DNase activity in human seminal plasma and follicular fluid and its inhibition by follicular fluid chelating agents.

RESEARCH QUESTION: What is the mechanism by which human follicular fluid inhibits seminal plasma DNase activity? DESIGN: Human genomic DNA was incubated with human follicular fluid and seminal plasma (reaction mixture) under different experimental conditions; increasing volumes of human follicular fluid; proteinase K digested or heat inactivated human follicular fluid; and the addition of Ca2+ or Mg2+ to the reaction mixture. RESULTS: Increasing volume of human follicular fluid resulted in a dose-dependent inhibition of seminal plasma DNase activity. Inhibition was not caused by proteins in the human follicular fluid as digestion with proteinase K or heat inactivation of human follicular fluid failed to abolish its inhibitory effect. Addition of divalent cations resulted in a reversion of the inhibitory effect, providing evidence that human follicular fluid inhibition of seminal plasma DNase activity seems to be mediated by a compound with chelating activity. Furthermore, incubation of genomic DNA with human follicular fluid in the presence of divalent cations served to elicit the existence of DNase activity. CONCLUSIONS: Human follicular fluid seems to contain a molecule or molecules with chelating capacity that inhibits DNase activity of both follicular fluid and seminal plasma. Our findings provide new insight to understanding sperm preservation and the physiology of fertilization biology.

Co-incubation of spermatozoa with human follicular fluid reduces sperm DNA fragmentation by mitigating DNase activity in the seminal plasma.

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.

Magnetic-activated cell sorting is not completely effective at reducing sperm DNA fragmentation.

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.

Magnetic cell sorting of semen containing spermatozoa with high DNA fragmentation in ICSI cycles decreases miscarriage rate.

This retrospective cohort study investigated whether reproductive outcome could be improved in couples presenting with a high level of sperm DNA fragmentation (SDF) by treating the ejaculate with the magnetic cell sorting (MACS) sperm selection procedure in combination with prior density gradient centrifugation (DGC). Only men presenting with ≥30% sperm DNA in the ejaculate were included because these patients can be potentially treated with MACS to reduce the proportion of sperm presenting DNA damage. In total, 305 couples were included in this study, and from these, 216 women underwent autologous ICSI (AUTO-ICSI), whereas the remaining 89 participated in oocyte donor ICSI (DONOR-ICSI). Ejaculates were collected and DGC treated with and without MACS. Live birth and miscarriage rates resulting from ICSI observed after clinical pregnancy were determined. Sperm selection using DGC or a combination of DGC and MACS did not show any statistical difference with respect to live birth rate of couples undergoing either AUTO-ICSI or DONOR-ICSI, irrespective of whether the couples had a moderate (≥30 to <50%) or high (≥50%) level of SDF. Remarkably, there was no evidence of miscarriage in either cohort of patients (AUTO-ICSI or DONOR-ICSI) following the MACS procedure.

Hydroxypropyl cellulose supplementation in vitrification solutions: a prospective study with donor oocytes.

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.

Thermal and clinical performance of a closed device designed for human oocyte vitrification based on the optimization of the warming rate.

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.

Intra-individual variation of sperm DNA fragmentation in the Human ejaculate.

This retrospective study assessed the biological intra-individual variability of the percentage of sperm with DNA damage (SDF) observed in subsequent ejaculates of the same individual. Variation in SDF was analyzed using the Mean Signed Difference (MSD) statistic based on 131 individuals, comprising 333 ejaculates. Either two, three or four ejaculates were collected from each individual. With this cohort of individuals two main questions were addressed; (1) does the number of ejaculates analyzed influence the variability in the level of SDF associated with each individual? and (2) is the variability observed in SDF similar when individuals are ranked according to their level of SDF? Results showed that the variation observed in mean SDF was not different when 2, 3 or 4 ejaculates were analyzed; consequently, we suggest that the assessment of SDF based on two ejaculates is likely to be representative of the mean SDF expected for the individual. In parallel, it was determined that the variation in SDF increased as SDF increased; in individuals presenting with an SDF value of lower than 30% (potentially fertile), only 5% possessed levels of MSD that could be considered as variable as that presented by individuals presenting with a recurrent high SDF. Finally, we showed that a single assessment of SDF in individuals with medium SDF (20-30%) was less likely to be predictive of the SDF value in the next ejaculate, and therefore, less informative of the patient's SDF status.

GPX3 Overexpression in Cumulus Cells Entails a Poor Prognosis for Uterine Implantation of Morphotype A Embryos.

Morphological embryo quality is an accurate prognostic tool for the success of assisted reproduction implantation, although complete certainty cannot be guaranteed. The transcriptome of the cumulus cells could be monitored as a faithful reflex of the physiological state of the oocytes, given the molecular crosstalk between both types of cells. Here, we compare the expression of specific genes related to oocyte competence, such as hyaluronic acid synthase 2 (HAS2), cell division control protein 42 (CDC42), connexin 43 (CX43), and glutathione peroxidase 3 (GPX3), in cumulus cells from implanted versus non-implanted embryos in 25 women, using RT-qPCR. After embryo transfer, two cohorts were differentiated: the pregnant group (women with the implantation of 100% of embryos transferred) versus the non-pregnant group (with an absence of embryo implantation), aiming to compare the possible differential expression of the selected genes in the cumulus cells of embryos from each group. HAS2, CDC42 and CX43 did not reveal differential expression between the two cohorts. However, GPX3 showed significantly reduced expression in the cumulus belonging to the pregnant group. Interestingly, even cumulus cells belonging only to morphotype A embryos showed a significantly lower expression of GPX3 in the pregnancy group. GPX3 overexpression in cumulus cells could be a poor prognostic indicator of implantation, discriminating beyond the capacity of the morphokinetic score. Unveiling the cumulus transcriptome could improve successful implantation in assisted reproduction treatments.

Decreased Expression of Sam68 Is Associated with Insulin Resistance in Granulosa Cells from PCOS Patients.

BACKGROUND AND OBJECTIVE: Polycystic ovary syndrome (PCOS) is a complex metabolic disorder associated with ovulatory dysfunction, hyperandrogenism, obesity, and insulin resistance, which leads to subfertility. PCOS is the most frequent metabolic disorder in women and the major cause of infertility. Susceptibility to developing PCOS is determined by a complex interaction between environmental and genetic factors. Although different mechanisms have been proposed to explain PCOS manifestations, defects in insulin actions or in the insulin signaling pathways are central in the pathogenesis of the syndrome. However, the mechanisms (molecular players and signaling pathways) underlying its primary origin still remain an unsolved issue. Current research is increasingly focusing on the discovery of novel biomarkers to further elucidate the complex pathophysiology of PCOS. Sam68, an RNA-binding protein, is recruited to insulin signaling, mediating different insulin actions. We aimed to investigate the role of Sam68 in insulin signaling and the possible implications of Sam68 in the insulin resistance in PCOS. MATERIALS AND METHODS: Granulosa cells were taken from women with PCOS (n = 25) and healthy donors (n = 25) and, within the age range of 20 to 42 years, from GINEMED, Assisted Reproduction Centre, Seville, Spain. The Sam68 expression level was analyzed both by qPCR and immunoblot. Statistical significance was assessed by one-way ANOVA, followed by a post-hoc test. A p value of < 0.05 was considered statistically significant. RESULTS: We found that insulin stimulation increases the phosphorylation and expression level of Sam68 in granulosa cells from normal donors. The downregulation of Sam68 expression resulted in a lower activation of both the MAPK and the PI3K pathways in response to insulin. Moreover, the granulosa cells from the women with PCOS presented a lower expression of Sam68, as well as insulin receptor and insulin receptor substrate-1 (IRS-1). In these cells, the overexpression of Sam68 resulted in an increased activation of both the MAPK and the PI3K pathways in response to insulin. CONCLUSIONS: These results suggest the participation of Sam68 in insulin receptor signaling, mediating the insulin effect in granulosa cells, and they suggest the possible role of Sam68 in the insulin resistance of PCOS.

The Effect of Copper on Endometrial Receptivity and Induction of Apoptosis on Decidualized Human Endometrial Stromal Cells.

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.

Live birth after replacement of an embryo obtained from a spontaneously in vitro matured metaphase-I oocyte.

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.

The influence of placental perfusion on birthweight in women with gestational diabetes.

OBJECTIVE: To evaluate the influence of both uterine and umbilical arteries Doppler pulsatility indexes (PI) and metabolic control on birthweight in pregnant women with gestational diabetes mellitus. METHODS: One hundred sixty-nine women with gestational diabetes were evaluated. Doppler measurements of umbilical artery and mean uterine arteries PI were recorded and the corresponding Z-score values by gestational age calculated. Maternal pregestational body mass index (BMI) and the levels of glycosylated hemoglobin were also recorded. The relationships between these studied variables and customised birthweight centiles according to sex and gestational age were analyzed using Spearman's correlation coefficient and linear regression. RESULTS: There was a significant correlation between birthweight centiles and Z-score values of the umbilical artery PI (r = -0.25, p = 0.001), but not with the Z-score values of the uterine artery PI (r = -0.12, p = 0.43). Third trimester maternal glycosylated hemoglobin was also positively correlated to birthweight (r = 0.29, p = 0.01). When using stepwise linear regression both maternal glycosylated hemoglobin and the Z-score of umbilical artery PI were included as independent variables in the predictive model of birthweight centile (p = 0.0002, p = 0.001 respectively, R(2)( )= 0.27). CONCLUSIONS: Umbilical artery PI predicts birthweight in women with gestational diabetes. However, metabolic control is the only important determinant of fetal macrosomia in these mothers.

Comparison of reproductive outcome in oligozoospermic men with high sperm DNA fragmentation undergoing intracytoplasmic sperm injection with ejaculated and testicular sperm.

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.

[Nurses as a support to improve the quality of life during assisted reproduction]. / La enfermera como apoyo en la calidad de vida en la reproducción asistida. (corrected).

OBJECTIVE: The aim of this study was to measure how infertility and assisted reproduction treatments (including artificial insemination) could affect the quality of life, and to evaluate how nurses could be helpful in this process, by alleviating anxiety and increasing the quality of life. METHOD: A cross-sectional observational study was conducted on 48 patients (26 cycles) in an Assisted Reproduction Unit from 2nd December 2013 to 30th April 2014. Socio-demographic data were obtained, with the quality of life being assessed using the FertiQoL questionnaire before and after the treatment, and the consultations with a nurse by telephone or e-mail of these patients were also analyzed. RESULTS: The study results show a decreased quality of life in these patients, which was worse in men and in couples who had no previous children. Patient-centered care improved quality of life and tolerability to the assisted reproduction treatment. Patients frequently telephoned the nurse to solve their doubts and problems. CONCLUSION: The present study suggests that nurses can play an important role in improving the quality of life of patients undergoing assisted reproduction treatment.

Increased pregnancy after reduced male abstinence.

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.

Shorter abstinence decreases sperm deoxyribonucleic acid fragmentation in ejaculate.

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 dispersión 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.

Two sets of monozygotic twins after intracytoplasmic sperm injection and transfer of two embryos on day 2.

OBJECTIVE: To report a case of quadruple gestation (two sets of monozygotic twins) after intracytoplasmic sperm injection (ICSI) and transfer of two embryos. DESIGN: Case report. SETTING: Tertiary clinical and academic medical center. PATIENT(S): Thirty-four-year-old patient who underwent an ICSI cycle. INTERVENTION(S): After 7 years of primary sterility for andrologic subfertility, the patient underwent an ICSI cycle, with transfer of two embryos on day 2. MAIN OUTCOME MEASURE(S): Transvaginal sonogram performed at the 36th day of gestational age showed a quadruple gestation (dichorionic-quadramniotic). RESULT(S): After receiving extensive counseling, the couple decided to proceed to a nonselective reduction of a set of monozygotic twins. Final outcome was complete loss of pregnancy. CONCLUSION(S): Several factors have been involved in the etiopathogenesis of monozygotic twins in assisted reproductive technology: maternal age, extended embryo culture and in vitro culture condition, blastocyst-stage transfer, ICSI, and assisted hatching. One of the most important objectives in assisted reproductive technology is to reduce the multiple-gestation rate; therefore, it is necessary to determine the optimum number of embryos to be transferred in each case. In addition, couples must be informed that monozygotic-twin pregnancies could be an important complication in IVF-ICSI cycles.