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PURPOSE: To examine the effect of co-incubating spermatozoa with human follicular fluid (HFF) on the rate of sperm DNA fragmentation. METHODS: This prospective study used semen (n = 23) and HFF from oocyte donors (n = 23). Liquified semen was divided into four aliquots: (1) neat semen (NEAT), (2) seminal plasma removed and replaced with sperm media (HTF) containing 0% (FF0), (3) 20% (FF20), or (4) 50% (FF50) HFF. Sperm motility and DNA fragmentation (SDF) were assessed following 24 h of incubation at 37 °C. Pro-oxidant capacity of HFF and seminal plasma and the effect of HFF on seminal plasma DNase activity was assessed in a sub-sample of 10 ejaculates. RESULTS: Sperm motility was higher after 3 h of incubation in media that contained HFF compared to the NEAT sample or when sperm was diluted in media without HFF. r-SDF (increase of SDF per time unit) values after 24 h of incubation for NEAT, FF0, FF20 and FF50 were 0.91, 0.69, 0.25 and 0.36, respectively. While pro-oxidant capacity of seminal plasma samples showed large variation (mean: 94.6 colour units; SD 65.4), it was lower and more homogeneous in FF samples (mean: 29.9 colour units; SD: 6.3). Addition of HFF to seminal plasma appeared to inhibit DNase activity. CONCLUSION: While differences exist in the pro-oxidant capacity of seminal plasma of patients, sperm DNA integrity was preserved with addition of HFF to sperm media, irrespective of the level of pro-oxidant capacity. DNase activity in the original seminal plasma was abolished after HFF co-incubation.

PURPOSE: To determine whether there is a homogeneous reduction of sperm DNA fragmentation (SDF) in sperm samples recovered from the MACS procedure, compared to spermatozoa in the initial ejaculate (NEAT) and those retained in the column. METHODS: This study investigated the relative change in sperm DNA quality (SDF) of neat ejaculates (10 idiopathic infertile and 10 normozoospermic patients) to subpopulations of spermatozoa that had passed through the column (MACS−) and those retained (MACS+) by the annexin-V conjugated microbeads. RESULTS: While the MACS protocol was capable of reducing the mean proportion of SDF (59.2%; P = 0.000) and sperm with highly degraded DNA (SDD; 65.7%, P = 0.000) in all patients, the reduction was not homogeneous across the patient cohort. A significant positive correlation (r = 0.772, P = 0.000) was apparent between the level of SDF in the NEAT ejaculate and the efficacy of SDF reduction observed in the MACS− fraction. CONCLUSION: MACS is capable of reducing the proportion of SDF, especially spermatozoa with a highly degraded DNA molecule. However, this reduction did not preclude the presence of a small subpopulation of spermatozoa with damaged DNA in the MACS− fraction. The MACS protocol was two- to threefold more efficient when the SDF in NEAT ejaculate was equal to or greater than 30%. In 4 of 20 individuals, the level of SDF after MACS resulted in semen for ICSI with a higher or non-significant reduction when compared to SDF observed in the NEAT ejaculate.

Intrauterine devices (IUDs) have been widely used to prevent pregnancies with great efficacy during decades. It has been demonstrated that IUD alters the endometrial gene expression, but there is no scientific data about how copper, a metal commonly used in these devices, by itself, is able to influence the processes of endometrial receptivity and apoptosis in decidualized human endometrial stromal cells. Five endometrial samples were obtained from fertile women and processed by a standard protocol to obtain human endometrial stromal cells for in vitro studies. Stromal cells were cultured in vitro and decidualized for 8 days. At day 6, copper was added to the treatment group or camptothecin as positive control for apoptosis until day 8. Five endometrial samples were used in each group. The aim of this study was to analyze the effect of copper in apoptosis and necrosis by flow cytometry, to visualize the apoptotic microtubule network during apoptosis by immunofluorescence, and finally to determine the gene expression profile of a panel of 192 genes related to endometrial receptivity and immune system by quantitative reverse transcription PCR (RT-qPCR). Copper, compared to the decidualized group, induced changes in the gene expression by an order of magnitude in 49 genes (42 up- and 9 downregulated). This alteration in the decidualization gene signature by copper includes 19 genes involved in the endometriosis pathology and others related to other gynecological disorders such as preeclampsia and infertility. Our results indicate that copper does not increase the apoptosis level induced by the decidualization treatment. However, copper alters the gene expression of some biomarkers of endometrial receptivity and immune response.

Purpose: Hydroxypropyl cellulose (HPC), a polysaccharide that forms a viscous gel under low temperatures, is a promising substitute of the blood-derived macromolecules traditionally used in cryopreservation solutions. The performance of a protein-free, fully synthetic set of vitrification and warming solutions was assessed in a matched pair analysis with donor oocytes. Methods: A prospective study including 219 donor MII oocytes was carried out, comparing the laboratory outcomes of oocytes vitrified with HPC-based solutions and their fresh counterparts. The primary performance endpoint was the fertilization rate. Secondary parameters assessed were embryo quality on days 2 and 3. Results: 70/73 (95.9%) vitrified MII oocytes exhibited morphologic survival 2 h post-warming, with 49 (70.0%) presented normal fertilization, compared to 105 of 146 (71.9%) MII fresh oocytes. Similar embryo quality was observed in both groups. A total of 18 embryos implanted, out of 38 embryos transferred (47.3%), resulting in 13 newborns.

OBJECTIVE To compare the results obtained with two different GnRH agonist dosages: 0.3mg versus 0.4mg to trigger ovulation in oocyte donor cycles. METHODS Experimental controlled randomized trial including 40 patients from a private practice center. The patients were randomized into two groups. Group A received a single dose of Triptorelin 0.3mg (Decapeptyl®) 36hours before pick-up. Group B patients received Triptorelin 0.4mg (Decapeptyl®) before pick-up to final oocyte maturation. We evaluated the total number of oocytes collected, the number of mature oocytes and total days of ovarian stimulation. RESULTS The average of total collected oocytes were 16 (Group A) versus 15 (Group B), and the mean number of mature oocytes were 13 versus 12 respectively. The only variable showing a difference was the percentage of mature oocytes, which was greater in Group A, resulting in 84.6%, in contrast with those treated with 0.4mg of Triptorelin (78.6%), although these differences were not statistical significant (p=0.35). Days of stimulation did not differ between groups. No cases of empty follicle syndrome were reported. CONCLUSIONS We found that an increase from 0.3 to 0.4mg of triptorelin in an oocyte donation program might not improve outcomes. Nevertheless, more studies might be necessary, not only in oocyte donors but in sterile women as well, to evaluate how GnRH agonist dosage could affect the results among other factors.

This case report describes a live birth after the fresh replacement of an embryo obtained from a spontaneously in vitro matured oocyte. The patient was subjected to controlled ovarian stimulation for IVF treatment, obtaining two oocytes. One was found to be immature at the time of denudation, at metaphase-I. This immature oocyte was kept in culture overnight in standard conditions along with the second oocyte - which was mature but failed to fertilize - spontaneously achieving metaphase-II, and was subjected to ICSI. The resulting embryo was replaced on the second day of development, producing a pregnancy that resulted in a healthy live birth. Post-denudation in vitro maturation could be considered as a tool to improve reproductive outcomes in selected patients, such as poor responders.

Although it was qualitatively pointed out by Fahy et al. (1984), the key role of the warming rates in non-equillibrium vitrification has only recently been quantitatively established for murine oocytes by Mazur and Seki (2011). In this work we study the performance of a closed vitrification device designed under the new paradigm, for the vitrification of human oocytes. The vitrification carrier consists of a main straw in which a specifically designed capillary is mounted and where the oocytes are loaded by aspiration. It can be hermetically sealed before immersion in liquid nitrogen for vitrification, and it is warmed in a sterile water bath at 37 °C. Measured warming rates achieved with this design were of 600.000 ºC/min for a standard DMEM solution and 200.000 ºC/min with the vitrification solution for human oocytes. A cohort of 143 donor MII sibling human oocytes was split into two groups: control (fresh) and vitrified with SafeSpeed device. Similar results were found in both groups: survival (97.1%), fertilization after ICSI (74.7% in control vs. 77.3% in vitrified) and good quality embryos at day three (54.3% in control vs. 58.1% in vitrified) were settled as performance indicators. The pregnancy rate was 3/6 (50%) for the control, 2/3 (66%) for vitrified and 4/5 (80%) for mixed transfers., The study received financial support from Fundación Ginemed, Junta de Andalucia (Excellence Project, 2008) and I.N.I.A. (RTA-2012).

Static assessment of sperm DNA Fragmentation (SDF at the time of ejaculation or sperm thawing when cryopreserved) and the dynamic assessment of SDF (SDF assessed after T2 hr, T6 hr and T24 hr of sperm thawing) were used to establish cut-off values associated with sperm donors when compared with closely related normozoospermic patients. Cryopreserved samples from donors revealed SDF levels two times lower in comparison with the patients. Donor sperm DNA exhibited a 2.5 times higher longevity when compared with the patients. Static values of SDF after thawing of approximately 11% identify the donors with a 71% of sensitivity and 84% specificity. With respect to the dynamic assessment, SDF increases of 2.3 per hr during the first 2 hr of incubation identify the donors with 70% of sensitivity and 66% of specificity. Creating the Rate of Combined Damage (RCD) defined as the product of SDF-T0 by the increase in the damage registered during the first 2 hr of incubation (r-SDF-T0-2 ), an index of RCD = 22.2 units has an identification capacity of donors with a 78% sensitivity and 77% specificity. Such cut-off values could be used to characterise donors with high chromatin resistance to damage when meeting the above-established criteria.

Objective To investigate the effectiveness of intracytoplasmic sperm injection (ICSI) using testicular sperm as a strategy to overcome infertility in men with high sperm DNA fragmentation (SDF). Design Prospective, observational, cohort study. Setting Private IVF centers. Patient(s) A total of 147 couples undergoing IVF-ICSI and day 3 fresh ETs whose male partner has oligozoospermia and high SDF. Intervention(s) Sperm injections were carried out with ejaculated sperm (EJA-ICSI) or testicular sperm (TESTI-ICSI) retrieved by either testicular sperm extraction (TESE) or testicular sperm aspiration (TESA). SDF levels were reassessed on the day of oocyte retrieval in both ejaculated and testicular specimens. Main Outcome Measure(s) Percentage of testicular and ejaculated spermatozoa containing fragmented DNA (%DFI) and clinical pregnancy, miscarriage, and live-birth rates. Result(s) The %DFI in testicular sperm was 8.3%, compared with 40.7% in ejaculated sperm. For the TESTI-ICSI group versus the EJA-ICSI group, respectively, the clinical pregnancy rate was 51.9% and 40.2%, the miscarriage rate was 10.0% and 34.3%, and the live-birth rate was 46.7% and 26.4%. Conclusion(s) ICSI outcomes were significantly better in the group of men who had testicular sperm used for ICSI compared with those with ejaculated sperm. SDF was significantly lower in testicular specimens compared with ejaculated counterparts. Our results suggest that TESTI-ICSI is an effective option to overcome infertility when applied to selected men with oligozoospermia and high ejaculated SDF levels.

This study was conducted to test the hypothesis that continuous epididymal sperm depletion after recurrent ejaculations (REC) in contrast to a period of abstinence (ABS) results in a decreased level of sperm DNA fragmentation (SDF) and a consequent increased rate of pregnancy. Forty couples undergoing intra-cytoplasmic injection (ICSI) were asked to abstain from ejaculation for a period of 4 days and then ejaculate once per day for a period of 4 days, followed by a period of abstinence for 12 hours; sperm samples obtained after ABS and REC were assessed for volume, concentration, motility, and SDF and compared in 25 of the patients. Additionally, and in a different experiment, the pregnancy rate of this experimental group (40 couples) was compared to a control group of 150 couples in which the males had abstained from ejaculation for 4 days prior to ejaculation. Sperm selection was performed using density gradient centrifugation prior to ICSI. Semen quality in the REC group that was assessed over the course of the ejaculation schedule showed a decrease in semen volume (67%) and SDF (27%) following sperm selection; there was no difference for sperm motility or sperm concentration. When the pregnancy rate between the 40 couples in the REC group and 150 couples in the control ABS group were compared, the REC group had a pregnancy rate of 56.4% (25/40), whereas the ABS rate was only 43.3% (65/150) (p = 0.030). We conclude that recurrent ejaculation every 24 hours for four days with a final abstinence of 12 hours, combined with sperm selection using density gradient centrifugation, produces a significant increase in pregnancy rate when using ICSI. As ICSI was the strategy selected for fertilization, we propose that the observed reduction in SDF was the primary factor leading to improved reproductive outcome.

Summary Sperm quality was assessed in normozoospermic human (n = 10) and Spanish breed stallion (n = 10) after sperm fractionation during ejaculation. The first ejaculated fraction was separated from the second. A third sample was reconstituted using equivalent proportion of both fractions (RAW). Fraction 1, Fraction 2 and RAW semen were incubated for 30 min at 37°C to homogenise the impact of iatrogenic damage between both species. Sperm concentration, motility and sperm DNA damage were assessed in each fraction and RAW semen. The results showed two important facts: (i) spermatozoa confined at Fraction 1 exhibit superior parameters than those included at Fraction 2 in both species, and (ii) there is a certain level of concordance between species in the proportion of benefit observed when Fraction 1 is compared to RAW semen. Altogether, these results call into question whether the standard practice of whole ejaculate collection can be considered the best strategy when using male gametes for artificial insemination. In fact, the reconstituted RAW semen exhibits poorer semen characteristics than those found in Fraction 1.

Objective To evaluate the relationship between duration of sexual abstinence and sperm selection on sperm DNA fragmentation (SDF). Design Prospective study based on normozoospermic individuals. Setting Fertility and IVF unit and university unit for research. Patient(s) Two cohorts of normozoospermic individuals: 21 men (aged 25–35 years) attending a clinic and with clearly adverse female factors; and a group of 12 selected donors (aged 20–25 years). Intervention(s) SDF assessment using the sperm chromatin the dispersion test (Halosperm) in two cohorts of normozoospermic men. Main Outcome Measure(s) SDF assessment after 24 hours of abstinence with recurrent ejaculation (one every 24 hours) using neat sperm samples; and SDF assessment before and after sperm selection with abstinence of 3 hours. Result(s) Lower baseline levels of SDF were observed after shorter periods of abstinence between ejaculations (24 hours and 3 hours) than those recommended. This effect is much more marked after quick repetitive ejaculation (3 hours of abstinence) and sperm selection. Conclusion(s) The present results challenge the role of abstinence in current male infertility treatments and suggest that SDF can be efficiently reduced by a biological practice consisting of short-term recurrent ejaculation coupled with effective sperm selection.