Interchromosomal effect in carriers of translocations and inversions assessed by preimplantation genetic testing for structural rearrangements (PGT-SR).

PURPOSE: Balanced carriers of structural rearrangements have an increased risk of unbalanced embryos mainly due to the production of unbalanced gametes during meiosis. Aneuploidy for other chromosomes not involved in the rearrangements has also been described. The purpose of this work is to know if the incidence of unbalanced embryos, interchromosomal effect (ICE) and clinical outcomes differ in carriers of different structural rearrangements. METHODS: Cohort retrospective study including 359 preimplantation genetic testing cycles for structural rearrangements from 304 couples was performed. Comparative genomic hybridisation arrays were used for chromosomal analysis. The results were stratified and compared according to female age and carrier sex. The impact of different cytogenetic features of chromosomal rearrangements was evaluated. RESULTS: In carriers of translocations, we observed a higher percentage of abnormal embryos from day 3 biopsies compared with day 5/6 biopsies and for reciprocal translocations compared with other rearrangements. We observed a high percentage of embryos with aneuploidies for chromosomes not involved in the rearrangement that could be attributed to total ICE (aneuploid balanced and unbalanced embryos). No significant differences were observed in these percentages between types of rearrangements. Pure ICE (aneuploid balanced embyos) was independent of female age only for Robertsonian translocations, and significantly increased in day 3 biopsies for all types of abnormalities. Furthermore, total ICE for carriers of Robertsonian translocations and biopsy on day 3 was independent of female age too. High ongoing pregnancy rates were observed for all studied groups, with higher pregnancy rate for male carriers. CONCLUSION: We observed a higher percentage of abnormal embryos for reciprocal translocations. No significant differences for total ICE was found among the different types of rearrangements, with higher pure ICE only for Robertsonian translocations. There was a sex effect for clinical outcome for carriers of translocations, with higher pregnancy rate for male carriers. The higher incidence of unbalanced and aneuploid embryos should be considered for reproductive counselling in carriers of structural rearrangements.

Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.

STUDY QUESTION: What is the origin and composition of cell-free DNA in human embryo spent culture media? SUMMARY ANSWER: Cell-free DNA from human embryo spent culture media represents a mix of maternal and embryonic DNA, and the mixture can be more complex for mosaic embryos. WHAT IS KNOWN ALREADY: In 2016, ~300 000 human embryos were chromosomally and/or genetically analyzed using preimplantation genetic testing for aneuploidies (PGT-A) or monogenic disorders (PGT-M) before transfer into the uterus. While progress in genetic techniques has enabled analysis of the full karyotype in a single cell with high sensitivity and specificity, these approaches still require an embryo biopsy. Thus, non-invasive techniques are sought as an alternative. STUDY DESIGN, SIZE, DURATION: This study was based on a total of 113 human embryos undergoing trophectoderm biopsy as part of PGT-A analysis. For each embryo, the spent culture media used between Day 3 and Day 5 of development were collected for cell-free DNA analysis. In addition to the 113 spent culture media samples, 28 media drops without embryo contact were cultured in parallel under the same conditions to use as controls. In total, 141 media samples were collected and divided into two groups: one for direct DNA quantification (53 spent culture media and 17 controls), the other for whole-genome amplification (60 spent culture media and 11 controls) and subsequent quantification. Some samples with amplified DNA (N = 56) were used for aneuploidy testing by next-generation sequencing; of those, 35 samples underwent single-nucleotide polymorphism (SNP) sequencing to detect maternal contamination. Finally, from the 35 spent culture media analyzed by SNP sequencing, 12 whole blastocysts were analyzed by fluorescence in situ hybridization (FISH) to determine the level of mosaicism in each embryo, as a possible origin for discordance between sample types. PARTICIPANTS/MATERIALS, SETTING, METHODS: Trophectoderm biopsies and culture media samples (20 µl) underwent whole-genome amplification, then libraries were generated and sequenced for an aneuploidy study. For SNP sequencing, triads including trophectoderm DNA, cell-free DNA, and follicular fluid DNA were analyzed. In total, 124 SNPs were included with 90 SNPs distributed among all autosomes and 34 SNPs located on chromosome Y. Finally, 12 whole blastocysts were fixed and individual cells were analyzed by FISH using telomeric/centromeric probes for the affected chromosomes. MAIN RESULTS AND THE ROLE OF CHANCE: We found a higher quantity of cell-free DNA in spent culture media co-cultured with embryos versus control media samples (P ≤ 0.001). The presence of cell-free DNA in the spent culture media enabled a chromosomal diagnosis, although results differed from those of trophectoderm biopsy analysis in most cases (67%). Discordant results were mainly attributable to a high percentage of maternal DNA in the spent culture media, with a median percentage of embryonic DNA estimated at 8%. Finally, from the discordant cases, 91.7% of whole blastocysts analyzed by FISH were mosaic and 75% of the analyzed chromosomes were concordant with the trophectoderm DNA diagnosis instead of the cell-free DNA result. LIMITATIONS, REASONS FOR CAUTION: This study was limited by the sample size and the number of cells analyzed by FISH. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to combine chromosomal analysis of cell-free DNA, SNP sequencing to identify maternal contamination, and whole-blastocyst analysis for detecting mosaicism. Our results provide a better understanding of the origin of cell-free DNA in spent culture media, offering an important step toward developing future non-invasive karyotyping that must rely on the specific identification of DNA released from human embryos. STUDY FUNDING/ COMPETING INTEREST: This work was funded by Igenomix S.L. There are no competing interests.

Confirmation rates of array-CGH in day-3 embryo and blastocyst biopsies for preimplantation genetic screening.

PURPOSE: The purpose of this study was to compare the confirmation rate of day-3 embryo biopsy (blastomere) and trophectoderm biopsy using array-comparative genomic hybridization (array-CGH) technology. METHODS: A blinded study was conducted to re-analyse 109 embryos previously diagnosed as chromosomally abnormal by array-CGH. Preimplantation genetic screening (PGS) was performed using array-CGH on day 3 (n = 50) or day 5 (n = 59). Partial chromosome gains or losses were excluded (n=6), and only whole chromosome aneuploidies were considered. Re-analysis of whole blastocysts was carried out following the same array-CGH protocol used for PGS. RESULTS: The PGS result was confirmed in the whole blastocyst in (a) 49/50 (98 %) abnormal embryos after day-3 biopsy and (b) 57/59 (96.6 %) abnormal embryos after trophectoderm biopsy. One embryo (1/50; 2 %) was diagnosed as abnormal, with monosomy 18, on day 3, and software analysis of the whole blastocyst gave a euploid result; however, a mosaic pattern was observed for monosomy 18 in the whole blastocyst. Two trophectoderm biopsy cases (3.4 %) did not have the abnormalities (trisomy 7, and trisomy 1 and 4, respectively) verified in the whole embryo. Concordance rates for both biopsy strategies and for individual chromosomes were evaluated by Fisher's exact test and showed no significant differences. CONCLUSIONS: Both types of biopsies showed similar high concordance rates with whole blastocyst results. Therefore, regarding the confirmation rates shown in this work, day-3 embryo biopsies can be representative of the whole embryo and both types of biopsy can be used for clinical analysis in PGS following the described array-CGH protocol.

Mosaic results after preimplantation genetic testing for aneuploidy may be accompanied by changes in global gene expression.

Aneuploidy in preimplantation embryos is a major cause of human reproductive failure. Unlike uniformly aneuploid embryos, embryos diagnosed as diploid-aneuploid mosaics after preimplantation genetic testing for aneuploidy (PGT-A) can develop into healthy infants. However, the reason why these embryos achieve full reproductive competence needs further research. Current RNA sequencing techniques allow for the investigation of the human preimplantation transcriptome, providing new insights into the molecular mechanisms of embryo development. In this prospective study, using euploid embryo gene expression as a control, we compared the transcriptome profiles of inner cell mass and trophectoderm samples from blastocysts with different levels of chromosomal mosaicism. A total of 25 samples were analyzed from 14 blastocysts with previous PGT-A diagnosis, including five low-level mosaic embryos and four high-level mosaic embryos. Global gene expression profiles visualized in cluster heatmaps were correlated with the original PGT-A diagnosis. In addition, gene expression distance based on the number of differentially expressed genes increased with the mosaic level, compared to euploid controls. Pathways involving apoptosis, mitosis, protein degradation, metabolism, and mitochondrial energy production were among the most deregulated within mosaic embryos. Retrospective analysis of the duration of blastomere cell cycles in mosaic embryos revealed several mitotic delays compared to euploid controls, providing additional evidence of the mosaic status. Overall, these findings suggest that embryos with mosaic results are not simply a misdiagnosis by-product, but may also have a genuine molecular identity that is compatible with their reproductive potential.

Improving FISH diagnosis for preimplantation genetic aneuploidy screening.

BACKGROUND: In our routine programme of preimplantation genetic aneuploidy screening (PGS) by fluorescence in situ hybridization (FISH), nine chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y) are analysed in two consecutive hybridization rounds. We also perform additional hybridization rounds for these chromosomes, using probes that bind to different loci, for non-conclusive results and for confirmation of certain aneuploidies. The aim of this study was to evaluate the impact of additional hybridization rounds on FISH accuracy. METHODS: This is a retrospective analysis of our FISH data from 1000 PGS cycles performed from December 2007 to December 2008 for various indications. In addition to the hybridization rounds described above, 132 of the embryos diagnosed as chromosomally abnormal were re-analysed on Day 5. RESULTS: A total of 2477 embryos were re-hybridized, 1496 due to non-conclusive results and 981 to confirm observed aneuploidies. After re-hybridization, 882 embryos (59%) were then diagnosed as normal, 600 embryos (40.1%) had a clear abnormality and only 14 embryos (0.9%) remained non-informative. From the 981 embryos in the latter group, 890 embryos had monosomies and, after re-hybridization 174 embryos (19.6%) were normal and 716 (80.5%) had confirmed monosomies. In contrast, re-hybridization confirmed 90 (98.9%) of the 91 observed trisomies. In addition, Day-5 re-analysis of abnormal embryos showed a higher rate of concordant diagnosis between Day 3 and Day 5 when re-hybridizations had been included on Day-3 (95 versus 82.7%; P= 0.0443), especially for the confirmation of monosomies (82.8 versus 61.0%; P = 0.0087). CONCLUSIONS: Our data indicate that additional hybridization rounds improve the accuracy of the diagnosis, increasing the number of chromosomally normal embryos available for transfer. Re-hybridization with additional probes as a standard approach to PGS could enhance the potential benefits of the technique.

Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst.

Embryonic implantation is a crucial event for the human reproductive function. Cytokines and paracrine molecules have been proposed as putative local regulators of this process. The leptin or the OB protein has been linked to the reproductive function and inflammatory response. In the present study, we describe for the first time the expression of leptin and leptin receptor (long form) in the secretory endometrium and that endometrial leptin secretion is regulated in vitro by the human blastocyst. Leptin and leptin receptor messenger RNA and protein were identified in secretory endometrium and in cultured endometrial epithelial cells (EECs) by RT-PCR, Western blot, and immunohistochemistry. The concentrations of immunoreactive leptin secreted by human embryos alone or cocultured with EECs were also assessed. We found that human blastocysts secrete significantly higher levels of leptin than arrested embryos. In contrast, leptin concentrations secreted by arrested embryos cocultured with EECs were significantly higher than blastocysts cocultured with EECs. These findings suggest that the human endometrium is a site for local production and a target tissue for circulating leptin. Expression of leptin and its functional receptor in the endometrium and regulation of endometrial leptin secretion by the human embryo suggests that the leptin system may be implicated in the human implantation process.

Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure.

We have developed a coculture system with autologous human endometrial epithelial cells (AEEC) that retained many features of human endometrial epithelium. Implantation failure (IF; >3 previous cycles failed with 3-4 good quality embryos transferred) is a distressing condition in which 2-day embryo transfer repetition is the routine option. The objective of this study was to investigate the basics and to evaluate prospectively the clinical value of embryo coculture on AEEC and blastocyst transfer with their own oocytes [in vitro fertilization (IVF) patients] or with donated oocytes (oocyte donation patients) compared to a routine day 2 embryo transfer for patients with IF. Scanning electron microscopy and mouse embryo assays demonstrate that EEC from fertile and IF patients were morphologically and functionally similar; similar findings were observed in EEC obtained from fresh or frozen endometria. Clinically, 168 IVF cycles were performed in 127 patients with 3.8+/-0.2 previously failed cycles, and 80 cycles were performed in 57 patients undergoing oocyte donation with 3.0+/-0.2 previously failed cycles. Twenty IVF patients and 15 ovum donation patients with 3 previously failed cycles in whom a 2-day embryo transfer was performed were used as controls. In 88% of ovum donation cycles, at least 2 blastocysts were available for transfer, with 60.1% blastocyst formation; 2.2+/-0.1 blastocysts were transferred/cycle, and 36 pregnancies (determined by fetal cardiac activity) were obtained (32.7% implantation and 54.5% pregnancy rates). In 168 IVF cycles, 8.1+/-0.2 embryos/cycle started coculture, resulting in 49.2% blastocyst formation; 2.3+/-0.2 blastocysts were transferred/cycle, and 29 clinical pregnancies were obtained (11.8% implantation and 20.2% pregnancy rates). Fifteen cycles were canceled (9%). In oocyte donation patients with IF undergoing 2-day embryo transfer, implantation and pregnancy rates were significantly lower (4.5% and 13.3%; P < 0.01) than with coculture; however, in IVF patients with IF, results with day 2 transfer (10.7% and 35%) were similar to those with coculture. The present study demonstrates that coculture of human embryos with AEEC and blastocyst transfer is safe, ethical, and effective and constitutes a new approach to improve implantation in patients with IF undergoing ovum donation, but not in IVF patients.

Embryonic regulation of integrins beta 3, alpha 4, and alpha 1 in human endometrial epithelial cells in vitro.

In the present study, we examined the embryonic regulation of beta 3 integrin in human endometrial epithelial cells (EEC) at the protein level and analyzed putative embryonic factors responsible for this regulation. The model employed is based on a clinical in vitro fertilization program in which single human embryos were cocultured with EEC until blastocyst stage and then transferred back to the uterus. After embryo transfer, EEC wells were divided according to the embryonic status reached: EEC with embryos that achieved the blastocyst stage, EEC with arrested embryos, and EEC without embryos. Immunostaining for beta 3 was positive in plasma membrane of EEC. Flow cytometry showed a mean percentage of beta 3-stained cells of 24.1 +/- 5.7 in EEC cocultured with embryos that achieved the blastocyst stage (n = 13) vs. 9.5 +/- 1.6 (P < 0.05) in those EEC cultured with arrested embryos (n = 12). Immunostaining for alpha 1 and alpha 4 integrins was negative in EEC monolayers studied, regardless of the presence or absence of embryos, and these findings were confirmed by flow cytometry. The possibility that the embryonic IL-1 system and leukemia inhibitory factor were involved in the endometrial beta 3 up-regulation was investigated by neutralizing experiments demonstrating a significant inhibition of beta 3-stained cells when EEC monolayers were cultured in the presence of EEC/blastocyst-conditioned media with (n = 4) vs. without (n = 8) antihuman interleukin (IL)-1 alpha + IL-1 beta (1.65% vs. 14.6%; P < 0.05). Dose-response experiments further demonstrated an up-regulation of beta 3 positive cells when IL-1 alpha + IL-1 beta were added to the medium at a concentration of 10 pg/mL compared with control medium without added cytokines (40% vs. 20%, n = 4). The functional relevance of the EEC beta 3 up-regulation was tested using a mouse blastocyst adhesion assay. More mouse blastocysts attached to EEC previously in contact with human blastocyst (72.7%) compared with those EEC previously in contact with arrested embryos (40%). Our results demonstrate the selective effect of a developing human embryo on EEC expression of beta 3, which is maximal when a human blastocyst instead of an arrested embryo is considered. Furthermore, the embryonic IL-1 system seems to be involved in the EEC beta 3 up-regulation, reinforcing the concept of precise paracrine cross-talk between blastocyst and endometrial epithelium during embryonic implantation.

Implantation rates after two, three, or five days of embryo culture.

Extended embryo culture together with amelioration of embryo selection methods and embryo culture conditions have allowed a substantial increase on both pregnancy and implantation rates. However, uterine embryo transfers are still performed after 2 to 6 days of egg retrieval. In this paper, we show the results of two studies, one prospective study comparing IVF outcome of day 2 and day 3 embryo transfers, and a retrospective study looking at blastocyst transfers versus day 3 embryo transfers in our egg donation program. Also, we test the predictive value of the presence of three or more seven cell-stage embryos on day 3 of development on blastocyst formation and pregnancy rates. No significant differences were found between day 2 and day 3 embryo transfers in terms of pregnancy, ongoing pregnancy, and implantation rates, as well as in multiple and in high order pregnancy. In general, day 6 embryo transfers resulted in significantly higher ongoing pregnancy and implantation rates compared with day 3 embryo transfers (41.1 per cent and 23.6 per cent versus 50.1 per cent and 38.1 per cent, respectively). No differences were found in terms of multiple gestations despite transferring significantly more embryos on day 3 compared with day 6 transfers. When less than three 7-cell embryos were present in the embryo cohort, day 6 embryo transfers did not improve the rates of ongoing pregnancy with regards to day 3 embryo transfer, although significant high implantation rates were obtained on the group of blastocyst transfer. The presence of three or more 7 cell-stage embryos improved significantly both ongoing pregnancy and rates on blastocyst transfers compared to day 3 embryo transfers (65.6 per cent versus 50.6 per cent and 37.4 per cent vs 24.7 per cent, respectively). In conclusion, at least in egg donation, day 3 embryo transfers do not improve either pregnancy or implantation rates when compared to day 2 transfers. Generally speaking blastocyst transfers give significantly higher chance of pregnancy and implantation rates per cycle and per transfer than early cleavage stage transfers. However, the absence of a good embryo cohort, that is having less than three 7 cell-stage embryos on day 3, blastocyst transfers will improve implantation rates but not ongoing pregnancy rates.

Hormonal regulation of serum and endometrial IL-1 alpha, IL-1 beta and IL-1ra: IL-1 endometrial microenvironment of the human embryo at the apposition phase under physiological and supraphysiological steroid level conditions.

We have investigated serum and intracavitary levels of IL-1 alpha, IL-1 beta and IL-1ra from agonadal women undergoing mock cycles (n = 20) of oocyte donation as a clinical model of controlled hormonal stimulation. Further, we compared the intracavitary IL-1 alpha, IL-1 beta and IL-1ra levels in the microenvironment of the human embryo at the apposition phase, day 5 after progesterone (P) administration using two different clinical models: oocyte donation (n = 20) which provides physiological steroid levels and a higher implantation rate per embryo, and in vitro fertilization (n = 6) with supraphysiological hormonal levels and a lower implantation rate.

The pathogenesis of ovarian hyperstimulation syndrome: in vivo studies investigating the role of interleukin-1beta, interleukin-6, and vascular endothelial growth factor.

OBJECTIVE: To evaluate systemic and ovarian changes in levels of interleukin (IL)-1beta, IL-6, and vascular endothelial growth factor (VEGF) in response to hCG administration to determine which may be the potential initiator of vascular effects and to identify the main source of the substance; to evaluate serum and follicular fluid levels of these cytokines as markers of ovarian hyperstimulation syndrome (OHSS), and to compare levels of these cytokines under basal conditions in women with normal ovulation and those with polycystic ovary syndrome (PCOS). DESIGN: Prospective controlled study. SETTING: In vitro fertilization program at the Instituto Valenciano de Infertilidad, Valencia, Spain. PATIENT(S): Women undergoing IVF, in whom the first two study objectives were analyzed, and women with normal ovulation and patients with PCOS undergoing retrieval of immature oocytes in natural cycles or cycles stimulated for IUI but cancelled during induction of ovulation, in whom the third study objective was analyzed. INTERVENTION(S): Serum was collected before and after hCG administration, and follicular fluid was collected at ovum pick-up. MAIN OUTCOME MEASURE(S): Serum and follicular fluid levels of IL-1beta, IL-6, and VEGF. RESULT(S): There was a significant increase in serum VEGF levels after hCG administration in patients who were at risk for OHSS compared with those who were not at risk for OHSS. Significantly lower VEGF levels were found in the follicular fluid of patients who were at risk; this decrease was the only useful marker to discriminate between the two groups. Moreover, both groups had similar cytokine production under basal conditions. An increase in serum E2 occurred coincident with a decrease in IL-1beta, IL-6, and VEGF in patients with PCOS. CONCLUSION(S): Vascular endothelial growth factor seems to be the mediator of hCG on the vascular tree. There was an early systemic increase in VEGF that may have significance in the development of OHSS. A decrease in the follicular fluid VEGF concentration is a valid marker to identify women in whom OHSS will develop. The pattern of cytokine release in patients with PCOS under basal conditions was not different from that in women with normal ovulation.

The follicular and endocrine environment in women with endometriosis: local and systemic cytokine production.

OBJECTIVE: To assess the endocrine, paracrine, and autocrine milieu in patients with endometriosis on the basis of the measurement of several cytokines in serum and follicular fluid (FF) and in vitro culture of granulosa luteal cells. DESIGN: Case-control study. SETTING: In vitro fertilization program at the Instituto Valenciano de Infertilidad. PATIENT(S): Twenty patients with laparoscopically documented endometriosis and 18 controls. Fifteen subjects were studied in a natural cycle and 23 were investigated in a stimulated cycle while undergoing IVF. INTERVENTION(S): Individual follicle aspiration, oocyte isolation, FF storage, and preparation of luteinized granulosa cell cultures. Diagnostic laparoscopy in natural cycles. MAIN OUTCOME MEASURE(S): Serum (day of ovum pick-up or laparoscopy) and FF measurement of interleukin (IL)-1beta, IL-6, and vascular endothelial growth factor (VEGF). Secretion of IL-1beta, IL-6, and VEGF in the cell-conditioned medium. Results were compared between patients with endometriosis and controls. RESULT(S): Interleukin-6 levels in serum were increased in the natural cycles of patients with endometriosis and modulated by ovarian stimulation, showing a significant decrease in hMG- and FSH-stimulated cycles and a significant increase after hCG administration. In addition, IL-6 levels were increased in the FF of patients with endometriosis and released in higher amounts by their granulosa luteal cells. Vascular endothelial growth factor was accumulated in lesser concentrations in the FF of patients with endometriosis. Interleukin-1beta levels did not show significant changes. Implantation rates were decreased significantly in patients with endometriosis who were undergoing IVF. CONCLUSION(S): The data demonstrate that cytokines are regulated differently in patients with endometriosis, who have increased IL-6 production, and suggest that fine hormonal modulation of this cytokine occurs at the systemic and local (ovarian) levels. These changes show that the endocrine, paracrine, and autocrine milieu is different in patients with endometriosis and may be related to their lower implantation rates.

QSAR applications during last decade on inhibitors of acetylcholinesterase in Alzheimer's disease.

This article reviews multi-criteria QSAR applications on Acetylcholinesterase inhibitors as palliative drugs for Alzheimer's Disease, published in the period 2001-2011. It includes QSAR models for different series of compounds, comparative studies, and advances in methodologies. This period is marked by a shift in focus from palliative treatment to pathogenesis. However, we believe that research into palliative treatment should continue. More comparative studies are desirable. In order to facilitate comparative and general studies on Acetylcholinesterase inhibitors, a standard experimental protocol for measuring an inhibitor's potency is needed. Finally, we recommend chemists to work closely with system and molecular biologists.

Smoking habits of parents and male: female ratio in spermatozoa and preimplantation embryos.

BACKGROUND: Previous observations have addressed a decreased male:female ratio associated with smoking. Our aim was to assess whether this effect is observed at the spermatozoa or at the early embryo development. METHODS: We retrospectively assessed smoking intake habits of 56 couples included in our preimplantation genetic diagnosis (PGD) program. Three groups were established according to male or female cigarette consumption per day: non-smokers, smokers (1-19 cigarettes per day) and heavy smokers (> or =20 cigarettes per day). Fluorescence in-situ hybridization (FISH) was performed on ejaculated sperm samples to analyse chromosomes X and Y. On day 3, embryos were also analysed. Additionally, sperm samples from four heavy smoking and four non-smoking donors were prospectively analysed before and after capacitation. RESULTS: FISH on spermatozoa revealed no statistical differences in the Y:X ratio between the three groups. However, in the PGD study, in male heavy smokers, the XY:XX embryo ratio was decreased compared with non-smokers (22:47 versus 80:71; P = 0.0057). The smoking condition of the female partner had no significant effect on embryo XY:XX ratio, but for non-smoking females with a heavy smoking partner, the ratio was decreased (P = 0.0018) compared with non-smoking males. In heavy smoking donors a decreased of Y:X ratio was observed after swim-up with a statistically significant difference of ratios (P = 0.021). CONCLUSIONS: Smoking habits of males do not have an effect on the percentage of X- and Y-bearing spermatozoa on ejaculated samples. However, male heavy smokers produce an increased incidence of female embryos that could be related to an enrichment of X spermatozoa after swim-up in patients with high tobacco consumption.

The interleukin-1 system and human implantation.

PROBLEM: Cytokines and growth factors are increasingly implicated in embryonic implantation. In the present study, we focus on the interleukin-1 system as an example of local regulator in human implantation. METHOD: Three different approaches are considered. First, we present evidence demonstrating its presence, regulation, and relevance on the human endometrium. Second, we demonstrate the presence of IL-1 system in the human embryo and the selective IL-1 release only when embryos were co-cultured with human endometrial epithelial cells (EEC) or EEC-conditioned media, indicating an obligate role of the endometrium in the regulation of the embryonic IL-1 system. Finally, we show data regarding the presence, hormonal regulation, and endometrial origin of IL-1 alpha, IL-beta, and IL-1ra levels in the endometrial fluid inside the endometrial cavity. Specifically, we present the IL-1 intracavitary microenvironment surrounding the human embryo at the apposition phase. RESULTS: This work suggests that the IL-1 system seems to be relevant for human endometrial and embryonic physiology. Furthermore, this family of molecules must be considered as a relevant paracrine language displayed by both partners that may be important to endometrial and embryonic crosstalk during embryonic implantation.

Clinical experience employing co-culture of human embryos with autologous human endometrial epithelial cells.

Attempts to improve the outcome in IVF and related techniques has drawn our attention to the development of culture systems that can grow embryos up to the blastocyst stage. We have developed a co-culture system with autologous human endometrial epithelial cells (EEC) that retained many features of the endometrium. In this review, we analyse our experience over the last 4 years; in particular, we address the question of whether the system would be safe and useful in cases of IVF and oocyte donation with previous implantation failure, and which factors may contribute to the failure of human embryos to develop in vitro up to the blastocyst stage. In all, 168 IVF cycles were carried out in 127 patients with 3.8+/-0.2 previous implantation failure, and 80 cycles in 57 women having oocyte donation with 3.0+/-0.2 previous implantation failure. In 168 IVF cycles, a 48.9% blastocyst formation rate was recorded; 2.3+/-0.1 blastocysts were transferred per cycle and 30 clinical pregnancies obtained (11.9% implantation and 19.6% pregnancy rates). A total of 20 IVF and 15 oocyte donation patients with three previous implantation failures in whom a day 2 embryo transfer was performed were the controls. In 88% of oocyte donation cycles, a 61.0% blastocyst formation rate was observed; 2.3+/-0.1 blastocysts were transferred per cycle and resulted in 38 clinical pregnancies (32.7% implantation and 54.3% pregnancy rates). In all, 15 cycles were cancelled (9%). In oocyte donation patients with implantation failure undergoing day 2 embryo transfer, implantation and pregnancy rates were significantly lower (4.5 and 13.3%; P < 0.01) than with co-culture; however, in IVF patients with implantation failure with day 2 transfer, results (10.7 and 35%) were similar to those with co-culture. A second question addressed was whether chromosomal abnormalities may contribute to the failure of human embryos to develop in vitro. We observed the performance of human embryos from our preimplantation diagnosis programme, which were biopsied and subsequently cultured in EEC before transfer. Out of 68 chromosomally normal embryos, 37 reached the blastocyst stage (54.4%) compared with 35 out of 104 abnormal embryos (33.6%). The present study demonstrates that co-culture of human embryos with EEC and blastocyst transfer is safe, effective, and may be a new approach to improve implantation in patients with implantation failure undergoing oocyte donation, but not in IVF patients. The system shows that abnormal embryos can also grow to the blastocyst stage, although to a lower rate. Prospective randomized studies are needed to confirm the preliminary conclusion that co-culture is an acceptable system to select good quality embryos, and the endometrium is a limiting factor in implantation that needs to be carefully managed.

An analysis of spontaneous hatching in a human endometrial epithelial coculture system: is assisted hatching justified?

PURPOSE: To evaluate spontaneous embryo hatching in an endometrial epithelial coculture system, and compare it with cases where coculture was performed because of maternal age, previous repeated implantation failures, or both. To clarify in which cases assisted hatching would be appropriate. METHODS: Individual human embryos were cocultured on an endometrial epithelial cell monolayer until Day 6. RESULTS: Blastocyst hatching rate at Day 6, depending on maternal age, was 9.1% (age <37 years) and 3.4% (age > or = 37 years). However, blastocyst hatching rates depending on number of previous IVF failures were similar. CONCLUSIONS: Maternal age and previous implantation failures are factors affecting the ability of human embryos to reach the blastocyst stage in coculture. However, assisted hatching is not justified in these populations because of the absence of hatching rate differences between blastocysts obtained from these two groups and the control group.

[The interleukin-1 system during human implantation]. / Le système interleukine-1 au cours de l'implantation dans l'espèce humaine.

Although the immune and reproductive systems have been considered independent of each other, the cooperation of both systems are now known to be crucial for the initiation and maintenance of mammalian pregnancy. Nowadays cytokines and growth factors have became increasingly implicated in embryonic implantation. Endometrial functions, embryonic secretions and embryo-endometrial interactions require a continuous dialogue and synchronism between both partners (endometrium and embryo). The present review focuses on the Interleukin-1 system as an example of local regulator in embryonic implantation. Evidence demonstrating its presence and relevance on human endometrium physiology and human preimplantation embryonic development are presented. Furthermore, we described data suggesting the possible role of this cytokine in human implantation.

Immunohistochemical localization, identification and regulation of the interleukin-1 receptor antagonist in the human endometrium.

There is evidence suggesting the importance of the interleukin-1 receptor type I (IL-1Rtl) and interleukin-1 beta (IL-1 beta) as mediator in local intercellular interactions in endometrial tissue and embryonic implantation. To complete our understanding of the entire endometrial IL-1 system in humans, we have investigated the immunohistochemical distribution of IL-1 receptor antagonist (IL-1ra) in the human endometrium throughout the menstrual cycle. We have also identified the forms of IL-1ra present in human endometrial cells. Immunoreactive IL-1ra was found in both cryostat and paraffin-embedded sections of human endometrium using the alkaline phosphatase-peroxidase (A-P) method with two different IL-1ra antibodies. IL-1ra was present throughout the entire menstrual cycle, located primarily in the endometrial epithelium. However, IL-1ra staining was significantly higher during follicular phase in comparison with early and mid-late luteal phases. Reverse transcriptase polymerase chain reaction of cultured stromal and glandular cells showed that these cells express the intracellular form of IL-1ra mRNA (icIL-1ra). Our results demonstrate the regulated presence of the icIL-1ra in the human endometrium. This finding supports a possible autocrine-paracrine role for the IL-1 system in the human endometrium and embryonic implantation.

The role of endothelial cells in the pathogenesis of ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of treatment with fertility drugs. Using human lung microvascular endothelial cells (HUMEC-L) as an in-vitro model of OHSS, we have tested the hypothesis that the endothelium is a target of HCG in the pathogenesis of OHSS. Since OHSS is characterized by increased capillary permeability, we have investigated the production and action of vasoactive agents. When HUMEC-L were cultured with high doses of estradiol (E(2)), no significant changes were observed in the secretion of vascular endothelial growth factor (VEGF), interleukin (IL)-6 or IL-1 beta. However, the addition of HCG resulted in a significant increase in the secretion of VEGF and IL-6. Time-course experiments showed that VEGF was secreted within minutes of HCG addition, whereas IL-6 was significantly increased only after 48 h in culture. The secretion of IL-1 beta was unchanged by these hormonal conditions. The presence of HCG receptors was demonstrated in HUMEC-L in basal conditions as well as after the addition of E(2). The expression of VEGF receptors was also investigated. High doses of E(2) were unable to increase the expression of KDR, flt-1 and sfl-t, but the addition of HCG significantly upregulated the KDR concentration in endothelial cells, while no change was observed for flt. Permeability assays demonstrated that while E(2) alone did not change the arrangement of HUMEC-L in vitro, the presence of HCG caused changes in the actin fibres corresponding to increased capillary permeability. Anti-human VEGF antibodies were able to overcome these changes. In conclusion, these experiments show that the endothelium may be a primary target of HCG, causing an acute release of VEGF and a significant increase in IL-6 and resulting in an autocrine-paracrine action that may increase vascular permeability.