 hours before the ET. G-CSF (300mg/0.5ml) started simultaneously to PRP and was administered subcutaneously every week. RESULTS: Regarding implantation, clinical pregnancy and miscarriage rates, we found no statistically significant difference (18.2% versus 17.6%, p=0.90; 36.4% versus 30.3%, p=0.61 and 25.0% versus 9.0%, p=0.43, respectively). The use of PRIMER enabled RIF patients (previous ET µ: 4.0±1.5) to reach similar ongoing pregnancy and live birth rates like those patients who had their first IVF/ICSI cycle attempt (27.3% versus 27.3%, p=0.99). CONCLUSIONS: Our results showed, for the first time, evidence that this therapeutic protocol (PRIMER) could be used as a feasible treatment based on biological rationale for patients with RIF, considering its promising outcomes, it is a simple procedure and not associated with patient complications.

Randomized comparison of two commercial culture media (Cook and Vitrolife) for embryo culture after IMSI.

OBJECTIVE: A variety of studies randomizing women/cycles or oocytes/embryos has been carried out to compare different culture media for culturing embryos up to cleavage or blastocyst stages showing controversial results. A recent systematic review suggested that data in the literature are insufficient to conclude the best culture medium for embryo quality, pregnancy and implantation. The objective of this study was to evaluate whether there is any difference between two commercial culture media regarding clinical outcomes after IMSI cycles. METHODS: A total of 120 patients, ≤39 years of age, undergoing ART treatment submitted to the IMSI program were prospectively broken down and randomized into two groups: Group I (Cook media) and Group II (Vitrolife media). RESULTS: Our data demonstrated that there was no difference using all the media from Cook or all the media from Vitrolife, for culturing embryos till day 2, in the bench incubator at low O2 concentration, in relation to fertilization, embryo quality, pregnancy and implantation rates (p>0.05). CONCLUSION: Both culture media used, Cook medium and Vitrolife medium, for the IMSI procedure and for later embryo culture with transfer on the second day, are equally effective and can be used depending on the ease and availability of acquisition.

The effects of male age on sperm DNA damage: an evaluation of 2,178 semen samples.

OBJECTIVE: This study aimed to evaluate the effects of male age on sperm DNA damage. METHODS: This cross-sectional study included semen samples collected from 2,178 men seen at an infertility clinic. For DNA integrity analysis, the proportions of spermatozoa showing DNA fragmentation (TUNEL assay), abnormal chromatin packaging/underprotamination (chromomycin A3), abnormal mitochondrial membrane potential (MMP/MitoTracker Green), and apoptosis (annexin V) were recorded. For group comparisons, enrolled subjects were divided into three groups based on their ages: ≤35 years; 36-44 years; and ≥45 years. The associations between age and sperm parameters were assessed using Spearman's rank correlation coefficient. RESULTS: Although aging did not affect sperm apoptosis (p>.05), sperm DNA fragmentation and MMP deteriorated significantly with age (p<.05). Chromatin packaging/protamination improved significantly with age (p<.05). CONCLUSION: Sperm DNA fragmentation worsened with age and was apparently associated with mitochondrial damage. The age-related increase in sperm DNA damage suggests that delaying childbearing, not only in women but also in men, might jeopardize a couple's reproductive capacity. The increase seen in chromatin packaging might represent a protective feature for DNA. However, additional studies must be performed to confirm the results concerning chromatin packaging/protamination.

Single versus sequential culture medium: which is better at improving ongoing pregnancy rates? A systematic review and meta-analysis.

This study aimed to evaluate if single medium is better than sequential medium at improving ongoing pregnancy rates in patients undergoing assisted reproductive technology (ART) procedures. The data featured in this meta-analysis were extracted from four randomized controlled trials yielded from a systematic search carried out on electronic databases. The primary endpoint was ongoing pregnancy rate. Secondary endpoints included clinical pregnancy and miscarriage rates. The endpoints for ongoing pregnancy rate were also analyzed based on the time at which the embryo transfers were performed: cleavage stage (day 2/3) and/or blastocyst stage (day 5/6). There were no significant differences between single and sequential medium for clinical pregnancy (RR=1.09; 95%CI=0.83-1.44; p=0.53), ongoing pregnancy (RR=1.11; 95%CI=0.87-1.40; p=0.39), or miscarriage rates (RR=0.89; 95%CI=0.44-1.81; p=0.74). No significant difference was found for ongoing pregnancy rate (RR=1.29; 95%CI=0.93-1.78; p=0.12) between single and sequential medium when only trials in which embryos were transferred at the blastocyst stage were included. In conclusion, the choice of embryo culture approach - single or sequential medium - did not affect the ongoing pregnancy rates of patients undergoing ART cycles.

Fresh embryos versus freeze-all embryos - transfer strategies: Nuances of a meta-analysis.

OBJECTIVE: The present meta-analysis aimed to evaluate whether the freeze-all strategy (Freeze/All-ET) could bring about improvements in the clinical assisted reproductive technique (ART) outcomes when compared with the fresh embryo transfer strategy (Fresh-ET) in patients undergoing an ART cycle in accordance with the mean number of oocytes collected. METHODS: A systematic review based on electronic searches in databases (PubMed, EMBASE, Web of Science, SCOPUS, and Cochrane Central Register of Controlled Trials) was carried out to identify randomized controlled trails (RCTs) comparing ART outcomes between fresh-embryo transfers versus elective frozen-embryo transfers up to February of 2017. Four reviewers independently evaluated abstracts, validity assessment and data extraction. Odds Ratio (OR) values with a 95% confidence interval (CI), and heterogeneity were evaluated. RESULTS: Five RCTs were included as targets for data extraction and meta-analysis purposes. The results of this meta-analysis were divided into two parts (Freeze/All-ET versus Fresh-ET): Part I- All trials in which the mean number of collected oocytes was >12 and <21 for ongoing pregnancy rate (OR=1.24; 95%CI=1.06-1.44), clinical pregnancy rate (OR=1.19; 95%CI=0.98-1.43), live birth rate (OR= 1.39; 95%CI=0.99-1.95), and miscarriage rate (OR=0.68; 95%CI=0.46-1.00); Part II- Three studies where the mean number of oocytes retrieved was >12 and <15 for ongoing pregnancy rate (OR=1.17; 95%CI=1.00-1.38), clinical pregnancy rate (OR=1.34; 95%CI=0.79-2.28), live birth rate (OR= 1.24; 95%CI=1.00-1.55), and miscarriage rate (RR=0.68; 95%CI=0.46-1.02). CONCLUSIONS: The freeze-all strategy could be favorable when high numbers of oocytes are collected, signaling an association between higher ovarian stimulation and consequent impairment of endometrial receptivity. However, when the mean number of oocytes collected is <15, the freeze-all strategy does not appear to be advantageous.