A large observational data study supporting the PROsPeR score classification in poor ovarian responders according to live birth outcome.

STUDY QUESTION: Can the Poor Responder Outcome Prediction (PROsPeR) score identify live birth outcomes in subpopulations of patients with poor ovarian response (POR) defined according to the ESHRE Bologna criteria (female age, anti-Müllerian hormone (AMH), number of oocytes retrieved during the previous cycle (PNO) after treatment with originator recombinant human follitropin alfa? SUMMARY ANSWER: The PROsPeR score discriminated the probability of live birth in patients with POR using observational data with fair discrimination (AUC ≅ 70%) and calibration, and the AUC losing less than 5% precision compared with a model developed using the observational data. WHAT IS KNOWN ALREADY: Although scoring systems for the likelihood of live birth after ART have been developed, their accuracy may be insufficient, as they have generally been developed in the general population with infertility and were not validated for patients with POR. The PROsPeR score was developed using data from the follitropin alfa (GONAL-f; Merck KGaA, Darmstadt, Germany) arm of the Efficacy and Safety of Pergoveris in Assisted Reproductive Technology (ESPART) randomized controlled trial (RCT) and classifies women with POR as mild, moderate or severe, based upon three variables: female age, serum AMH level and number of oocytes retrieved during the previous cycle (PNO). STUDY DESIGN, SIZE, DURATION: The external validation of the PROsPeR score was completed using data derived from eight different centres in France. In addition, the follitropin alfa data from the ESPART RCT, originally used to develop the PROsPeR score, were used as reference cohort. The external validation of the PROsPeR score l was assessed using AUC. A predetermined non-inferiority limit of 0.10 compared with a reference sample and calibration (Hosmer-Lemeshow test) were the two conditions required for evaluation. PARTICIPANTS/MATERIALS, SETTING, METHODS: The observational cohort included data from 8085 ART treatment cycles performed with follitropin alfa in patients with POR defined according to the ESHRE Bologna criteria (17.6% of the initial data set). The ESPART cohort included 477 ART treatment cycles with ovarian stimulation performed with follitropin alfa in patients with POR. MAIN RESULTS AND THE ROLE OF CHANCE: The external validation of the PROsPeR score to identify subpopulations of women with POR with different live birth outcomes was shown in the observational cohort (AUC = 0.688; 95% CI: 0.662, 0.714) compared with the ESPART cohort (AUC = 0.695; 95% CI: 0.623, 0.767). The AUC difference was -0.0074 (95% CI: -0.083, 0.0689). This provided evidence, with 97.5% one-sided confidence, that there was a maximum estimated loss of 8.4% in discrimination between the observational cohort and the ESPART cohort, which was below the predetermined margin of 10%. The Hosmer-Lemeshow test did not reject the calibration when comparing observed and predicted data (Hosmer-Lemeshow test = 1.266688; P = 0.260). LIMITATIONS, REASONS FOR CAUTION: The study was based on secondary use of data that had not been collected specifically for the analysis reported here and the number of characteristics used to classify women with POR was limited to the available data. The data were from a limited number of ART centres in a single country, which may present a bias risk; however, baseline patient data were similar to other POR studies. WIDER IMPLICATIONS OF THE FINDINGS: This evaluation of the PROsPeR score using observational data supports the notion that the likelihood of live birth may be calculated with reasonable precision using three readily available pieces of data (female age, serum AMH and PNO). The PROsPeR score has potential to be used to discriminate expected probability of live birth according to the degree of POR (mild, moderate, severe) after treatment with follitropin alfa, enabling comparison of performance at one centre over time and the comparison between centres. STUDY FUNDING/COMPETING INTEREST(S): This analysis was funded by Merck KGaA, Darmstadt, Germany. P.L. received grants from Merck KGaA, outside of the submitted work. N.M. reports grants, personal fees and non-financial support from Merck KGaA outside the submitted work. T.D.H. is Vice President and Head of Global Medical Affairs Fertility, Research and Development at Merck KGaA, Darmstadt, Germany. P.A. has received personal fees from Merck KGaA, Darmstadt, Germany, outside the submitted work. C.R. has received grants and personal fees from Gedeon Richter and Merck Serono S.A.S., France, an affiliate of Merck KGaA, Darmstadt, Germany, outside the submitted work. P.S. reports congress support from Merck Serono S.A.S., France (an affiliate of Merck KGaA, Darmstadt, Germany), Gedeon Richter, TEVA and MDS outside the submitted work. C.A., J.P., G.P. and R.W. declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Impact of the polycarbonate strippers used in assisted reproduction techniques on embryonic development.

STUDY QUESTION: Do daily manipulations of preimplantation embryos with polycarbonate (PC)-made bisphenol A (BPA)-releasing strippers influence embryo development? SUMMARY ANSWER: Compared to glass strippers, PC strippers enhance the blastocyst development rate but this does not seem to be BPA-related. WHAT IS KNOWN ALREADY: PC strippers have been shown to release tiny amounts (around 0.5 ng/ml BPA) of BPA in routine human IVF procedures. A chronic exposure to BPA either in vivo or in vitro during the preimplantation period can impact post-implantation and post-natal development. BPA can act rapidly by binding to membrane receptors and inducing rapid non-genomic effects. STUDY DESIGN, SIZE, DURATION: This experimental study using mouse embryos had a balanced design and blinded evaluations of the endpoints. PARTICIPANTS/MATERIALS, SETTING, METHODS: In vivo fertilized zygotes were obtained from outbred Swiss CD1 mice crossings after an ovarian stimulation. The zygotes were allocated to three daily handling conditions (HCs) and cultured until Day 4 in a single human commercial medium. Each day, the embryos were handled for 20 s either in a PC stripper (HC1) or in a glass stripper (HC2). In HC3, the embryos were pre-exposed to 0.5 ng/ml BPA before being handled for 20 s in a glass stripper. Handling operations were repeated on Days 1, 2 and 3. Embryo development was assessed blindly on Day 4. Expanded blastocysts were selected for a transcriptomic analysis using Agilent Sureprint G3 Mouse GE v2 microarrays and the retrotransposon LINE1-Orf2 expression was analysed using qRT-PCR, as a proxy for a global evaluation of the epigenetic status. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to the embryos manipulated in HC2 (n = 243), those in HC1 (n = 228) developed significantly more often to the blastocyst stage (55 vs 46%; P < 0.05). It appears the effect of these PC strippers was not BPA-related because embryos pre-exposed to BPA (HC3, n = 230) showed no difference in the blastocyst rate when compared to HC2 (43 vs 46%). When analysing same-stage blastocysts, we noticed no difference in the embryo gene expression between the three HC groups. LARGE SCALE DATA: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE148868. LIMITATIONS, REASONS FOR CAUTION: Our results using a mouse model designed to mimic human conditions (outbred strain, human commercial IVF dishes and a unique commercial human embryonic culture media) are reassuring since no gene was found to be differentially expressed, including LINE-1 genes, as a proxy for a global evaluation of the epigenetic status. However, no global epigenetic analysis of the genome has been performed. Furthermore, we did not evaluate post-implantation events, although BPA exposure during peri-conception could affect foeto-placental and post-natal development. WIDER IMPLICATIONS OF THE FINDINGS: Based on the precautionary principle, several European countries banned the use of BPA in baby bottles and food packaging several years before European Agencies took an official position. The question of applying this principle to plastics in closed contact with human embryos is raised. Further studies are needed for a decision to be made. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a grant from the Agence de Biomédecine (AOR 2016). The authors declare no competing interest.

[Regional state-of-the-art of the access to oncofertility consultation for young women with breast cancer]. / État des lieux régional de l'accès à une consultation d'oncofertilité chez les femmes jeunes ayant un cancer du sein.

OBJECTIVES: According to the 2004 Bioethics Act, oncofertility counselling must be systematically offered to all women of childbearing age before they are exposed to potentially gonadotoxic treatment. The main objective of this study was to evaluate the proportion of women under 40 years of age treated with chemotherapy for breast cancer in Midi-Pyrénées who have received an oncofertility consultation. A secondary objective was to assess practitioners' knowledge on the subject. METHODS: A cross-reference was made between the databases of the oncology network in Midi-Pyrénées and the two approved centres for the preservation of fertility in the region. A computerized practitioner questionnaire was sent to all surgeons and oncologists who could manage these patients. RESULTS: From 2012 and 2017, 667 women aged≤40 years received (neo)adjuvant chemotherapy treatment: only 156 (23.4%) had access to an oncofertility consultation and 58 (8.7%) received preservation. This rate (23.4%) varied according to the age of the patients, ranging from 56.9% for those aged 25-29 to 13.4% for those aged 35-39 and the managing institution. Of the 85 practitioners surveyed, 45 (55%) responded to the questionnaire, and of these 20 (44%) knew that ovarian stimulation treatment could be used even in hormone-dependent breast cancer situations and 13 (29%) of practitioners believed that the time required to preserve fertility was more than 1 month. CONCLUSION: Our study revealed a significant disparity in access to oncofertility consultation. It is essential to set up information and awareness-raising actions on the subject.

Expression of phospholipase PLC Zeta in human spermatozoa: impact of cryopreservation.

BACKGROUND: Cryopreservation is used for infertility treatment and for fertility preservation. The results of the use of frozen spermatozoa for ART (Assisted Reproductive Technology) are lower than those of fresh spermatozoa. The phospholipase C Zeta (PLCζ) protein is involved in oocyte activation. OBJECTIVES: The aim of this study was to compare the percentage of spermatozoa expressing phospholipase C Zeta protein before and after a frozen-thawing cycle. MATERIALS AND METHODS: Samples were provided after at least 2 days of sexual abstinence. A part of the fresh ejaculate (200 µL) was recovered for the study. Fifty microliters was necessary to carry out the technique before freezing. The remaining 150 µl was frozen according to a slow manual freezing technique. The samples were treated based on the procedure described by Yelumalai et al. (Fertil. Steril., 104, 2015, 561-568.e4) and Grasa et al. (Hum. Reprod. Oxf. Engl. 23, 2008, 2513-2522). RESULTS: Freezing was associated with a decrease in the percentage of spermatozoa exhibiting PLCζ (44 ± 22% before vs 31 ± 19% after, p < 0.05). The percentage of spermatozoa exhibiting PLCζ at post-acrosomal position was significantly greater before freezing (8% vs 5%, p < 0.05). There was no significant difference for the percentage of spermatozoa exhibiting PLCζ at equatorial position (15% before freezing versus 12% after thawing, NS). DISCUSSION: The results of the present study show that the presence of PLCζ on spermatozoa is decreased after freezing-thawing procedures. PLCζ is a soluble cytosolic protein (Nomikos et al., ), so it can be lost during cryopreservation. These membrane alterations are probably multifactorial. CONCLUSION: Our results, in agreement with other studies, raise the hypothesis that cryopreservation reduces spermatic PLCζ expression.

Sperm morphology: assessment, pathophysiology, clinical relevance, and state of the art in 2017.

For over 30 years, sperm morphology assessment has been one of the most common tests in evaluation of fertility. This review examines the clinical relevance of sperm morphology assessment in the diagnosis of infertility and in assisted reproductive technology, as well as its analytical reliability. Publications on the pathophysiology, the analytical reliability of the test and its clinical relevance in diagnosis and in Assisted Reproductive Technology (ART) were evaluated. This review compared and discussed study methodologies and results, including patient characteristics, preparation, smear staining methods and classification systems. The assessment of the percentage of some abnormalities such as for example thin head, amorphous head, or bent or asymmetrical neck is of little clinical use, and their pathophysiology is not well explained as most are physiological traits. Some studies have highlighted correlations between the percentage of normal forms and functional sperm abnormalities, as well as correlations with ability to conceive in vivo and, in some situations, with the success of intra-uterine insemination (IUI) or conventional IVF. However, except in the case of some specific sperm defects (easy to detect with 99 or 100% of spermatozoa affected) and which are often linked to genetic disorders (globozoospermia, macrocephaly, decapitated sperm syndrome and fibrous sheath dysplasia), sperm morphology assessment has very poor sensitivity and specificity in the diagnosis of infertility. Moreover, there is very little evidence that indices of multiple sperm defects [sperm deformity index (SDI), teratozoospermia index (TZI), and multiple abnormalities index (MAI)] are relevant. Above all, many publications report a major lack of analytical reliability of this test, mainly in assessment of the details of sperm abnormalities. Many questions arise concerning how and when sperm morphology should be assessed, and how to interpret the thresholds of normal forms. Questions are raised on the real clinical impact of this test.

Bisphenol A in culture media and plastic consumables used for ART.

STUDY QUESTION: Do the embryo culture media and plastic materials used during assisted reproductive technology (ART) laboratory procedures expose embryos to bisphenol A (BPA)? SUMMARY ANSWER: BPA was not detected in embryo culture media or protein supplements at concentrations above those encountered in normal patient serum and follicular fluids. WHAT IS KNOWN ALREADY: BPA is strongly suspected of altering the epigenome during mammalian development. Medical devices have been shown to be a source of BPA exposure in adult and neonatal intensive care units. STUDY DESIGN, SIZE, DURATION: An analytical study of ART culture media and plastic labware products was performed under conditions close to routine practice and if BPA was detected, tests were carried out under more stringent conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two single-step embryo culture media, two sequential media and three different protein supplements [a purified human serum albumin (HSA), a synthetic serum substitute, and a recombinant HSA] were tested for BPA. Thirty-three different plastic consumables, used from oocyte collection through to embryo transfer, were tested for their ability to leach BPA into their surrounding environment.BPA concentrations were measured according to a previously described liquid chromatography/mass spectrometry method. This method is linear over the calibration range from 0.5 to 100 ng/ml using a linear model weighted by 1/X² and validated in terms of selectivity, linearity, repeatability, reproducibility and limit of quantification (0.5 ng/ml). MAIN RESULTS AND THE ROLE OF CHANCE: Neither the culture media nor the protein supplements were shown to contain detectable levels of BPA. None of the plastic materials leached BPA into the surrounding medium at levels higher than the upper limit detected previously in serum and follicular fluids in women (about 2 ng/ml). However, the plastic of the three tested strippers used for oocyte denudation/embryo handling did contain BPA. Two of these strippers are made with polycarbonate, a plastic whose synthesis is known to require BPA. LIMITATIONS, REASONS FOR CAUTION: This study is limited to the ART media and materials tested here and using a BPA assay with a limit of quantification at 0.5 ng/ml. A minimum volume was required for testing, and one type of plastic labware could not be tested in conditions identical to those in routine use. WIDER IMPLICATIONS OF THE FINDINGS: Although we demonstrated that some plastic materials used in ART contain BPA, under routine conditions none appear capable of leaching BPA at levels higher than those from maternal internal exposure. However, BPA is strongly suspected of altering the epigenome. Since important epigenetic modifications occur in the early embryonic stage, it is questionable whether plastics that contain BPA, polycarbonate in particular, should be used in the manufacture of plastic consumables for ART procedures. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a grant from the Agence de Biomédecine (AOR 2012) and by a grant from the French Ministry of Health (Clinical Research Hospital Program 2012; no.12-018-0560). The authors declared no competing interest.

Intracytoplasmic morphologically selected sperm injection (IMSI) does not improve outcome in patients with two successive IVF-ICSI failures.

PURPOSE: Assessment of sperm morphology has been reconsidered since 2001 with the development of motile sperm organelle morphology examination (MSOME). This observation technique that combines high magnification microscopy and the Nomarski interference contrast makes it possible to select spermatozoa with as few vacuoles as possible before microinjection into the oocyte (intracytoplasmic morphologically selected sperm injection, IMSI). More than 10 years after the development of IMSI, the indications of the IMSI technique and its ability to increase pregnancy and/or birthrates (compared with conventional ICSI) are still subject to debate. We aimed to better define the interest of IMSI in the third attempt. METHODS: We assessed the benefit of IMSI by carrying out a retrospective comparative study between IMSI and conventional ICSI during a third ART attempt. Two hundred sixteen couples with two previous ICSI failures were studied between February 2010 and June 2014. RESULTS: IMSI did not significantly improve the clinical outcomes compared with ICSI, either for implantation (12 vs 10%), clinical pregnancy (23 vs 21%), or live birth rates (20 vs 19%). CONCLUSION: This study provides supplementary arguments for not achieving IMSI procedure in the third attempt after two previous ICSI failures.

Sperm freezing to address the risk of azoospermia on the day of ICSI.

STUDY QUESTION: In which cases is freezing of ejaculated sperm indicated before ICSI? SUMMARY ANSWER: Sperm freezing should be performed only when out of two analyses at least one total sperm count in the ejaculate is lower than 10(6). WHAT IS KNOWN ALREADY: Due to variations in individual sperm parameters, in cases of severe oligozoospermia there is a risk of absence of spermatozoa on the day of ICSI, leading to cancellation of the attempt. Sperm freezing can avoid this problem but little is known of the parameters governing the decision to freeze sperm or not. STUDY DESIGN, SIZE, DURATION: This retrospective study included 247 men who underwent sperm cryopreservation to prevent the risk of azoospermia on the day of ICSI, from 2000 to 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Receiver operating characteristic curve analysis was used to define the threshold value. The lowest total sperm count per ejaculate was studied as a predictive factor for the use of frozen sperm in a total of 593 ICSI attempts. Moreover, 2003 patients who had at least 4 semen analyses for andrological diagnosis have been studied to evaluate the reproducibility of sperm count. To evaluate the psychological impact of sperm freezing, a questionnaire was administered to 84 men who attended for sperm cryopreservation between June and December 2014. The cost of sperm freezing was analysed according to the French prices. MAIN RESULTS AND THE ROLE OF CHANCE: When at least one total sperm count was <10(5) the risk of azoospermia in at least one ICSI attempt was 52% (34/66) versus 3% (5/181) when all counts were ≥10(5) (P < 0.0001). However, the study of the reproducibility of pre-ICSI semen analyses has shown wide variations among ejaculates, and therefore sperm freezing is recommended when one analysis from at least two, showed a sperm count <10(6). Such a policy could allow a saving of about €70 000 by avoiding unnecessary sperm freezings. The psychological impact of sperm freezing was good since >70% of men had positive feelings about this technique. LIMITATIONS, REASONS FOR CAUTION: This was a fairly short-term study and preservation of future fertility was not assessed. It appeared impossible to find a threshold that would predict the risk of azoospermia with 100% accuracy. Therefore there is still a risk of absence of spermatozoa on the day of ICSI despite a good negative predictive value when no total sperm count was lower than 10(5). WIDER IMPLICATIONS OF THE FINDINGS: These data suggest that sperm freezing should be proposed when total sperm count is lower than 10(6) to avoid cancellation of the ICSI attempt due to azoospermia.

Sperm vacuoles cannot help to differentiate fertile men from infertile men with normal sperm parameter values.

STUDY QUESTION: Can the assessment of sperm vacuoles at high magnification contribute to the explanation of idiopathic infertility? SUMMARY ANSWER: The characteristics of sperm head vacuoles (number, area, position) are no different between fertile controls and patients with unexplained infertility. WHAT IS KNOWN ALREADY: Until now, the assessment of sperm head vacuoles has been focused on a therapeutic goal in the intracytoplasmic morphologically selected sperm injection (IMSI) procedure, but it could be pertinent as a new diagnostic tool for the evaluation of male fertility. STUDY DESIGN, SIZE, DURATION: This diagnostic test study with blind assessment included a population of 50 fertile men and 51 men with idiopathic infertility. They were selected from September 2011 to May 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fertile men were within couples who had a spontaneous pregnancy in the last 2 years. Infertile men were within couples who had unexplained infertility and were consulting in our centre. After analysis of conventional sperm parameters, we investigated the number, position and area of sperm head vacuoles at high magnification (×6000) with interference contrast using an image analysis software. We also carried out a nuclear status analysis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay (TUNEL), sperm chromatin structure assay (SCSA) and aniline blue staining. MAIN RESULTS AND THE ROLE OF CHANCE: Concerning the vacuoles data, we did not find any significant difference between the two populations. We found no significant correlation between the vacuolar parameters (mean number of vacuoles, relative vacuole area and percentage of spermatozoa with large vacuoles) and either conventional semen parameters, male age or the data from the aniline blue staining, SCSA assay and TUNEL assay. LIMITATIONS, REASONS FOR CAUTION: Despite the fact all of the vacuole parameters values were identical in fertile and infertile men, we cannot totally exclude that a very small cause of unexplained infertilities could be related to an excess of sperm vacuoles. WIDER IMPLICATIONS OF THE FINDINGS: In line with its widely debated use as a therapeutic tool, sperm vacuole assessment for diagnostic purposes does not seem useful. STUDY FUNDING/COMPETING INTERESTS: The study was funded by a grant from Association pour la Recherche sur les Traitements de la Stérilité. There are no competing interests to declare.

Is intracytoplasmic morphologically selected sperm injection (IMSI) beneficial in the first ART cycle? a multicentric randomized controlled trial.

Intracytoplasmic morphologically selected sperm injection (IMSI), by selecting spermatozoa at high magnification improves the outcome of intracytoplasmic sperm injection (ICSI) mainly after several failures. However, only few monocentric randomized studies are available and they do not analyse results as a function of sperm characteristics. In 255 couples attempting their first assisted reproductive technology (ART) attempt for male infertility (motile sperm count <1×106 after sperm selection, but at least 3×106 spermatozoa per ejaculate to allow a detailed analysis of sperm characteristics), a prospective randomized trial was performed to compare the clinical outcomes of IMSI and ICSI and to evaluate the influence of sperm characteristics on these outcomes. IMSI did not provide any significant improvement in the clinical outcomes compared with ICSI neither for implantation (24% vs. 23%), nor clinical pregnancy (31% vs. 33%) nor live birth rates (27% vs. 30%). Moreover, the results of IMSI were similar to the ICSI ones whatever the degree of sperm DNA fragmentation, nuclear immaturity and sperm morphology. These results show that IMSI instead of ICSI has no advantage in the first ART attempts. However, this does not rule out IMSI completely and more randomized trials must be performed especially regarding patients carrying severe teratozoospermia, or high sperm DNA fragmentation levels or having previous ICSI failures.

Sperm cephalic vacuoles: new arguments for their non acrosomal origin in two cases of total globozoospermia.

To add new arguments concerning the origin of the sperm-head vacuoles observed under high magnification with interference contrast microscopy, we carried out in two patients with total globozoospermia confirmed using transmission electron microscopy (TEM), a detailed sperm morphometric analysis with high magnification (×6000) under Nomarski contrast, an acrosomal status analysis (using fluorescent labelling with peanut agglutinin (PNA) lectins and anti-CD46 antibodies) and a nuclear status analysis (using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay TUNEL, sperm chromatin structure assay SCSA and aniline blue staining). Our two patients with globozoospermia had relative sperm vacuole areas of 6.3% and 5%, similar to those observed in a reference population of 12 fertile men (5.9%). TUNEL and SCSA assays gave normal results in both patients, although the percentage of immature nuclei using aniline blue staining was increased (27 and 46% for patient 1 and 2 respectively). Cytofluorescence and TEM analysis evidence differences between the two patients: although no acrosomal neither Golgi residue could be detected in patient 1, patient 2 had positive PNA lectin labelling for 9% of spermatozoa and Golgi residues were seen using electron microscopy. Unlike patient 1, a live birth could be obtained after intracytoplasmic sperm injection (ICSI) for patient 2. This descriptive study of two patients with total globozoospermia confirmed using TEM argue in favour of a deep analysis of total globozoospermia before assisted reproductive technology and provides further information on the non-acrosomal origin of the sperm-head vacuoles observed under high magnification.

Transmissions of hepatitis C virus during the ancillary procedures for assisted conception.

Since mother to child transmissions of hepatitis C virus (HCV) have been reported to be low, teams involved in assisted reproductive technologies have accepted HCV positive patients into their programmes. We report in the present paper two cases of undoubted patient to patient HCV transmission while patients were attending for assisted conception. In both cases, HCV genotyping and sequencing of the first hypervariable region of the HCV genome provided molecular evidence for nosocomial transmission. Investigations made to elucidate the route of contamination have shown that the most likely route of contamination is through healthcare workers. Such nosocomial HCV infection has been reported in other healthcare situations, mainly in dialysis units, and physical proximity was also suspected to be at the origin of the infection. We conclude that assisted reproduction teams must be very prudent when including such patients in their programmes.

Enhancement of zona binding using 2-hydroxypropyl- beta-cyclodextrin.

Since membrane cholesterol depletion is known to play an important role in sperm capacitation, we have investigated the effect of 2-hydroxypropyl-beta-cyclodextrin, a cyclic oligosaccharide that mediates cholesterol efflux, on sperm functions. Sperm treatment with cyclodextrin did not affect the motility patterns but induced an increase in sperm binding to zona pellucida (24 +/- 5 versus 13 +/- 4 in control, P < 0.01). Cyclodextrin treatment was associated with an increase in spontaneous acrosome reaction (32 +/- 8% versus 22 +/- 4% in controls after a 4 h incubation, P < 0.05; 61 +/- 10% versus 50 +/- 11% in controls after a 24 h incubation, NS) but with a decrease in acrosome response to ionophore challenge (44 +/- 5% versus 51 +/- 3% in controls, P < 0.05). Concerning cell sterols, cyclodextrin induced a rapid and dramatic fall in the cholesterol and desmosterol content of spermatozoa. We conclude that cyclodextrin is a powerful capacitating agent, but since it induces an increase in spontaneous acrosome loss, it needs to be further evaluated before routine use in assisted reproductive technology media.

Computer-assisted assessment of sperm morphology: comparison with conventional techniques.

The aim of the present study was to compare conventional and computer-assisted morphology assessment of spermatozoa. Sixty-two semen samples from patients undergoing in vitro fertilization (IVF) and 40 samples from patients undergoing an intracytoplasmic sperm injection (ICSI) were studied using both techniques. The percentage of normal spermatozoa found was closely correlated between the techniques (r=0.788, p < 0.0001). The intra-operator variation was low for both techniques but the inter-operator variation was much higher with the conventional than with the computer-assisted method (coefficient of variation = 0.43 vs. 0.08, respectively, for conventional and computer-assisted assessments). The percentage of spermatozoa with normal morphology, as well as sperm motility, was significantly enhanced after PureSperm preparation, whatever the method used for assessment. In the IVF study, fertilization rate was poorly correlated with sperm morphology using both methods. However, combined with motility, morphology assessed with the computer allowed discrimination of two groups of patients with significantly different fertilization rates (30.5 +/- 5.4% vs. 63.1 +/- 5.4%, p < 0.0001). In contrast, the fertilization rate in ICSI was influenced neither by sperm morphology nor by motility. In conclusion, computer-assisted assessment of sperm morphology has a slightly better predictive value for ART than conventional assessment, but above all is much more reproducible, allowing standardization.

Use of a medium devoid of any human or animal compound (SMART2) for embryo culture in intracytoplasmic sperm injection.

PURPOSE: Our purpose was to evaluate the efficiency of a medium, devoid of any human or animal compound and specially designed for early embryo development (from the zygote to the eight-cell stage), SMART2, in intracytoplasmic sperm injection (ICSI) and to compare it with a medium containing human serum albumin (EllioStep2). METHODS: Oocytes from 50 ICSI attempts were randomly placed, after sperm injection, into either SMART2 or EllioStep2. After a 48-hr incubation, the embryos were examined for quality scoring before transfer or freezing. RESULTS: The percentage of normally fertilized oocytes per intact oocytes was slightly higher using SMART2 (139/199 vs. 135/224, respectively, for SMART2 and EllioStep2; P < 0.05). The distribution of embryo scores and the percentage of embryos with a fair morphology (71/143 vs. 72/148, respectively, for SMART2 and EllioStep2; not significant) were identical in both media. CONCLUSIONS: These data show that SMART2 medium can be successfully used for early embryo growth and, because it is devoid of any human or animal compound, offers better safety for patients than conventional media.

Human sperm capacitation and in-vitro fertilization in a chemically defined and protein-free medium SMART1.

Media for sperm capacitation and in-vitro fertilization (IVF) are supplemented by proteins (albumin, globulins) extracted from human or animal sera, which raises the problem of potential contamination by pathogens. The present study aimed to evaluate the efficiency of a protein-free medium (SMART1, Bio-Media, Boussens, France) and to compare it with a human serum albumin (HSA) containing medium (FertiCult, FertiPro NV, Aalter, Belgium). In the first part of the study, media were compared for their ability to support human sperm functions. Total motility, progressive motility and rapid motility were no different between media after a 30 min and a 4 h incubation, but were significantly reduced using SMART1 after a 24 h incubation. However, the kinematic parameters (straight line velocity, mean path velocity, curvilinear velocity and mean amplitude of lateral head displacement) were significantly lower using SMART1, whatever the incubation time. The spontaneous acrosome reaction and the acrosome response to A23187 ionophore were similar in both media. In the second part of the study, media were compared in a randomized trial in 93 IVF attempts. No significant difference was found in the transfer per attempt rate (92 versus 87% respectively for SMART1 and FertiCult, NS) but the percentage of fertilized oocytes was significantly higher using SMART1 (65 versus 55% respectively for SMART1 and FertiCult, P < 0.01). The percentage of embryos with a fair morphology was identical in both media (30 versus 30% respectively for SMART1 and FertiCult, NS). In conclusion, despite a decrease in sperm kinematics, SMART1 medium allows an increase in fertilization rate and, since it is devoid of any human or animal compound, may be preferable for human use.

Use of a plant enzyme preparation (Coronase) instead of hyaluronidase for cumulus cell removal before intracytoplasmic sperm injection.

The aim of this study was to compare the efficiency of a plant enzyme preparation (Coronase) with animal extracted hyaluronidase to remove cumulus cells before intracytoplasmic sperm injection (ICSI). The first part of the study was performed on mouse oocytes and embryos. Coronase displayed a similar efficiency to that of hyaluronidase for removing cumulus cells and the same percentage of activated oocytes was obtained with both techniques. However, prolonged incubation in Coronase, 120 min, led to a degeneration of oocytes. Incubation of 2-cell mouse embryos for 10 min with Coronase did not affect their subsequent in-vitro development to blastocyst. Coronase was then compared to hyaluronidase in the treatment of human oocytes prior to ICSI. The time required for total denudation was slightly longer using Coronase (98 s +/- 25 s versus 84 s +/- 24 s respectively for Coronase and hyaluronidase; P < 0.01). However, the two pronuclear (2PN) fertilization rate (70/103 versus 63/107 respectively for Coronase and hyaluronidase, not significant) and the percentage of embryos with a good morphology (39/74 versus 32/67 respectively for Coronase and hyaluronidase, not significant) were identical with both treatments. In conclusion, Coronase displays an efficiency close to that of hyaluronidase, without any adverse effect on oocytes, and may be preferable for human use.

Randomized comparison of three media used for embryo culture after intracytoplasmic sperm injection.

Since the metabolic requirements of fertilization and early embryonic development are very different, we have tested a new culture medium (EllioStep2, Ellios Bio-Media, Paris, France) specially designed for the first cleavages and compared it with two conventional media: BM1 (Ellios Bio-Media, Paris, France) and IVF50 (Scandinavian IVF Science, Gothenburg, Sweden). In order to avoid any interference with fertilization, the test was performed as part of an intracytoplasmic sperm injection (ICSI) study. A total of 416 ICSI attempts were randomly performed using one or other of the media. After sperm injection, oocytes were incubated either in EllioStep2 or in BM1 or in IVF50. The embryo quality, pregnancy and implantation rates, number of frozen embryos were compared in the different media. The percentage of fair embryos (grades 4 and 3) was significantly higher when EllioStep2 was used than when oocytes was cultured in BM1 medium (54 versus 47%; P < 0.01) or in IVF50 (69 versus 61%; P < 0.01). The pregnancy rate per transfer and the implantation rate were not significantly higher with EllioStep2 than with BM1 or IVF50. However, the percentage of embryo freezings per attempt was significantly higher with EllioStep2 than with BM1 (47/105 versus 28/105; P < 0.01). In conclusion, the use of EllioStep2 is associated with an increase in embryo quality permitting a higher number of embryo freezings.