Early Spontaneous Twinning Recorded By Time-Lapse.

Monozygotic twins (MZT) are 2.5 times more frequent in ART than in natural conceptions. A number of ART-related mechanisms have been probably linked with MZT. Studies that retrospectively analyze the time-lapse (TL) records resulting in MZT suggest that some morphokinetic traits of the inner cell mass and the trophectoderm could be predictors of MZT, but results are controversial. We present the complete TL record of one case of MZT that split itself at the very moment of the division into two cells, with one of the cells coming out through a hole in the zona pellucida (ZP). Both resulting embryos developed normally, and were vitrified. It is suggested that the hole in the ZP may facilitate the extrusion of some cells of the

Sperm aminopeptidase N identifies the potential for high-quality blastocysts and viable embryos in oocyte-donation cycles.

STUDY QUESTION: Is there a relationship between human sperm aminopeptidase N (APN) and embryo development in humans? SUMMARY ANSWER: Human sperm APN could possibly become a new molecular biomarker for identifying the potential for high-quality and usable embryos. WHAT IS KNOWN ALREADY: The diagnosis of male fertility is one of the major concerns of reproductive medicine. Approximately 30-40% of men with otherwise normal fertility parameters are still unable to achieve pregnancy. The predictive clinical value of semen analysis to identify fertile or infertile males is limited; therefore, new diagnostic methodologies for sperm are urgently required. Sperm APN may be a relevant molecular marker due to its high concentration in sperm cells and its important roles in sperm physiology, such as its functions in motility, acrosome reaction and embryo development. STUDY DESIGN, SIZE, DURATION: This study included 81 couples who underwent oocyte-donation cycles at Clínica IVI Bilbao (Spain), yielding 611 embryos, between September 2014 and July 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted in an assisted reproduction unit and an academic research laboratory. All the semen samples were examined and classified following World Health Organization guidelines. Spermatozoa were isolated from semen using the discontinuous colloidal silica gradient (45-90%) technique. Embryo quality and development were determined according to the Spanish Association of Reproduction Biology Studies (ASEBIR) criteria. Human sperm APN levels were analyzed by quantitative and semiquantitative flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: The most well-developed and usable blastocysts were associated with low sperm APN levels. Semen samples that had lower APN levels generated more expanded, hatched and usable blastocysts and fewer early, arrested and non-usable blastocysts. The cumulative probability of having well-developed blastocysts increased by 1.38-fold at Day 5 and 1.90-fold at Day 6 of embryo development, and the likelihood of having usable embryos increased by 1.48-fold, when semen samples with low APN levels were used during the ICSI technique. LIMITATIONS, REASONS FOR CAUTION: The data were obtained from a single fertility clinic. A multicentre study will be required to confirm the results. WIDER IMPLICATIONS OF THE FINDINGS: Human sperm APN has the potential to become a new molecular biomarker to help identify the potential for high-quality embryos and diagnose male infertility, especially when seminal parameters are close to the threshold values. It could be a crucial tool for couples for whom the number of usable blastocysts is critical for ART success. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Basque Government (GIC15/165) and the University of the Basque Country (UPV/EHU) (EHUA14/17). The authors declare that they have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

A propensity score-based, comparative study assessing humid and dry time-lapse incubation, with single-step medium, on embryo development and clinical outcomes.

STUDY QUESTION: Does culture in a high relative humidity atmosphere improve clinical outcomes when using a time-lapse integrated incubator and single-step culture medium? SUMMARY ANSWER: Using an integrated time-lapse system and single-step culture medium, culture in a high relative humidity atmosphere increases the likelihood of embryos, especially those subjected to preimplantation genetic testing for aneuploidies, to achieve a pregnancy compared to those cultured in dry conditions. WHAT IS KNOWN ALREADY: The use of a humid atmosphere inside incubators can reduce changes in culture media osmolality, which has been reported to have a significant effect on embryo quality and morphokinetics. Studies assessing the effect of humid culture (HC) in clinical outcomes are, however, scarce and inconclusive, mostly due to a high variability in culture conditions and reduced sample size. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study performed over 1627 ICSI cycles performed during 3 consecutive years in which embryo cohorts were cultured in a time-lapse incubator with three dry and three humidified chambers, and using single-step culture medium. Clinical outcomes were compared between treatments in which embryo cohorts were cultured in either humid (n = 833) or dry (n = 794) conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study includes autologous treatments, with (N = 492) and without (N = 372) preimplantation genetic testing for aneuploidies (PGT-A) and ovum donation treatments (N = 763), performed in three university-affiliated private IVF centres. Stimulation, oocyte pickup and fertilization were performed according to the standard procedures of the clinic. All embryo cohorts were cultured in the same model of time-lapse incubator, distributed to either a dry or humidified chamber, while the rest of the culture variables remained equal. The population was weighted by the inverse probability of treatment to control for all measured confounders. The association between HC and the main outcome was assessed by logistic regression over the weighted population. The E-value was reported as a way of considering for unmeasured confounders. Differences in embryo development and other secondary outcomes between the study groups were assessed by Pearson Chi-squared test, ANOVA test and Kaplan-Meier survival analysis. MAIN RESULTS AND THE ROLE OF CHANCE: An univariable logistic regression analysis, weighted by the inverse probability of treatment, determined that embryos cultured in humid conditions are more likely to achieve a clinical pregnancy than those cultured in dry conditions (odds ratio (OR) = 1.236 (95% CI 1.009-1.515), P = 0.041, E = 1.460). Through stratification, it was determined that said effect is dependent on the type of treatment: no improvement in clinical pregnancy was present in ovum donation or autologous treatments, but a statistically significant positive effect was present in treatments with preimplantation genetic testing (OR = 1.699 (95% CI 1.084-2.663), P = 0.021, E = 1.930). Said increase does not relate with an improvement in later outcomes. Differences were also found in variables related to embryo developmental morphokinetics. LIMITATIONS, REASONS FOR CAUTION: The retrospective nature of the study makes it susceptible to some bias linked to the characteristics of the treatments. To lessen the effect of possible biases, cases were weighted by the inverse probability of treatment prior to the evaluation of the outcome, as means to assess for measured confounders. In addition, the E-value of the weighted OR was calculated as a sensitivity analysis for unmeasured confounders. A randomized prospective study could be performed for further assessing the effect of humid conditions in clinical outcome. WIDER IMPLICATIONS OF THE FINDINGS: These results support that embryo culture under conditions of high relative humidity contributes to optimize clinical results in undisturbed culture in a time-lapse incubator with single-step medium. To our knowledge, this is the largest study on the matter and the first performing a propensity score-based analysis. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the ''Centro para el Desarrollo Tecnologico Industrial'' from the Spanish Ministry of Science, Innovation, and Universities (CDTI-20170310) and Generalitat Valenciana and European Social Fund (ACIF/2019/264). None of the authors have any competing interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Evidence of Paraoxonases 1, 2, and 3 Expression in Human Ovarian Granulosa Cells.

Increasing evidence suggests that the antioxidant paraoxonase proteins, PON1, PON2, and PON3, have a role in reproduction and may be synthesized by ovarian cells. The aim of this work was to investigate whether human ovarian granulosa cells (GC) express paraoxonases 1, 2, and 3 (PON1, PON2, and PON3) at both the transcriptional and protein levels. Cells were purified from follicle samples of women undergoing ovarian stimulation at oocyte retrieval. We analyzed mRNA by polymerase chain reaction using specific primers for the different variants and quantified the proteins by Western blot using commercially available human recombinant PON proteins as standards. The protein subcellular distribution was determined by immunofluorescence and confocal microscopy and the cell cycles by flow cytometry. Thymidine was used for cellular synchronization at G1/S. Human hepatoma HepG2 and immortalized granulosa COV434 cell lines were used to optimize methodologies. mRNAs from PON1, the two variants of PON2, and PON3 were detected in GC. The cells actively secreted PON1 and PON3, as evidenced by the protein detection in the incubation medium. PON1 and PON3 were mainly distributed in the cytoplasm and notably in the nucleus, while PON2 colocalized with mitochondria. Subcellular nucleo-cytoplasmic distribution of PON1 was associated with the cell cycle. This is the first evidence describing the presence of mRNAs and proteins of the three members of the PON family in human ovarian GC. This study provides the basis of further research to understand the role of these proteins in GC, which will contribute to a better understanding of the reproduction process.

(Pro)renin Receptor Is Present in Human Sperm and It Adversely Affects Sperm Fertility Ability.

Sperm fertility ability may be modulated by different molecular systems, such as the renin-angiotensin system (RAS). Although renin is one of its most relevant peptides, the presence and role of the (pro)renin receptor (PRR) is completely unknown. We have proved for the first time the existence of PRR and its transcript in human sperm by western blot and RT-PCR. Immunofluorescence studies showed that this receptor is mainly located in the apical region over the acrosome and in the postacrosomal region of the sperm head and along the sperm tail. In addition, this prospective cohort study also proves that semen samples with higher percentages of PRR-positive spermatozoa are associated with poor sperm motility, worse blastocyst development and no-viable blastocysts. Our results provide insight into how PRR play a negative role in sperm physiology that it may condition human embryo quality and development. An in-depth understanding of the role of PRR in sperm fertility can help elucidate its role in male infertility, as well as establish biomarkers for the diagnosis or selection of sperm to use during assisted reproductive techniques.

Oocytes of women who are obese or overweight have lower levels of n-3 polyunsaturated fatty acids compared with oocytes of women with normal weight.

OBJECTIVE: To ascertain whether the oocytes of women who are obese or overweight have a different fatty acid (FA) profile than women with normal weight. DESIGN: Prospective case-control study. SETTING: Two IVF centers. PATIENT(S): A total of 205 women undergoing IVF and intracytoplasmic sperm injection (ICSI) were included in the study, totaling 922 oocytes. INTERVENTION(S): The unfertilized and the immature oocytes from the women who underwent IVF/ICSI were subjected to FA analysis with capillary gas chromatography. Women were classified according their body mass index (BMI) as normal, overweight, or obese. Germinal vesicle oocytes, metaphase I oocytes, and unfertilized metaphase II oocytes were analyzed separately. MAIN OUTCOME MEASURE(S): Fatty acid profile. RESULT(S): A very different oocyte FA pattern was observed for each BMI. Women with normal weight had higher levels of saturated FAs, and lower levels of monosaturated FAs. Women who were obese had lower levels of n-3 polyunsaturated FA, and the lowest n-6:n-3 ratios. Regarding specific FAs, docosahexaenoic acid levels were lower in women with normal weight than in those who are overweight, and in women who are overweight than in those who are obese. The opposite occurred with eicosapentaenoic acid, with the highest levels in women who have normal weight followed by those who are overweight and lower levels in those women who were obese. When FA analysis was restricted to a subset of oocytes, many of these differences persisted. CONCLUSION(S): Our study shows that oocytes from women who are obese or overweight have a different FA composition. This difference in levels could be related to the IVF poor outcome in these women. Therefore, this different composition could suggest that offspring of women who are obese or overweight have an unfavorable milieu even before conception.

Ovarian stimulated cycles reduce protection of follicular fluid against free radicals.

Controlled ovarian hyperstimulation cycle with exogenous gonadotropins (COH) is associated with clinical complications. The aim of this work was to determine whether COH alters the physiological antioxidant status of follicular fluid in women with no reproductive dysfunction, compared to the natural cycle (NC). In this longitudinal study, forty-one women (oocyte donors) consecutively underwent NC and COH. Follicular fluid was collected at oocyte retrieval and different redox biomarkers were determined: total antioxidant activity (TAA), oxygen radical absorbance capacity (ORAC), nitric oxide, α- and γ-tocopherol, the fatty acid composition, activities of superoxide dismutase, catalase, total and Se-dependent glutathione peroxidases, and the antioxidant paraoxonase (PON) family. Results showed that TAA (1.70 ±â€¯0.03 mM versus 1.86 ±â€¯0.03 mM, p < 0.05), α-tocopherol (4.37 ±â€¯0.26 µM versus 5.74 ±â€¯0.30 µM, p < 0.05), PON1 paraoxonase (245 ±â€¯24 nmol/min/ml versus 272 ±â€¯27 nmol/min/ml, p < 0.05), PON1 arylesterase (87.2 ±â€¯4.6 µmol/min/ml versus 99.3 ±â€¯4.8 µmol/min/ml, p < 0.05), and PON3 simvastatinase (13.48 ±â€¯0.52 nmol/min/ml versus 16.29 ±â€¯0.72 nmol/min/ml, p < 0.001) were significantly lower in COH versus NC. Fatty acids from COH were more saturated, increasing palmitate and decreasing the n-6 and total polyunsaturated fatty acids (PUFAs). Docosahexaenoic acid also increased (p < 0.05). Results suggest that COH could lead to premature ovarian aging and provide new insights into the possible prevention of the adverse effects of ovarian hyperstimulation by directing therapeutic applications to the maintenance of the redox balance and fatty acid status, with special attention to paraoxonase proteins and docosahexaenoic acid.

NGS Analysis of Human Embryo Culture Media Reveals miRNAs of Extra Embryonic Origin.

Our objective in this work was to isolate, identify, and compare micro-RNAs (miRNAs) found in spent culture media of euploid and aneuploid in vitro fertilization (IVF) embryos. Seventy-two embryos from 62 patients were collected, and their spent media were retained. A total of 108 spent conditioned media samples were analyzed (n = 36 day 3 euploid embryos, n = 36 day 3 aneuploid embryos, and n = 36 matched control media). Fifty hed-control media embryos were analyzed using next-generation sequencing (NGS) technology. We detected 53 known human miRNAs present in the spent conditioned media of euploid and aneuploid IVF embryos. miR-181b-5p and miR-191-5p were found the most represented. We validated our results by quantitative polymerase chain reaction (qPCR), but no significant results were obtained between the groups. In conclusion, we obtained the list of miRNAs present in the spent conditioned media from euploid and aneuploid IVF embryos, but our data suggest that these miRNAs could have a nonembryonic origin.

Lower follicular n-3 polyunsaturated fatty acid levels are associated with a better response to ovarian stimulation.

OBJECTIVES: To analyze in detail the fatty acid (FA) composition of follicular fluid (FF) from two-sized follicles at oocyte retrieval and to determine associations of the FAs from large follicles with the woman's age and the response to ovarian stimulation. DESIGN: Observational study. SETTING: University and fertility clinic. PATIENTS: Sixty-four women (age 19-46), consisting of unfertile patients and oocyte donors, undergoing controlled ovarian stimulation. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): FF from small (< 12 mm) and large (≥ 18 mm) follicles was collected at oocyte retrieval. FAs by gas chromatography-flame ionization detection. RESULT: Thirty-two FAs with chain lengths ranging from 14 to 25 carbons were identified. There was a readjustment in FA distribution as follicle size increased, raising very long-chain saturated FAs, nervonic (24:1n-9), arachidonic (20:4n-6), and n-3 polyunsaturated FAs (PUFA, P < 0.001), the latter mainly due to an increase in docosahexaenoic acid (22:6n-3, DHA). In large follicles, double bond and peroxidizability indices and total n-3 PUFA, particularly DHA, correlated positively with the woman's age and negatively with the number of total and mature oocytes, total and top-quality embryos, and fertilization rate. CONCLUSIONS: We have described 32 FAs in ovarian FF, of which 16 changed their distribution with follicle size. The results also indicate that lower n-3 PUFA levels in large follicles, which are associated with younger women, predict a better response to ovarian stimulation based on the recovery of total and mature oocytes, total and top-quality embryos, and fertilization rate per cycle. KEY MESSAGE: The fatty acid profile of ovarian FF changes as the follicle grows and lower n-3 PUFA levels in large follicles, associated with younger women, predict a better response to ovarian stimulation.

Analysis of Protein Oxidative Modifications in Follicular Fluid from Fertile Women: Natural Versus Stimulated Cycles.

Oxidative stress is associated with obstetric complications during ovarian hyperstimulation in women undergoing in vitro fertilization. The follicular fluid contains high levels of proteins, which are the main targets of free radicals. The aim of this work was to determine specific biomarkers of non-enzymatic oxidative modifications of proteins from follicular fluid in vivo, and the effect of ovarian stimulation with gonadotropins on these biomarkers. For this purpose, 27 fertile women underwent both a natural and a stimulated cycle. The biomarkers, glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), Nε-(carboxymethyl)lysine (CML), and Nε-(carboxyethyl)lysine (CEL), were measured by gas-liquid chromatography coupled to mass spectrometry. Results showed that follicular fluid contained products of protein modifications by direct metal-catalyzed oxidation (GSA and AASA), glycoxidation (CML and CEL), and lipoxidation (CML). GSA was the most abundant biomarker (91.5%). The levels of CML amounted to 6% of the total lesions and were higher than AASA (1.3%) and CEL (1.2%). In the natural cycle, CEL was significantly lower (p < 0.05) than in the stimulated cycle, suggesting that natural cycles are more protected against protein glycoxidation. These findings are the basis for further research to elucidate the possible relevance of this follicular biomarker of advanced glycation end product in fertility programs.

Human sperm testicular angiotensin-converting enzyme helps determine human embryo quality.

Angiotensin-converting enzyme functions in the male reproductive system, but the extent of its function in reproduction is not fully understood. The primary objective of this work was to investigate the relationship between the testicular isoform of angiotensin-converting enzyme present in human spermatozoa and semen parameters, human embryo quality, and assisted reproduction success. A total of 81 semen samples and 635 embryos from couples undergoing oocyte donation cycles at the IVI Bilbao Clinic were analyzed. Semen parameters, embryos quality, and blastocyst development were examined according to the World Health Organization standards and the Spanish Association of Reproduction Biology Studies criteria. The percentage of testicular angiotensin-converting enzyme-positive spermatozoa and the number of molecules per spermatozoon were analyzed by flow cytometry. Both parameters were inversely correlated with human sperm motility. Higher percentages of testicular angiotensin-converting enzyme-positive spermatozoa together with fewer enzyme molecules per spermatozoon were positively correlated with better embryo quality and development. Our results suggest that embryos with a higher implantation potential come from semen samples with higher percentages of testicular angiotensin-converting enzyme-positive cells and fewer enzyme molecules per spermatozoon. Based on these findings, we propose that testicular angiotensin-converting enzyme could be used to aid embryologists in selecting better semen samples for obtaining high-quality blastocysts during in vitro fertilization procedures.

Obstetric and perinatal outcomes of pregnancies conceived with embryos cultured in a time-lapse monitoring system.

OBJECTIVE: To compare obstetric and perinatal outcomes of singleton pregnancies resulting from embryos incubated in a time-lapse system (TLS) with those of embryos grown in standard IVF incubators (SI). DESIGN: Retrospective description of a cohort of patients who conceived during a randomized, controlled trial. SETTING: Private university-affiliated IVF center. PATIENT(S): Of 856 randomized patients, 378 gave birth to a live-born infant: 216 of the deliveries originated from embryos incubated in TLS, and 162 deliveries were from embryos cultured in SI. INTERVENTION(S): Embryo incubation and selection in TLS. MAIN OUTCOME MEASURE(S): Delivery and neonatal outcomes. RESULT(S): No significant differences were observed in the baseline characteristics of the study population. The delivery rate was 49.3% (TLS) vs. 40.0% (SI), and multiple deliveries were higher in the TLS group: 31.0% (67 of 216) vs. 24.7% (40 of 162) in the SI group. When singleton pregnancies were analyzed no differences were found between the two groups in the rate of obstetric problems with respect to weeks at delivery: 38.8 (95% confidence interval [CI] 38.4-39.1) (TLS) vs. 39.5 (95% CI 38.0-39.9) (SI); preterm births (<37 weeks): 10.7% (TLS) vs. 12.3% (SI); and very preterm births (<34 weeks): 2.9% (TLS) vs. 3.3% (SI). No statistical differences were found in neonatal outcomes such as birth weight: 3,163 g (95% CI 3,035-3,292 g) (TLS) vs. 3,074 (95% CI 2,913-3,236) (SI); low birth weight (<2,500 g): 12.8% (TLS) vs. 12.3% (SI); very low birth weight (<1,500 g): 2.0% (TLS) vs. 2.4% (SI); or height: 50.3 cm (95% CI 49.6-50.9 cm) (TLS) vs. 49.7 (95% CI 48.9-50.4 cm) (SI). No major malformations or perinatal mortality were found in either of the two groups. CONCLUSION(S): No detrimental effects were observed in obstetric and perinatal outcomes when a time-lapse incubator was used rather than a more widely used conventional incubator. As far as we know this is the first report from a randomized study of the neonatal outcomes of time-lapse monitoring. Our results suggest that this technology is an effective and safe alternative for embryo incubation, though trials of larger numbers of patients are required to further confirm our conclusions. CLINICAL TRIAL REGISTRATION NUMBER: NCT01549262.

Paraoxonase activities in human follicular fluid: role in follicular maturation.

The paraoxonases (PONs) are antioxidant enzymes associated with beneficial effects against several diseases and some exposures. Little is known, however, about the role of PONs in human reproduction. This work was conducted to investigate whether any association existed between the activities of the PON enzymes (1, 2, and 3) with the follicular size and fertility parameters in assisted reproduction. The study included 100 subfertile women (patients) and 55 proven fertile women (oocyte donors), all undergoing an ovarian stimulation cycle. Follicular fluid from small (diameter <12 mm) and large (diameter ≥18 mm) follicles was collected from each woman. The PONs were quantified in follicular fluid by immunoblotting. PON1 arylesterase and paraoxonase, PON2 methyl paraoxonase and PON3 simvastatinase activities from both donors and patients were significantly higher (P < 0.001) in follicular fluid from large follicles compared with small ones. In large follicles, PON3 activity was significantly higher (P < 0.01) in donors compared with patients. Follicular fluid PON1 arylesterase and paraoxonase activity was positively correlated with the number of retrieved oocytes in donors. This study shows an increase in the activities of PONs with follicle size, thus providing indirect evidence for the role of PONs in follicle maturation.

Biological effects of plasma rich in growth factors (PRGF) on human endometrial fibroblasts.

OBJECTIVES: To evaluate the biological outcomes of plasma rich in growth factors (PRGF) on human endometrial fibroblasts in culture. STUDY DESIGN: PRGF was obtained from three healthy donors and human endometrial fibroblasts (HEF) were isolated from endometrial specimens from five healthy women. The effects of PRGF on cell proliferation and migration, secretion of vascular endothelial growth factor (VEGF), procollagen type I and hyaluronic acid (HA) and contractility of isolated and cultured human endometrial fibroblasts (HEF) were analyzed. Statistical analysis was performed in order to compare the effects of PRGF with respect to control situation (T-test or Mann-Whitney U-test). RESULTS: We report a significantly elevated human endometrial fibroblast proliferation and migration after treatment with PRGF. In addition, stimulation of HEF with PRGF induced an increased expression of the angiogenic factor VEGF and favored the endometrial matrix remodeling by the secretion of procollagen type I and HA and endometrial regeneration by elevating the contractility of HEF. These results were obtained for all PRGF donors and each endometrial cell line. CONCLUSIONS: The myriad of growth factors contained in PRGF promoted HEF proliferation, migration and synthesis of paracrine molecules apart from increasing their contractility potential. These preliminary results suggest that PRGF improves the biological activity of HEF in vitro, enhancing the regulation of several cellular processes implied in endometrial regeneration. This innovative treatment deserves further investigation for its potential in "in vivo" endometrial development and especially in human embryo implantation.

Angiotensin II type 2 receptor is expressed in human sperm cells and is involved in sperm motility.

OBJECTIVE: To investigate the presence of angiotensin II type 2 receptor (AT2R) in human spermatozoa and its implication in sperm fertility status. DESIGN: We carried out expression assays for AT2R by reverse transcription-polymerase chain reaction, Western blot, immunofluorescence, and flow cytometry techniques in human sperm cells. Percentage of AT2R-positive sperm cells was analyzed by flow cytometry. SETTING: Assisted reproduction unit and academic research laboratory. PATIENT(S): Ninety-seven human semen samples from the Clínica IVI Bilbao. INTERVENTION(S): All samples were examined and classified according to World Health Organization guidelines. Spermatozoa were isolated from semen on discontinuous colloidal silica gradient (45%-90%) and swim-up techniques. MAIN OUTCOME MEASURE(S): Presence and location of the AT2R in spermatozoa and percentage of AT2R-positive sperm cells measured by flow cytometry. RESULT(S): We demonstrated the existence of AT2R and its transcript in human sperm by Western blot and reverse transcription-polymerase chain reaction. Immunofluorescence studies showed that AT2R is mainly located at the equatorial segment of the sperm head. The AT2R levels were associated with sperm motility parameters. Particularly, we found a significant positive correlation between AT2R and spermatozoa with progressive motility grade and a significant negative correlation with immotile spermatozoa, both in fresh semen samples and in prepared sperm cells. Regarding pathologic studies, the levels of AT2R measured by flow cytometry were lower in spermatozoa of asthenozoospermic men than in normozoospermic controls. CONCLUSION(S): Angiotensin II type 2 receptor is present in human semen and may be involved in the control of sperm motility. In-depth understanding of the proteins involved in sperm motility can help to elucidate the role of these proteins in male infertility as well as to establish new biomarkers for male infertility.

Clinical validation of embryo culture and selection by morphokinetic analysis: a randomized, controlled trial of the EmbryoScope.

OBJECTIVE: To determine whether incubation in the integrated EmbryoScope time-lapse monitoring system (TMS) and selection supported by the use of a multivariable morphokinetic model improve reproductive outcomes in comparison with incubation in a standard incubator (SI) embryo culture and selection based exclusively on morphology. DESIGN: Prospective, randomized, double-blinded, controlled study. SETTING: University-affiliated private in vitro fertilization (IVF) clinic. PATIENT(S): Eight hundred forty-three infertile couples undergoing intracytoplasmic sperm injection (ICSI). INTERVENTION(S): No patient intervention; embryos cultured in SI with development evaluated only by morphology (control group) and embryos cultured in TMS with embryo selection was based on a multivariable model (study group). MAIN OUTCOME MEASURE(S): Rates of embryo implantation, pregnancy, ongoing pregnancy (OPR), and early pregnancy loss. RESULT(S): Analyzing per treated cycle, the ongoing pregnancy rate was statistically significantly increased 51.4% (95% CI, 46.7-56.0) for the TMS group compared with 41.7% (95% CI, 36.9-46.5) for the SI group. For pregnancy rate, differences were not statistically significant at 61.6% (95% CI, 56.9-66.0) versus 56.3% (95% CI, 51.4-61.0). The results per transfer were similar: statistically significant differences in ongoing pregnancy rate of 54.5% (95% CI, 49.6-59.2) versus 45.3% (95% CI, 40.3-50.4) and not statistically significant for pregnancy rate at 65.2% (95% CI, 60.6-69.8) versus 61.1% (95% CI, 56.2-66.1). Early pregnancy loss was statistically significantly decreased for the TMS group with 16.6% (95% CI, 12.6-21.4) versus 25.8% (95% CI, 20.6-31.9). The implantation rate was statistically significantly increased at 44.9% (95% CI, 41.4-48.4) versus 37.1% (95% CI, 33.6-40.7). CONCLUSION(S): The strategy of culturing and selecting embryos in the integrated EmbryoScope time-lapse monitoring system improves reproductive outcomes. CLINICAL TRIAL REGISTRATION NUMBER: NCT01549262.

Quality of additional embryos transferred on pregnancy outcomes in IVF: predictions using a mathematical approach.

This study assessed the influence of adding embryos with different embryo quality on pregnancy rate and multiple pregnancy rate (MPR). The study included 1891 IVF transfers performed at two centres with different embryo transfer policies. Pregnancy rate and MPR were analysed following three models and then including embryo quality. A predictive mathematical model and two scatter plots were constructed. The model based on embryo independence was incompatible with the observed data, while both the ground and collaborative models provided excellent fits. The collaborative model, however, predicted multiple pregnancies, especially triplets, more accurately. Transfer of additional embryos, irrespective of embryo quality, always increased pregnancy rate and MPR. When implantation rate was low, there was a marked increase in pregnancy rate but only a relatively small increase in MPR. In contrast, with higher implantation rates, the increase in pregnancy rate was mainly due to the increase in MPR, with the same singleton pregnancy rate. Transfer of additional embryos, irrespective of embryo quality, follows a collaborative pattern and always results in an increase in pregnancy rate and MPR. The scatter plots accurately predicted the influence of the different combinations of number and embryo quality on pregnancy rate and MPR.

Glutathione S-transferase activity in follicular fluid from women undergoing ovarian stimulation: role in maturation.

Female infertility involves an emotional impact for the woman, often leading to a state of anxiety and low self-esteem. The assisted reproduction techniques (ART) are used to overcome the problem of infertility. In a first step of the in vitro fertilization therapy women are subjected to an ovarian stimulation protocol to obtain mature oocytes, which will result in competent oocytes necessary for fertilization to occur. Ovarian stimulation, however, subjects the women to a high physical and psychological stress, thus being essential to improve ART and to find biomarkers of dysfunction and fertility. GSH is an important antioxidant, and is also used in detoxification reactions, catalysed by glutathione S-transferases (GST). In the present work, we have investigated the involvement of GST in follicular maturation. Patients with fertility problems and oocyte donors were recruited for the study. From each woman follicles at two stages of maturation were extracted at the preovulatory stage. Follicular fluid was separated from the oocyte by centrifugation and used as the enzyme source. GST activity was determined based on its conjugation with 3,4-dichloronitrobenzene and the assay was adapted to a 96-well microplate reader. The absorbance was represented against the incubation time and the curves were adjusted to linearity (R(2)>0.990). Results showed that in both donors and patients GST activity was significantly lower in mature oocytes compared to small ones. These results suggest that GST may play a role in the follicle maturation by detoxifying xenobiotics, thus contributing to the normal development of the oocyte. Supported by FIS/FEDER (PI11/02559), Gobierno Vasco (Dep. Educación, Universiades e Investigación, IT687-13), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01).

Characterization of the paraoxonase system in follicular fluid of women subjected to an ovarian stimulation cycle.

Reactive oxygen species (ROS) and antioxidants are involved in the regulation of reproductive processes. Previous studies in infertile women undergoing an ovarian stimulation cycle have suggested a possible role for ROS in the occurrence of conception in In Vitro Fertilization. In this context the control of the redox balance of follicular fluid becomes essential for reproduction, so that the presence of enzymes with antioxidant activities, such as the paraoxonase (PON) system, would play a role in maintaining this balance. The objective of this work was a) to characterize the paraoxonase system in follicular fluid of women undergoing a controlled ovarian stimulation cycle, analysing the associated PON1, PON2, and PON3 activities, and b) to study the possible involvement of the PON system in follicular maturation. The enzyme activities were quantified in follicular fluid from large and small follicles from women undergoing an ovarian stimulation cycle in the IVI-Bilbao clinic. PON activities were quantified using spectrophotometric and HPLC techniques. Statistical comparisons were performed using the Student's t-test for paired data. Results indicate that follicular fluid presents paraoxonase activities which are detectable by the methods developed in this study. PON activities were associated with follicular maturation, suggesting that the PON system plays a role in oocyte maturation. This work was supported by research grants from the Ministry of Health and Consumption (FIS/FEDER PI11/02559), the Basque Country Government (Dep. Education, Universities and Research ref. IT687-13, and DCIT ref. S-PE13UN063), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01).