ESHRE good practice recommendations on recurrent implantation failure.

STUDY QUESTION: How should recurrent implantation failure (RIF) in patients undergoing ART be defined and managed? SUMMARY ANSWER: This is the first ESHRE good practice recommendations paper providing a definition for RIF together with recommendations on how to investigate causes and contributing factors, and how to improve the chances of a pregnancy. WHAT IS KNOWN ALREADY: RIF is a challenge in the ART clinic, with a multitude of investigations and interventions offered and applied in clinical practice, often without biological rationale or with unequivocal evidence of benefit. STUDY DESIGN SIZE DURATION: This document was developed according to a predefined methodology for ESHRE good practice recommendations. Recommendations are supported by data from the literature, if available, and the results of a previously published survey on clinical practice in RIF and the expertise of the working group. A literature search was performed in PubMed and Cochrane focussing on 'recurrent reproductive failure', 'recurrent implantation failure', and 'repeated implantation failure'. PARTICIPANTS/MATERIALS SETTING METHODS: The ESHRE Working Group on Recurrent Implantation Failure included eight members representing the ESHRE Special Interest Groups for Implantation and Early Pregnancy, Reproductive Endocrinology, and Embryology, with an independent chair and an expert in statistics. The recommendations for clinical practice were formulated based on the expert opinion of the working group, while taking into consideration the published data and results of the survey on uptake in clinical practice. The draft document was then open to ESHRE members for online peer review and was revised in light of the comments received. MAIN RESULTS AND THE ROLE OF CHANCE: The working group recommends considering RIF as a secondary phenomenon of ART, as it can only be observed in patients undergoing IVF, and that the following description of RIF be adopted: 'RIF describes the scenario in which the transfer of embryos considered to be viable has failed to result in a positive pregnancy test sufficiently often in a specific patient to warrant consideration of further investigations and/or interventions'. It was agreed that the recommended threshold for the cumulative predicted chance of implantation to identify RIF for the purposes of initiating further investigation is 60%. When a couple have not had a successful implantation by a certain number of embryo transfers and the cumulative predicted chance of implantation associated with that number is greater than 60%, then they should be counselled on further investigation and/or treatment options. This term defines clinical RIF for which further actions should be considered. Nineteen recommendations were formulated on investigations when RIF is suspected, and 13 on interventions. Recommendations were colour-coded based on whether the investigations/interventions were recommended (green), to be considered (orange), or not recommended, i.e. not to be offered routinely (red). LIMITATIONS REASONS FOR CAUTION: While awaiting the results of further studies and trials, the ESHRE Working Group on Recurrent Implantation Failure recommends identifying RIF based on the chance of successful implantation for the individual patient or couple and to restrict investigations and treatments to those supported by a clear rationale and data indicating their likely benefit. WIDER IMPLICATIONS OF THE FINDINGS: This article provides not only good practice advice but also highlights the investigations and interventions that need further research. This research, when well-conducted, will be key to making progress in the clinical management of RIF. STUDY FUNDING/COMPETING INTERESTS: The meetings and technical support for this project were funded by ESHRE. N.M. declared consulting fees from ArtPRED (The Netherlands) and Freya Biosciences (Denmark); Honoraria for lectures from Gedeon Richter, Merck, Abbott, and IBSA; being co-founder of Verso Biosense. He is Co-Chief Editor of Reproductive Biomedicine Online (RBMO). D.C. declared being an Associate Editor of Human Reproduction Update, and declared honoraria for lectures from Merck, Organon, IBSA, and Fairtility; support for attending meetings from Cooper Surgical, Fujifilm Irvine Scientific. G.G. declared that he or his institution received financial or non-financial support for research, lectures, workshops, advisory roles, or travelling from Ferring, Merck, Gedeon-Richter, PregLem, Abbott, Vifor, Organon, MSD, Coopersurgical, ObsEVA, and ReprodWissen. He is an Editor of the journals Archives of Obstetrics and Gynecology and Reproductive Biomedicine Online, and Editor in Chief of Journal Gynäkologische Endokrinologie. He is involved in guideline developments and quality control on national and international level. G.L. declared he or his institution received honoraria for lectures from Merck, Ferring, Vianex/Organon, and MSD. He is an Associate Editor of Human Reproduction Update, immediate past Coordinator of Special Interest Group for Reproductive Endocrinology of ESHRE and has been involved in Guideline Development Groups of ESHRE and national fertility authorities. D.J.M. declared being an Associate Editor for Human Reproduction Open and statistical Advisor for Reproductive Biomedicine Online. B.T. declared being shareholder of Reprognostics and she or her institution received financial or non-financial support for research, clinical trials, lectures, workshops, advisory roles or travelling from support for attending meetings from Ferring, MSD, Exeltis, Merck Serono, Bayer, Teva, Theramex and Novartis, Astropharm, Ferring. The other authors had nothing to disclose. DISCLAIMER: This Good Practice Recommendations (GPR) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation. ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type. Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE.

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.

A new system of sperm cryopreservation: evaluation of survival, motility, DNA oxidation, and mitochondrial activity.

BACKGROUND: Sperm vitrification (V) is a method for cryopreservation, without the use of conventional cryoprotectants, by plunging the sperm suspension directly into liquid nitrogen (LN25). OBJECTIVE: This study aimed to compare the new system of V with conventional freezing (CF) protocol using fresh spermatozoa as reference (C). MATERIAL AND METHODS: Prospective cohort study. A total of 47 sperm samples from men attending the infertility clinic at Instituto Valenciano de Infertilidad Valencia. The sperm V solution was 0.3 M trehalose-sucrose and plunged directly in liquid nitrogen in microdroplets of 5-10 lL, using a new system collector of V. Sperm viability indicators such as sperm motility, vitality rates, mitochondrial function, and sperm DNA oxidation were assessed before and after cryopreservation. Sperm motility and vitality analysis were performed according to published guidelines of the World Health Organization (WHO, 2010). Mitochondrial function was evaluated using JC-1 (fluorescent cationic dye, 5,50,6,60-tetrachloro-1-10,3,30-tetraethyl-benzamidazolocarbocyanin iodide). Sperm DNA oxidation was determined using a fluorescent assay (Oxy-DNA test) for the detection of 8-oxoguanine. The evaluation was carried out before and after cryopreservation using flow cytometry. Statistical analysis was performed using ANOVA and chi-square test, and p < 0.05 was considered statistically significant. RESULT(S): Sperm parameters, including progressive motility, total motility, and viability, observed after cryopreservation were as follows: C = 74.9% [1] 12.3, CF = 27.2% [1] 8.4, V = 42.3% [1] 9.3, p < 0.001; C = 90.1 [1] 6.8, CF = 42.0 [1] 12.9, V = 61.4 [1] 11.8, p < 0.001; C = 90.0% [1] 7.4, CF = 42.5% [1] 14.6, V = 70.9% [1] 6.5, p < 0.001, respectively. Regarding Oxy-DNA and mitochondrial activity, they were significantly affected in both groups (V and CF) when compared to the control group. DISCUSSION: The sperm V and CF have negative impact on sperm parameters as well as DNA integrity and mitochondrial activity. However, sperm V presented improved sperm motility recovery, similar levels of DNA oxidation, and, moreover, a slightly increase in mitochondrial activity when compared to the conventional method. CONCLUSION(S): V as an optimal protocol for sperm cryopreservation.

Understanding the role of killer cell immunoglobulin-like receptors in pregnancy complications.

Pregnancy is a unique immunological situation in which a fetus-bearing paternal histocompatibility antigens can survive in a maternal environment without apparent rejection. To face this challenge, cells of the uterine immune system show characteristic changes in absolute number and composition during pregnancy. Particularly relevant to this process are uterine natural killer (uNK) cells and their cell surface receptors, killer immunoglobulin-like receptors (KIRs). The main purpose of this review is to outline the current body of knowledge on the involvement of KIRs in the complications of pregnancy. Implantation depends on the invasion of embryonic trophoblast cells into maternal uterine tissue and remodeling of the uterine spiral arterioles, which is essential for placental perfusion and successful pregnancy. The proper interaction between maternal KIRs and their ligands human leukocyte antigen (HLA) class I molecules, expressed by the extravillous trophoblast cells, is crucial in this process. KIRs are a complex family that includes both activator and inhibitory receptors. The activation profile is genetically determined in each individual and leads to diverse levels of functionality for NK and T cells on engagement with specific HLA class I molecules. An association between different KIR alleles and HLA molecules has been reported in pregnancy complications, supporting the idea of a relevant role of these receptors in successful pregnancy.

The educational and professional status of clinical embryology and clinical embryologists in Europe.

STUDY QUESTION: What is the recognition of clinical embryology and the current status of clinical embryologists in European countries, regarding educational levels, responsibilities and workload, and need for a formal education in assisted reproductive technology (ART)? SUMMARY ANSWER: It is striking that the profession of clinical embryology, almost 40 years after the introduction of IVF, is still not officially recognized in most European countries. WHAT IS KNOWN ALREADY: Reproductive medicine has developed into a sophisticated multidisciplinary medical branch since the birth of Louise Brown 37 years ago. The European Board & College of Obstetrics and Gynaecology (EBCOG) has recognized reproductive medicine as a subspeciality and has developed a subspeciality training for gynaecologists in collaboration with the European Society for Human Reproduction and Embryology (ESHRE). However, nothing similar exists for the field of clinical embryology or for clinical embryologists. STUDY DESIGN, SIZE, DURATION: A questionnaire about the situation in clinical embryology in the period of 2012-2013 in the respective European country was sent to ESHRE National representatives (basic scientists only) in December 2013. At this time, 28 European countries had at least one basic scientist in the ESHRE Committee of National Representatives. PARTICIPANTS/MATERIALS, SETTING, METHODS: The survey consisted of 46 numeric, dichotomous (yes/no) or descriptive questions. Answers were obtained from 27 out of 28 countries and the data were tabulated. Data about the numbers of 'ESHRE Certified Embryologists' were taken from the ESHRE Steering Committee for Embryologist Certification. MAIN RESULTS AND THE ROLE OF CHANCE: In 2012, more than 7000 laboratory staff from 1349 IVF clinics in 27 European countries performed over 700 000 fresh and frozen ART cycles. Despite this, clinical embryology is only recognized as an official profession in 3 out of 27 national health systems. In most countries clinical embryologists need to be registered under another profession, and have limited possibilities for organized education in clinical embryology. Mostly they are trained for practical work by senior colleagues. ESHRE embryologist certification so far constitutes the only internationally recognized qualification; however this cannot be considered a subspecialization. LIMITATIONS, REASONS FOR CAUTION: Data were obtained through different methods, by involving national embryologist societies and cycle registers, collecting information from centre to centre, and in some cases by individual assessment of the situation. For these reasons, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: This paper presents the current status of clinical embryology and clinical embryologists in Europe and is an important step towards implementation of clinical embryology as an officially recognized profession. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: No.

Congenital malformations, chromosomal abnormalities and perinatal results in IVF/ICSI newborns resulting from very poor quality embryos: a case-control study.

AIMS: To explore whether the transfer of very poor quality (VPQ) embryos is associated with an increase in congenital malformations or perinatal problems. METHODS: In this retrospective case-control study, 74 children conceived by in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI) resulting exclusively from the transfer of VPQ embryos were compared with 1,507 children born after the transfer of top morphological quality (TQ) embryos over the same period of time in the same centers. RESULTS: The prevalence of birth defects in children resulting from VPQ embryos was 1.35% (1/74), similar to the 1.72% (26/1,507) when only TQ embryos were transferred; the rate of chromosomal abnormalities detected was also similar (0.0 vs. 0.4%), as was perinatal mortality. After correcting for multiplicity (higher in the TQ group), the aforementioned parameters remained similar in the two groups. CONCLUSION: Congenital malformations and perinatal complications do not seem to be more common in children born after transfer of VPQ embryos in IVF/ICSI cycles. Given our preliminary data, which need to be confirmed in much larger studies, when only VPQ embryos are available for transfer in IVF/ICSI cycles, we do not believe that they should be discarded with the intention of avoiding birth defects or perinatal complications.

The follicular hormonal profile in low-responder patients undergoing unstimulated cycles: Is it hypoandrogenic?

STUDY QUESTION: What is the final hormonal milieu of pre-ovulatory follicles of low-responder (LR) patients undergoing unstimulated cycles? SUMMARY ANSWER: Neither androgen secretion nor LH was impaired in pre-ovulatory follicles of LR women. WHAT IS KNOWN ALREADY: Therapies currently used to improve ovarian response in LR women have an impact on the final hormonal follicular milieu, and these changes are believed to be partially responsible for determining the success rate in these women. Surprisingly, as far as we know, there is no report of the final hormonal profile of LR women undergoing unstimulated cycles or evidence that follicular androgen secretion in LR women is impaired. STUDY DESIGN, SIZE AND DURATION: A prospective case-control study including 94 women, 36 normal controls and 58 LR patients (19 Young ≤ 35 years LR and 39 Aged >35 years LR) from 2009 to 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Fifty-eight LR women were divided into two groups: Young LR (age ≤ 35; n = 19) and Aged LR (ALR; age >35; n = 39). The control group (group C) comprised 36 egg donors undergoing an unstimulated cycle in our IVF unit. Serum and follicular fluid hormonal concentrations for estradiol (E2), progesterone, testosterone and androstendione were measured. The spindle parameters of metaphase II oocytes generated from these groups were also analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-ovulatory follicles from LR patients had similar androgenic and LH concentrations to those observed in the control group. However, higher intrafollicular concentrations of FSH and progesterone were observed in ALR. Moreover, no differences were found for the spindle evaluation of oocytes between groups by the Oosight technology. LIMITATIONS, REASONS FOR CAUTION: The controls were younger and had a lower BMI than the LR women. The sample size available restricted statistical power. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that the problem with LR women is not the final pre-ovulatory follicular androgen concentration since this is similar to normal responders, but in the ability to respond to controlled ovarian stimulation protocols. Therefore, efforts should be focused on long-interval androgen priming to potentially increase the recruitment of small antral follicles rather than increasing the intraovarian androgen levels within the current cycle. STUDY FUNDING/COMPETING INTEREST: The present project has been supported by the R+D programme from the Generalitat Valenciana (Regional Valencian Government) IMPIVA MIDTF/2010/95. The authors have no conflict of interest to declare.

Oxygen consumption is a quality marker for human oocyte competence conditioned by ovarian stimulation regimens.

OBJECTIVE: To evaluate the effect of different ovarian stimulation protocols on oocyte respiration and to investigate the relationship between oocyte oxygen consumption and reproductive outcome. DESIGN: Prospective observational cohort study. SETTING: Infertility clinic in a university hospital. PATIENT(S): A total of 349 oocytes from 56 IVF treatment cycles in our oocyte donation program. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Average oocyte oxygen consumption rate in fmol/s. We correlated oxygen consumption values with ovarian stimulation features, fertilization, embryo quality on days 2 and 3, and implantation. RESULT(S): Differences in the measured oxygen consumption rates were found depending on which type of gonadotropins were used in the stimulation protocol. Higher consumption rates were found for oocytes that underwent normal fertilization compared with rates from nonfertilized or abnormal oocytes (odds ratio = 1.340; 95% confidence intervals = 1.037-1.732). Furthermore, higher oxygen consumption was observed for those oocytes which generated embryos that implanted compared with those that did not implant (6.21 ± 0.849 fmol/s vs. 5.23 ± 0.345 fmol/s. CONCLUSION(S): Measurement of oxygen consumption rates for individual oocytes before fertilization provides a noninvasive marker of oocyte quality and hence a quantitative assessment of the reproductive potential for the oocyte.

An in vivo culture system for human embryos using an encapsulation technology: a pilot study.

BACKGROUND: Animal studies have demonstrated better embryo development in vivo than in vitro. This pilot study tested the feasibility of using a novel in utero culture system (IUCS) to obtain normal human fertilization and embryo development. METHODS: The IUCS device comprised a perforated silicone hollow tube. The study included 13 patients (<36 years) undergoing a first intracytoplasmic sperm injection (ICSI) treatment and 167 metaphase II oocytes in three groups. In Group 1, 1-2 h after ICSI, sibling oocytes were assigned to IUCS or conventional in vitro culture. The device was retrieved on Day 1, and all zygotes were cultured in vitro till Day 5. In Group 2, fertilized oocytes were assigned on Day 1, embryos retrieved on Day 3 and all embryos cultured till Day 5. In Group 3, after Day 0 assignment, embryos were retrieved on Day 3 for blastomere biopsy and fluorescence in situ hybridization (FISH) and cultured until Day 5. The highest quality blastocysts were transferred on Day 5. RESULTS: Fertilization and embryo development were comparable in the in vitro and IUCS arms, with a tendency towards better embryo quality in the IUCS. FISH analysis in Group 3 revealed more normal embryos using the IUCS (P = 0.049). Three clinical pregnancies and live births were obtained: two from the IUCS arm and one from the in vitro arm. CONCLUSIONS: Our pilot study shows that this new IUCS appears to be feasible and safe, supporting normal fertilization, embryo development and normal chromosomal segregation. Furthermore, live births are possible after the transient presence of a silicone device in the uterus. Clinicaltrials.gov: NCT00480103.

Effect of different cryopreservation protocols on the metaphase II spindle in human oocytes.

The aim of this study was to evaluate the impact of different cryopreservation protocols on the repolymerization of metaphase (M)II spindles in human oocytes. Fresh aspirated donor oocytes were cryopreserved 3-4 h after retrieval using four different protocols: slow freezing using 1.5 mol/l 1,2-propanediol (PROH) + 0.2 mol/l sucrose (n = 36); 1.5 mol/l PROH + 0.3 mol/l sucrose (n = 34); 1.5 mol/l PROH + 0.3 mol/l sucrose with Na(+) depleted-choline replaced media (n = 27), and vitrification by the Cryotip method (n = 23). The control group comprised 34 fresh oocytes. Three hours after thawing, surviving and control oocytes were fixed for meiotic spindle/chromatin assessment. Survival rates were 63.8, 73.5, 74.1 and 86.9% respectively for the four protocols described above. Survival for vitrified oocytes was higher than that observed for slow freezing with 0.2 and 0.3 mol/l sucrose (P < 0.05). The proportion of oocytes showing normal spindle configuration was similar for the four protocols (81, 73.9, 88.9 and 81.3% respectively) and 88.5% for controls, showing that the MII spindle returns to its normal configuration after 3 h of post-thawing incubation under standard conditions, irrespective of the cryopreservation technique used.

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.

T helper 1-type immunity to trophoblast antigens in women with a history of recurrent pregnancy loss is associated with polymorphism of the IL1B promoter region.

Recurrent pregnancy loss (RPL) is a common disorder during early gestation. Recent evidence suggests that T helper 1 (Th1)-type immunity is associated with unsuccessful pregnancy especially in women with RPL of otherwise unknown etiology, while Th2-type immunity is associated with pregnancy success. Interleukin (IL)-1 may influence Th1/Th2 immune responsiveness and has been implicated in the establishment of successful pregnancy. In the present study, we investigated polymorphism of the IL-1beta gene (IL1B) in women with a history of RPL. Significant increases in the frequencies of IL1B promoter region variants IL1-511C and IL1B-31T were found in women with a history of RPL. Increased frequencies of these two variants and their homozygotes were found only in cases having evidence of Th1 immunity to trophoblast as determined by IFN-gamma production of peripheral blood mononuclear cells (PBMCs) stimulated with a trophoblast cell-line extract. Significantly higher IFN-gamma production by PBMCs in response to trophoblast correlated with variant IL1B-511C and its homozygocity in women with RPL. These results suggest that variants -511C and -31T in the IL1B promoter region confer risk for RPL associated with Th1 immunity to trophoblast antigens.

Expression of interleukin-1 system genes in human gametes.

There is considerable evidence that the interleukin-1 (IL-1) system plays an important role in ovarian and testicular physiology, implantation, and other reproductive events. Human embryos express IL-1beta, IL-1 receptor type I (IL-1RtI), and IL-1 receptor antagonist (IL-1RA) at both the mRNA and protein levels. The presence of IL-1alpha and IL-1beta in oocyte-conditioned media and on the surface of human oocytes suggests that these cells may also produce this cytokine; however, whether the IL-1 system gene products are present as stable mRNAs in human gametes (oocytes and spermatozoa) has not yet been demonstrated. We used stringent cell separation techniques combined with reverse transcription-polymerase chain reaction to investigate the expression of various IL-1 system genes (IL-1alpha, IL-1beta, IL-1RtI, and IL-1RA) in human gametes and cumulus cells. Our results indicate that freshly isolated cumulus cells express all these IL-1 system components. On the other hand, IL-1alpha, IL-1beta, and IL-1RtI mRNAs were not found in either unfertilized or fertilized human oocytes, and a very few metaphase II human oocytes had transcripts for either secreted (10%) or intracellular (17%) IL-1RA. Mature spermatozoa did not contain mRNA for any of the of the IL-1 system components. The absence of informational RNA for the IL-1 system components in human unfertilized and polyploid oocytes and fresh immature oocytes suggests that maternal transcripts for these genes do not contribute to early embryo development. The presence of IL-1 components at the protein level in human oocytes may be due to binding of IL-1 produced by cumulus cells or other cell types, or to prior intrafollicle transcription and translation. Likewise, IL-1 system components do not appear to have a physiological role in mature spermatozoa since none of these components are present at the mRNA or protein levels, and important functional parameters such as motility and acrosome reaction appear not to be affected by IL-1beta in vitro. However, the abundant expression of IL-1alpha, IL-1beta, the IL-1RtI, and its antagonist IL-1RA by human cumulus cells provides further evidence that the IL-1 system plays a role in human ovarian physiology.

Oocyte donation in women with recurrent pregnancy loss.

The prognosis of couples with recurrent miscarriage is controversial despite efforts made during this century to learn about the physiopathology and treatment of this troublesome condition. Here we present our experiences of employing oocyte donation in eight couples in whom the woman was a low responder to gonadotrophin stimulation and had a previous history of recurrent abortion with negative routine infertility work-up for repeated pregnancy loss. Patients were desensitized with gonadotrophin-releasing hormone analogues and supplemented with oestradiol valerate for a minimum of 15 days until oocytes were donated from in-vitro fertilization and fertile donors. Then, progesterone was added until day 100 of pregnancy. A total of 12 oocyte donation cycles were performed in these patients. Clinical pregnancy and delivery rates per cycle were 75.0 and 66.6% respectively. The delivery rate per patient was 85.7% in this series, and the miscarriage rate per cycle was 11.1%. The results of ovum donation compared favourably with low responders without a history of recurrent abortion undergoing this treatment during the study period. These results strongly suggest that the oocyte may be the origin of infertility in women with idiopathic recurrent miscarriages. In addition, the results question the role of maternal local and systemic factors in early recurrent pregnancy loss, as well as the paternal contribution to its aetiology.

Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa.

In 25 patients (14 suffering from obstructive azoospermia, six from non-obstructive azoospermia, three from asthenoazoospermia and two from absence of ejaculation) spermatozoa were extracted from testicular biopsies. Intracytoplasmic sperm injection (ICSI) with fresh testicular spermatozoa was performed in 18 cases; spermatozoa in excess were cryopreserved in pills. No pregnancies were achieved. In the remaining seven patients, testicular spermatozoa were retrieved and cryopreserved during a diagnostic testicular biopsy. After thawing, sperm motility was assessed in 17 cases (68%), and 18 ICSI with cryopreserved testicular spermatozoa were performed. The mean two-pronuclear (2PN) fertilization rate was 59%, the mean cleavage rate was 92%, and six clinical pregnancies were achieved, all of them still ongoing (pregnancy rate 33%). A comparison of the results of ICSI carried out with fresh or cryopreserved testicular spermatozoa showed that the mean 2PN fertilization rates per cycle (53 compared with 55%), mean cleavage rates per cycle (99 compared with 96%) and embryo quality were not significantly different. In conclusion, cryopreservation of testicular spermatozoa is feasible, even in patients with non-obstructive azoospermia, and the results of ICSI with frozen-thawed testicular spermatozoa are similar to those obtained using fresh testicular spermatozoa. Cryopreservation of testicular spermatozoa may avoid repetition of testicular biopsies to retrieve spermatozoa for successive ICSI cycles in patients in whom the only source of motile spermatozoa is the testicle.

Role of endometrial factors in regulating secretion of components of the immunoreactive human embryonic interleukin-1 system during embryonic development.

In the study reported here, we localized at the protein level the major components of the interleukin (IL)-1 system in the human embryo, and we investigated the endometrial factors influencing their secretion during embryonic development. To localize these components, we performed immunohistochemical experiments in 44 oocytes and 78 embryos. The following primary antibodies were used: monoclonal mouse anti-human IL-1 receptor type I (IL-1R tl), monoclonal mouse anti-human IL-1 beta, and polyclonal rabbit anti-human IL-1 receptor antagonist (IL-1ra). For embryo culture, human embryos at different developmental stages were cultured in 100-microliters drops of Ham's F-10 medium + 4 mg/ml BSA (n = 33), in 100-microliters drops of Menezo B2 culture medium (n = 18), or in wells with 1 ml of Menezo B2 culture medium (n = 8). For embryo coculture, endometrial stromal cells (ESC) and endometrial epithelial cells (EEC) were isolated from human secretory endometrium and cultured until confluence in 75% Dulbecco's Modified Eagle's Medium and 25% MCDB-105 containing antibiotics and supplemented with 10% charcoal-Dextran-treated fetal bovine serum. Individual human embryos were cocultured with experimental EEC and ESC (n = 23 and n = 4, respectively) for 5 days in 600-microliters drops of Menezo B2 medium, and conditioned medium was removed every 24 h. Human embryos were also cultured with EEC-conditioned medium (n = 9). IL-1 alpha, IL-1 beta, and IL-1ra levels were determined by ELISA in the 24-h culture- or coculture-conditioned media. Immunostaining confirmed the presence of IL-1 beta, IL-1ra, and IL-1R tl in oocytes and embryos in all stages analyzed, with no statistical differences. IL-1 alpha, IL-1 beta, and IL-1ra were absent in conditioned media of cultured embryos and embryos cocultured with ESC. However, when human embryos were cocultured with EEC or with EEC-conditioned medium alone, two different populations of embryos were observed: IL-1 producers (57% and 56%) and IL-1 nonproducers (43% and 44%, respectively). Finally, the IL-1 profile of a single human embryo cocultured with maternal EEC which successfully implanted and developed is presented, this pattern being similar to that described in the IL-1 producer population. These results demonstrate the presence of the IL-1 system in the human embryo. However, the selective release of IL-1 only when embryos were cocultured with EEC or EEC-conditioned medium indicates an obligatory role of the endometrium in the regulation of the embryonic IL-1 system. Furthermore, the differential embryonic production of IL-1 may be related to the implantation capability of the embryos.

Efficacy of intracytoplasmic sperm injection using testicular spermatozoa.

In patients with obstructive azoospermia in whom standard microsurgical procedures fail or are unfeasible, the only source of spermatozoa is the testicle. In addition, in some azoospermic patients with severe spermatogenic failure, a few spermatozoa may be present in testicular biopsy specimens despite high serum follicle stimulating hormone concentrations. In all these cases, intracytoplasmic sperm injection (ICSI) with testicular biopsy-extracted spermatozoa may offer the chance of pregnancy. To assess the efficacy of this procedure, we compared the results of two series of ICSI cycles performed during the same time period: 21 cycles using testicular biopsy-extracted spermatozoa and 83 cycles using ejaculated spermatozoa. Mean fertilization rates (59% with testicular and 68% with ejaculated spermatozoa), mean cleavage rates (93% with testicular and 90% with ejaculated spermatozoa), embryo quality (77% good quality embryos in the testicular sperm group and 77% in the ejaculated sperm group) and clinical pregnancy rates (36.8% in the testicular sperm group and 28% in the ejaculated sperm group) were not significantly different in both groups. We conclude that high fertilization, cleavage and pregnancy rates can be achieved with intracytoplasmic testicular sperm injection, reaching levels comparable with those of ICSI using ejaculated spermatozoa.

Ascorbate-supplemented media in short-term cultures of human embryos.

The present study aimed to evaluate whether ascorbate, a reactive oxygen species (ROS) scavenger, can improve fertilization and development of human embryos in vitro when added to the simple salt solution human tubal fluid (HTF) or the complex tissue culture medium Ham's F-10, which contains iron and copper in its formulation. Human oocytes, spermatozoa and embryos from 83 infertile IVF couples were randomly allocated and cultured in the presence or absence of 62.5 microM ascorbate in HTF medium (39 couples) or Ham's F-10 medium (44 couples). No significant effect of ascorbate on fertilization, number of cells and embryo grade per embryo on days 2 and 3 after insemination, or percentage of embryos showing developmental block on day 3 (those embryos that were still at the 2-cell stage) was observed when data were analysed together or divided into several groups according to the cause of infertility, quality of semen sample used for insemination and women's age in either of the two media tested. Despite these results, a positive effect of ascorbate on fertilization and embryo development in vitro cannot be totally ruled out until the effects of other, non-physiological concentrations of ascorbate and longer-term embryo cultures (to the blastocyst stage) have been tested.

Outcome of patients with endometriosis in assisted reproduction: results from in-vitro fertilization and oocyte donation.

A retrospective analysis of our in-vitro fertilization (IVF) and oocyte donation programmes was carried out in order to gain clinical knowledge of the factors involved in the aetiology of the endometriosis-associated infertility. Comparison between the IVF outcomes from 96 cycles in 78 patients with tubal infertility and from 96 cycles in 59 women with endometriosis indicates that endometriosis patients have a poor IVF outcome in terms of reduced pregnancy rate per cycle (P < 0.0004), reduced pregnancy rate per transfer (P < 0.002), and reduced implantation rate (P < 0.003). The analysis of patients undergoing oocyte donation for different reasons, including low response with or without endometriosis, showed that patients with this disease have the same chances of implantation and pregnancy as other recipients when the oocytes came from donors without known endometriosis. However, when the results of oocyte donation were classified according to the origin of the oocytes donated, patients who received embryos derived from endometriotic ovaries showed a significantly (P < 0.05) reduced implantation rate as compared to the remaining groups. Taken together, all these observations suggest that infertility in endometriosis patients may be related to alterations within the oocyte, which in turn result in embryos with decreased ability to implant.