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Choosing spermatozoa with an optimum fertilizing potential is one of the major challenges in assisted reproductive technologies (ART). This selection is mainly based on semen parameters, but the addition of molecular approaches could allow a more functional evaluation. To this aim, we used sixteen fresh sperm samples from patients undergoing ART for male infertility and classified them in the high- and poor-quality groups, on the basis of their morphology at high magnification. Then, using a DNA sequencing method, we analyzed the spermatozoa methylome to identify genes that were differentially methylated. By Gene Ontology and protein-protein interaction network analyses, we defined candidate genes mainly implicated in cell motility, calcium reabsorption, and signaling pathways as well as transmembrane transport. RT-qPCR of high- and poor-quality sperm samples allowed showing that the expression of some genes, such as AURKA , HDAC4 , CFAP46 , SPATA18 , CACNA1C , CACNA1H , CARHSP1 , CCDC60 , DNAH2 , and CDC88B , have different expression levels according to sperm morphology. In conclusion, the present study shows a strong correlation between morphology and gene expression in the spermatozoa and provides a biomarker panel for sperm analysis during ART and a new tool to explore male infertility., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2021 Nino Guy Cassuto et al.)

Objective: To evaluate an embryo transfer strategy for difficult transfers (DiTs)., Design: Prospective, nonrandomized, observational, cohort study., Setting: A hospital fertility center in France., Patients: Data were collected on all embryo transfers conducted using the strategy between February 2014 and February 2020., Interventions: Anatomical characteristics that could cause DiT were identified by transvaginal ultrasound and the catheter was adapted accordingly. Transfer was guided by transvaginal ultrasound. After passage through the cervix, a rest period was introduced to allow any contractions to stop before embryo deposition in the uterus., Main Outcome Measures: The primary criterion was the percentage of pregnancies per transfer (P/T) after an easy transfer (EaT) or a DiT. The secondary criteria included the anatomical causes of DiT and the patients' levels of discomfort., Results: Of 2,046 transfers, 257 (12%) were DiTs: minor difficulties (n = 152; 7.4%), major difficulties (n = 96; 4.7%), very significant difficulties (n = 7; 0.3%), or impossible (n = 2; 0.1%). The most common causes of DiTs were endocervical crypts (54%), tortuous cervical canal (36%), and marked uterine anteversions (30%). Several causes were often responsible for DiTs. There was no statistically significant difference in the P/T between the EaTs (n = 1,789, 41%) and all degrees of DiT (n = 257, 37%). In addition, there was no statistically significant difference between the level of patient-reported discomfort in the EaT and DiT groups., Conclusions: This study demonstrated that an adapted embryo transfer strategy, monitored by transvaginal ultrasound, led to similar pregnancy rates regardless of whether the transfer was easy or difficult., (© 2020 The Author(s).)

Objectives: To determine whether transvaginal ultrasound-guided embryo transfer is a technique that can be used routinely, whether it improves IVF outcomes and whether it makes difficult transfers easier and more successful., Material and Method: Non-randomized retrospective study conducted between 2012 and 2016 in the fertility center of the Diaconesses-Croix St-Simon hospital group. The outcomes of 3910 transfers, performed by 5 senior operators, under transabdominal ultrasound guidance are compared with those of 800 transfers, performed by 1 senior operator under transvaginal ultrasound guidance. The criteria studied are the feasibility of the technique and the percentage of pregnancies per transfer in the two populations described, as well as in the difficult and very difficult transfer populations., Results: All the transfers were feasible under transvaginal ultrasound guidance without the use of forceps or additional instruments. The percentage of pregnancies per transfer is significantly increased, when the transfer is performed under transvaginal ultrasound guidance compared with that performed under transabdominal ultrasound guidance, in the general population (38%, n=800 vs 30%, n=3910; P 0.0004) and in the reference population characterized by age <38 years and >6 oocytes collected per puncture (45%, n=490 vs 36%, n=1968; P 0.002). The percentage of pregnancies per transfer (P/T) is not significantly different in the populations of easy transfers (n 695, 38% P/T), difficult transfers (n 58, 46% P/T; P=ns) and very difficult transfers (n 47, 34% P/T; P=ns)., Conclusions: Embryo transfer is a key stage in IVF, in which the quality of performance determines the outcome. In this study, transvaginal ultrasound guidance of the transfer, which is the reference procedure in gynaecological imaging, significantly increases the percentage of pregnancies per transfer, both in the general population and in the reference population, compared with transfers performed under transabdominal ultrasound guidance. Transvaginal ultrasound facilitates the performance of difficult transfers and in particular achieves outcomes in these situations that are not significantly different from those of easy transfers. Visual monitoring of transcervical passage, which is rendered more precise and less traumatic and precision of embryo deposition are the factors that probably account for the improvement in outcomes., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background: Embryo implantation remains the main limiting factor in IVF/ICSI program. Endometrial immune remodeling events begin before implantation and are a vital process for pregnancy, preparing future maternal immune tolerance and regulating the placentation process., Methods: Between 2012 and 2014, 193 patients (analyzed group) enrolled in our IVF program benefitted of an endometrial immune profiling to determine if their uterus was immunologically ready to accept an embryo and, if not, the specific immune mechanisms involved. Subsequently, they had an effective embryo transfer (ET) with personalization of their treatments if an immune deregulation has been diagnosed. Each analyzed patient was paired to the closest patient included in the IVF program according to biological criteria (age, number of mature oocytes, stage and number of transferred embryo), which had no endometrial immune profiling (193 patients, non-analyzed group)., Finding: 78% of analyzed patients had a uterine immune dysregulation and therefore care personalization. Their corresponding live birth rate (LBR) was twice higher than observed in the matched control group with conventional cares (30.5% versus 16.6%, OR: 2.2 [1.27-3.83] p=0.004) with a simultaneous drastic reduction of miscarriages per initiated pregnancy (17.9% versus 43.2%, OR: 0.29 [0.12-0.71], p=0.005). 22% of analyzed patients had no dysregulation. They did not differ from their matched controls for LBR and miscarriages., Conclusion: Uterine immune profiling enables an integrated approach of infertility that includes endometrial immunity as a key factor in planning personalized IVF/ICSI treatments. Personalization of treatment according to the woman's uterine immune balance produced a very significantly higher LBR., (Copyright © 2016. Published by Elsevier B.V.)

Objectives: Identify, define and validate through statistical analysis the anatomical causes of difficult embryo transfers (ET)., Materials and Methods: This observational study, carried out in 306 IVF candidates, compared the frequency of anatomical anomalies of the uterus and cervix in women who underwent an easy ET with that in women who underwent a difficult ET. Anatomical anomalies were detected during an assessment of the cervix and uterus including transvaginal ultrasound, hysteroscopy and a mock transfer. Ease of ET was determined during the actual transfer procedure., Results: An easy ET was achieved in 151 patients, whereas difficulties occurred in 155 patients, among whom 55 patients underwent a "very difficult" ET. The most common anatomical characteristics associated with difficult ET were abnormal crypts in the cervical canal (86%) and tortuosity of the cervical canal (68%). Less frequent causes included: internal os contractions (28%) and pronounced anteversion of the uterus (26%). Very difficult ETs were associated with the presence of several causes., Conclusions: ET is the clinical step that has the most effect on IVF outcome. Difficult transfers are associated with a fall in pregnancy rates. The anatomical causes of difficult transfer identified in this study led to major changes in transfer procedure in our department and to the development of more adapted catheters., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Objective. To analyze DNA methylation levels between two groups of spermatozoa taken from the same sample, following morphological selection by high magnification (HM) at 6100x microscopy. A prospective study was conducted and studied 876 spermatozoa from 10 randomly selected men. Sperm morphology was characterized at HM according to criteria previously established. High-scoring Score 6 and low-scoring Score 0 sperm were selected. Sperm DNA methylation level was assessed using an immunoassay method targeting 5-methylcytosine residues by fluorescence microscopy with imaging analysis system to detect DNA methylation in single spermatozoon. Results. In total, 448 S6 spermatozoa and 428 S0 spermatozoa were analyzed. A strong relationship was found between sperm DNA methylation levels and sperm morphology observed at HM. Sperm DNA methylation level in the S6 group was significantly lower compared with that in the S0 group (p < 10(-6)), OR = 2.4; and p < 0.001, as determined using the Wilcoxon test. Conclusion. Differences in DNA methylation levels are associated with sperm morphology variations as observed at HM, which allows spermatozoa with abnormal levels to be discarded and ultimately decrease birth defects, malformations, and epigenetic diseases that may be transmitted from sperm to offspring in ICSI.

Consistent evidence from meta-analysis has linked assisted conception by IVF, and particularly intracytoplasmic sperm injection (ICSI), with an increased risk of major birth defects. To compare the risk of major malformations of children born after standard ICSI and after intracytoplasmic injection of morphologically selected spermatozoa (IMSI), a prospective population-based study was conducted from 2005 to 2010. ICSI and IMSI were performed in only one assisted reproduction unit according to its classification of spermatozoa and using fresh semen. Medical data and follow up during 2 years of 1028 infants were collected. Major malformations were identified and classified by an external independent physician. The two groups were similar concerning the parents' age, treatment, number of oocytes recovered, days of transfer, gestational age and birthweight. However, major malformations were significantly lower with IMSI (6/450, 1.33%) versus ICSI (22/578, 3.80%; adjusted odds ratio 0.35, 95% confidence interval 0.14-0.87, P=0.014), mainly affecting boys (adjusted odds ratio 2.84, 95% confidence interval 1.24-6.53, P=0.009). In conclusion, the significantly decreased risk of major birth defects associated with IMSI remained decreased after multivariate adjustment and highlights the beneficial effect of sperm selection before ICSI., (Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

The utility of sperm DNA testing remains controversial. However, it may be helpful in couples with unexplained failures of multiple assisted reproductive techniques and/or recurrent abortions. This study analysed 10,400 spermatozoa of 26 patients for sperm-head morphology with high-magnification microscopy, DNA fragmentation and sperm chromatin decondensation. A significant negative correlation was demonstrated between sperm-parameters and abnormal sperm-head morphology as assessed by high magnification (score 0 according to this study's classification): concentration (r=-0.41; P=0.03), motility (r=-0.42; P=0.03), morphology (r=-0.63; P=0.0008). No correlation was found with DNA fragmentation. However, the sperm chromatin-decondensation rate of score-0 spermatozoa was twice as high as the controls (19.5% versus 10.1%; P<0.0001). This observation suggests that score-0 spermatozoa should not be selected for intracytoplasmic sperm injection., (Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: To evaluate whether observation of spermatozoa at × 6,100 magnification can distinguish between those with and without a balanced chromosomal content., Design: Retrospective research study., Setting: Genetics laboratory of a university hospital and in vitro fertilization center., Patient(s): Six men carrying a reciprocal translocation and three men with a Robertsonian translocation., Intervention(s): Sperm fluorescence in situ hybridization (FISH) with a specific set of three probes for each translocation for determining chromosomal content, performed on both unselected spermatozoa and on spermatozoa selected at × 6,100 magnification according to the Cassuto-Barak classification., Main Outcome Measure(s): Chromosomal content in unselected and selected spermatozoa., Result(s): Chromosomal translocations lead to gametes carrying either a balanced or an unbalanced karyotype in offspring and consequently to changes in chromosome position within sperm nucleus and potentially in nuclear morphology. In the unselected spermatozoa, the rate of chromosomally balanced nuclei ranged from 37.1% to 52.6% and from 70% to 88.6% in reciprocal and Robertsonian translocations, respectively, which is in agreement with published data. In selected spermatozoa, there was no statistically significant difference between the rates of segregation modes when compared with their frequencies in unselected sperm cells., Conclusion(s): The observation of spermatozoa at high-magnification in translocation carriers cannot be used to select sperm cells with a balanced chromosomal content., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To understand the correlation between normalcy of the sperm, fertilization, and early embryo development, and to establish a detailed classification scoring scale for the individual spermatozoon with the highest predictive fertilizing potential in real time during intracytoplasmic sperm injection (ICSI)., Design: A retrospective and analysis., Setting: Laboratory Drouot., Patient(s): 27 couples with male factor infertility referred for ICSI treatment., Intervention(s): Before ICSI, motile spermatozoa were scored after aspiration., Main Outcome Measure(s): Oocyte fertilization, embryo development and morphology, outcome of scored motile injected spermatozoa., Result(s): Our suggested formula is (Normal head score = 2) + (Lack of vacuole score = 3) + (Normal base score = 1) = (Total score = 6) for a morphologic "normal top" spermatozoon, calculated with the major criteria affecting the outcome of ICSI. We take into account the normalcy of head size and shape, the base of the head, and the lack of vacuoles. Our scoring of three classes of injected spermatozoa revealed a statistically significant difference in fertilization rate: 39 out of 46 (84%), 94 out of 128 (73%), and 27 out of 44 (61%), respectively. Our examination of the contribution of maternal age in correlation to sperm score revealed a distinction between oocytes originating from women younger than 30 years and oocytes from women aged 30 years and older., Conclusion(s): Our suggested classification provides allows the best spermatozoon to be chosen for ICSI, particularly for oocytes from women aged 30 years and older.

White Leghorn chicks homozygous for B19 MHC haplotype were selected for 18 generations on tumor regression after inoculation in the wing web with an SR-D strain of Rous sarcoma virus (RSV) at 4 wk of age. Each chick was assigned a tumor profile index (TPI) based on age at death and size of the tumor. During 18 generations, 2,010 birds were divergently selected on TPI for either progression or regression of the tumor (P and R lines). A Brody growth curve was fitted for each bird. Brody function parameters included the asymptotic tumor volume (A), the factor for increased growth in progression phase (K1), the factor for decreased growth in regression phase (K2), age at maximum volume (Tmax), and maximum volume of the tumor (Vmax). Tumor growth curves were found to be different according to line, sex, and restriction fragment pattern Y complex Rfp-Y MHC haplotype (Yw*15, Yw*16, and Yw*17). Within the P line, birds from the Yw*16 haplotype reached Vmax at an earlier age than Yw*15 and Yw*17, but with a lower Vmax value. Within the R line, tumor growth curves of birds from Yw*16 and Yw*17 haplotypes were similar. Rank correlations between the different parameters and TPI were low (between -0.26 and 0.36). Heritability estimated by the sire component was high for Vmax (0.73). Heritabilities of Tmax and K2 were moderate (0.20 to 0.23 for Tmax and 0.18 to 0.21 for K2) allowing these traits to be used as selection criteria. Heritabilities of A and K1 were lower than 0.12. Modeling the growth curve should contribute to better distinction between progressors and regressors.

Selection for disease resistance related traits is a tool of choice for evidencing and exploring genetic variability and studying underlying resistance mechanisms. In this framework, chickens originating from a base population, homozygote for the B19 major histocompatibility complex (MHC) were divergently selected for either progression or regression of tumors induced at 4 weeks of age by a SR-D strain of Rous sarcoma virus (RSV). The first generation of selection was based on a progeny test and subsequent selections were performed on full-sibs. Data of 18 generations including a total of 2010 birds measured were analyzed for the tumor profile index (TPI), a synthetic criterion of resistance derived from recording the volume of the tumors and mortality. Response to selection and heritability of TPI were estimated using a restricted maximum likelihood method with an animal model. Significant progress was shown in both directions: the lines differing significantly for TPI and mortality becoming null in the "regressor" line. Heritability of TPI was estimated as 0.49 +/- 0.05 and 0.53 +/- 0.06 within the progressor and regressor lines respectively, and 0.46 +/- 0.03 when estimated over lines. Preliminary results showed within the progressor line a possible association between one Rfp-Y type and the growth of tumors.

The chicken major histocompatibility complex ( MHC) genes are organized into two genetically independent clusters which both possess class I and class IIbeta genes: the classical B complex and the Restriction fragment pattern- Y ( Rfp-Y) complex. In this study, we have examined the role of Rfp-Y genes in transplantation immunity. For this we used three sublines, B19H1, B19H2 and B19H3, derived from a line fixed for B19. Southern blots, PCR-SSCP assays using primers specific for Rfp-Y genes, and Rfp-Y class I allele-specific sequencing show that the polymorphisms observed in B19H1, B19H2 and B19H3 are due to the presence of three different Rfp-Y haplotypes. The Rfp-Y class I ( YF) alleles in these three haplotypes are highly polymorphic, and RT-PCR shows that at least two YF loci are expressed in each subline. The three sublines show Rfp-Y-directed alloreactivity in that Rfp-Y-incompatible skin grafts are rejected within 15 days, a rate intermediate between that seen in B-incompatible rejection (7 days) and that observed for grafts within the sublines (20 days). We conclude that Rfp-Y has an intermediate role in allograft rejection, likely to be attributable to polymorphism at the class I loci within this region.

Background: Our objective was to use an adapted RT-PCR technique to assess the presence of hepatitis C virus (HCV) in semen and also in different density gradient semen fractions collected from men with chronic viral hepatitis participating in an assisted reproduction programme., Methods: This study included 50 semen samples from 35 HCV(+) men, with active viral replication assessed by RT-PCR, collected the day of oocyte retrieval and used for assisted reproduction. These samples were subjected to standard assisted reproduction sperm preparation conditions, using density-gradient centrifugation with 45 and 90% layers. Aliquots of semen, 45 and 90% fractions, and embryo culture media were frozen at -80 degrees C for subsequent virological analyses. All aliquots were tested with a commercially available HCV RNA assay, adapted for use with semen after a number of technical changes. This assay yielded a sensitivity of 50-100 HCV RNA copies/ml and strongly diminished the effect of seminal amplification inhibitors., Results: HCV RNA was detected in 7/50 (14%) semen samples tested, 5/35 (14.3%) men. HCV RNA was found in only 1/50 45% fractions but never in the 90% fraction or embryo culture media. Sera from 3/5 men contained 3.19-7.40 x 10(5) IU/ml, while the two others had 4.5 and 11.7 x 10(6) IU/ml. However, HCV RNA was quantified at <600 IU/ml in the HCV(+) semen of these five patients. The ongoing pregnancy rate was of 20% (10/50) with one delivery at the time of the present report. No anti-HCV antibody was found in any of the women or the newborn., Conclusions: Although HCV is present at low concentrations in the semen of a few HCV(+) patients, no purified sperm fraction (i.e. 90% fraction) used in assisted reproduction was HCV(+) and no seroconversion was observed in the women and the newborn, thereby suggesting a very low risk of virus transmission. Nevertheless, because the presence of HCV in semen implies a possible risk of nosocomial contamination, safety regulations must be strictly applied in assisted reproduction laboratories.