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.

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.

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.