Abstract Study question Does semen samples quality contribute to blastocyst diversity by correlating with the presence of DNA in the blastocoelic fluid (BF)? Summary answer Semen samples quality is associated with the detection of DNA in BFs from both euploid and aneuploid blastocysts What is known already DNA was detected by whole genome amplification (WGA) in BFs from expanded blastocysts dependently on the blastocyst chromosome condition (assessed by PGT-A on trophectoderm (TE) cells), being more frequent in aneuploid blastocysts. TE-euploid blastocysts with positive BF-WGA also seem to have less chances to implant compared to those with failed BF-WGA. The extrusion of abnormal cells from the embryo proper was proposed as a mechanism triggered by the mosaic embryo to eliminate abnormal cells. Severe male factor samples are at risk of causing embryo mosaicism. Possible association between semen quality and DNA in BFs has not yet been investigated. Study design, size, duration This retrospective cohort study included 115 patients (maternal age 37.9±4.1), which underwent PGT-A in the last three years. The aim of the study was to evaluate whether the detection of DNA in BFs was correlated with semen samples indices. Both TE-euploid and TE-aneuploid blastocysts were considered for the analysis and stratified according to the semen samples quality as normozoospermic (N), moderate oligoasthenoteratozoospermic (m-OAT) and severe OAT (s-OAT) following the 2021 WHO criteria. Participants/materials, setting, methods BF and trophectoderm (TE) biopsies were collected from high grade expanded blastocysts before vitrification, and submitted to WGA. Amplification after WGA was evaluated by loading an aliquot of the amplified product onto a 1.5% agarose gel. 24-chromosome analysis was performed on TE biopsies. To assess the impact of semen samples quality on the detection of DNA in BFs, a multiple logistic regression analysis was performed correcting for maternal age as a confounding factor. Main results and the role of chance The BF from 590 blastocysts (265 TE-euploid and 325 TE-aneuploid) were submitted to WGA. In TE-euploid blastocysts, 125 derived from N semen samples, 52 from m-OAT, and 88 from s-OAT. The incidence of positive BF-WGA was proportional to the severity of the male factor condition and occurred in 48.0% of blastocysts originated from N samples, in 55.8% of blastocysts derived from m-OAT, and in 60.2% of blastocysts from s-OAT. In TE-aneuploid blastocysts, 178 originated from N semen samples, 80 from m-OAT, and 67 from s-OAT. The incidence of positive BF-WGA showed an opposite trend compared with the TE-euploid group being detected in 64.0% of blastocysts originated from N samples, in 66.3% of blastocysts derived from m-OAT, and in 52.2% of blastocysts obtained from s-OAT. The relationship between semen quality and positive BF-DNA amplification was confirmed by a multiple logistic regression model setting the BF-WGA as the outcome variable. In the TE-euploid blastocyst cohort, s-OAT showed an odd ratio (OR) of 1.78 (95% CI: 1.02-3.14; p=0.045), whereas in the TE-aneuploid blastocyst group, s-OAT displayed an OR of 0.55 (95% CI: 0.31-0.98; p=0.043). Limitations, reasons for caution This is a retrospective cohort study including a limited number of cases where the different categories of sperm samples were not equally represented. Although the results of BF-WGA were evaluated per single blastocyst, they cannot be considered independent variables. Wider implications of the findings In s-OAT samples, abnormal cell elimination is especially active in TE-euploid blastocysts, whereas in TE-aneuploid blastocysts the efficiency of this process is reduced suggesting the difficulty of eliminating abnormal cells in an aneuploid background. BF-DNA amplification is a minimally invasive tool contributing additional knowledge to elucidate blastocyst diversity. Trial registration number None

Research question: In sperm samples with complete asthenozoospermia, pregnancies are achieved by intracytoplasmic sperm injection (ICSI), but this condition has a negative impact on fertilization and embryo development owing to the difficulty of identifying viable cells for oocyte injection. Is the selection of sperm cells with head birefringence properties under polarizing light a successful strategy to identify viable spermatozoa? Design: This study included 192 ICSI cycles with complete asthenozoospermia (83 ejaculated and 109 testicular samples) performed under polarized light. Two types of sperm head birefringence were distinguished: partial (presumably reacted spermatozoa) and total (presumably intact acrosome). In some sperm cells, no birefringence was present. The main outcome of the study was the cumulative live birth rate (cLBR) per ICSI cycle. Results: Seventy-three deliveries resulted with 38.0% cLBR per ICSI cycle. The injection of birefringent spermatozoa led to significantly higher rates of fertilization, embryo development and implantation compared with the absence of birefringence (P < 0.001). Similarly, the resulting cLBR were 53.6% and 9.0%, respectively (P < 0.001). Spermatozoa with partial head birefringence yielded significantly higher fertilization and embryo utilization rates compared with total birefringence. The cLBR showed the same trend (62.7% and 46.7%, respectively, P = 0.048). Multiple logistic regression analysis showed the pattern of partial birefringence to be strongly associated with live birth rate. Conclusions: Immotile sperm cells with birefringence properties under polarized light have higher chances of inducing fertilization and embryo development compared with non-birefringent cells. In addition, a pattern of partial birefringence, associated with a reacted acrosome, is the strongest predictive factor for live birth delivery, both in ejaculated and testicular samples.

Amniotic membrane and amniotic fluid derived cells are regarded as a promising stem cell source for developing regenerative medicine techniques, although they have never been tested on male infertility diseases such as varicocele (VAR). The current study aimed to examine the effects of two distinct cell sources, human Amniotic Fluid Mesenchymal Stromal Cells (hAFMSCs) and amniotic epithelial cells (hAECs), on male fertility outcomes in a rat induced VAR model. To explain cell-dependent enhancement of reproductive outcomes in rats transplanted with hAECs and hAFMSCs, insights on testis morphology, endocannabinoid system (ECS) expression and inflammatory tissue response have been carried out alongside cell homing assessment. Both cell types survived 120 days post-transplantation by modulating the ECS main components, promoting proregenerative M2 macrophages (Mφ) recruitment and a favorable anti-inflammatory IL10 expression pattern. Of note, hAECs resulted to be more effective in restoring rat fertility rate by enhancing both structural and immunoresponse mechanisms. Moreover, immunofluorescence analysis revealed that hAECs contributed to CYP11A1 expression after transplantation, whereas hAFMSCs moved towards the expression of Sertoli cell marker, SOX9, confirming a different contribution into the mechanisms leading to testis homeostasis. These findings highlight, for the first time, a distinct role of amniotic membrane and amniotic fluid derived cells in male reproduction, thus proposing innovative targeted stem-based regenerative medicine protocols for remedying high-prevalence male infertility conditions such as VAR.

Journal Article Reply: Failure to detect DNA in blastocoel fluids after whole genome amplification—is it the next add-on? Get access L Gianaroli, L Gianaroli SISMER, Reproductive Medicine Unit, Bologna, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar D Perruzza, D Perruzza SISMER, Reproductive Medicine Unit, Bologna, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar C Albanese, C Albanese SISMER, Reproductive Medicine Unit, Bologna, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar S Azzena, S Azzena SISMER, Reproductive Medicine Unit, Bologna, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar C Tabanelli, C Tabanelli SISMER, Reproductive Medicine Unit, Bologna, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar A P Ferraretti, A P Ferraretti SISMER, Reproductive Medicine Unit, Bologna, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar M C Magli M C Magli SISMER, Reproductive Medicine Unit, Bologna, Italy Correspondence address. SISMER, Reproductive Medicine Unit, Bologna, Italy. E-mail: cristina.magli@sismer.it https://orcid.org/0000-0002-2889-8479 Search for other works by this author on: Oxford Academic PubMed Google Scholar Human Reproduction, dead141, https://doi.org/10.1093/humrep/dead141 Published: 19 July 2023

STUDY QUESTION Is the presence of DNA in the blastocoel fluid (BF) of expanded blastocysts, assessed by whole genome amplification (WGA), associated with the clinical outcome at the first transfer? SUMMARY ANSWER At the first transfer, blastocysts with negative BF-WGA have more chance to implant and to develop to term than those with positive BF-WGA results, both in preimplantation genetic testing for aneuploidies (PGT-A) cycles (where only euploid blastocysts resulting from the chromosomal analysis of trophectoderm (TE) biopsies were transferred) and in IVF/ICSI conventional cycles. WHAT IS KNOWN ALREADY Retrospective studies conducted in patients undergoing PGT-A have shown that the incidence of negative BF-WGA was significantly higher in TE-euploid blastocysts than in TE-aneuploid blastocysts. In addition, after the transfer of TE-euploid blastocysts, the ongoing clinical pregnancy rate was significantly higher in the group with negative BF-WGA compared with those with positive BF-WGA. STUDY DESIGN, SIZE, DURATION A prospective cohort study including 102 consecutive PGT-A patients (Group 1) and 88 consecutive conventional IVF/ICSI patients (Group 2), was conducted between January 2019 and December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS In both groups, BFs were collected from expanded blastocysts of high grade and processed for WGA. DNA amplification was evaluated by agarose gel electrophoresis for the presence (positive BF-WGA) or absence (negative BF-WGA) of a band. Directly after the BF retrieval, blastocysts from Group 1 underwent TE biopsy and vitrification. In Group 2, blastocysts were vitrified immediately after BF collection. In Group 1, only euploid blastocysts were considered for transfer according to the results of TE biopsies. In both groups, the selection of the blastocyst to be transferred was based on BF-WGA results giving priority, if available, to those with negative amplification. The primary outcome investigated was the live birth rate (LBR) at the first transfer. The main variable under investigation was the negative BF-WGA and results were corrected for confounders (maternal and paternal age, number of retrieved oocytes, male factor) by multiple logistic regression analysis. MAIN RESULTS AND THE ROLE OF CHANCE In Group 1, 60 patients transferred negative BF-WGA blastocysts and 42 positive BF-WGA blastocysts, and the LBR at the first transfer was 53.3% and 26.2%, respectively (P = 0.0081). After testing for selected confounders in a multiple logistic analysis, the transfer of blastocysts with negative BF-WGA resulted in an odds ratio of (OR) 3.52 (95% CI: 1.48-8.88, P = 0.0057) compared to transfer of positive BF-WGA blastocysts. In Group 2, at the first transfer 30 deliveries resulted from blastocysts with negative BF-WGA (48.4%) and three from the transfer of positive BF-WGA blastocysts in 26 patients (11.5%; P = 0.0014). Multiple logistic analysis indicated that the transfer of blastocysts with negative BF-WGA resulted in an OR 6.89 (95% CI: 1.98-32.95, P = 0.0056) compared to transfer of positive BF-WGA blastocysts. The LBR per transfer and the cumulative LBR per patient showed the same trend. LIMITATIONS, REASONS FOR CAUTION The study was performed in a single center. WIDER IMPLICATIONS OF THE FINDINGS The data from this study highlight the heterogeneity of blastocysts of similar morphology, even in those classified as euploid by TE analysis. Failure to detect DNA in BFs after WGA is associated with a significantly higher LBR at the first embryo transfer as well as per transfer and per patient. The processing of the BF by WGA is an easy and cost-effective tool that could become a valuable option to offer patients the highest chances of term pregnancy in the shortest time possible. STUDY FUNDING/COMPETING INTEREST(S) The study received no funding from external sources. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.

Abstract Study question Is the presence or absence of DNA in the blastocoelic fluid detected by whole genomic amplification (WGA) a valid method to prioritize embryos for transfer? Summary answer Blastocysts with DNA in the BF have lower ongoing pregnancy rates compared with blastocysts without DNA, both in PGT-A and in conventional IVF cycles What is known already The detection of DNA in BFs from expanded blastocysts has been reported in different studies. After amplification, this DNA can be analyzed to provide information on the blastocyst chromosome condition, but the degree to which it is representative of the corresponding embryo ploidy is still controversial. The reason of this divergence could reside in several factors, including the different status of the studied embryos. A recent study comparing euploid and aneuploid blastocysts reported a significantly higher incidence of failed BF-DNA amplification in euploid blastocysts compared with aneuploid blastocysts suggesting an effect of the embryo ploidy condition on BF content Study design, size, duration This prospective study included 142 cycles with PGT-A (Group-1; 24-chromosome analysis was performed on trophectoderm (TE) biopsies) and 121 conventional IVF consecutive cycles (Group-2) treated in the last three years. In both groups, the BF was collected from expanded blastocysts before vitrification, and submitted to WGA. Single blastocyst transfers were performed by selecting blastocysts based on BF-WGA results giving priority to those with failed amplification. In Group-1, only TE-euploid blastocysts were transferred Participants/materials, setting, methods Patients in Group-1 had a maternal age higher than in Group-2 (36.8±3 vs 34.1±3.5 years). The same protocol of vitrification was used for all patients, and only expanded blastocysts of high grade were included in the study. Amplification after WGA was evaluated by loading an aliquot of the amplified product onto a 1.5% agarose gel. An ongoing pregnancy was defined as a pregnancy regularly ongoing beyond the 16th week of gestation Main results and the role of chance In Group-1, a total of 622 blastocysts underwent trophectoderm (TE) biopsy and 261 were euploid. The BF was retrieved from 237 euploid blastocysts and submitted to WGA. Amplification failure resulted in 98 BFs, whereas 139 BF gave a positive amplification. In all, 57 clinical pregnancies resulted, 53 of which were regularly ongoing. 61 transfers were performed with euploid blastocysts with failed BF-WGA, and 81 with positive BF-WGA. When looking at the transfer outcome, the ongoing pregnancy rate was significantly higher for euploid blastocysts with failed BF-WGA (31/61, 50.8%) when compared to those with positive BF-WGA results (22/81, 27.2%, p<0.01). In Group-2, there were 62 clinical pregnancies, 52 of which were ongoing. In relation to the BF-WGA results, the ongoing pregnancy rate showed the same trend of Group-1, and was 20% (52/121) for blastocysts with failed BF-WGA and 59.1% (42/71, p<0.001) for blastocysts with positive BF-WGA Limitations, reasons for caution This is a prospective cohort study. The results should be confirmed by a prospectively randomized study Wider implications of the findings The presence of DNA in the BF could be indicative of an abnormal embryos that is trying to reach a viable condition. Therefore, failure to detect DNA after BF amplification could represent an additional criterion to select viable embryos for transfer both in PGT-A and in conventional IVF cycles Trial registration number Not applicable

RESEARCH QUESTION:The IVF Lite programme is based on mild ovarian stimulation including up to three fresh/frozen embryo transfers within 12 months. Is it effective and safe in good prognosis patients?DESIGN:Single-centre prospective study on infertile patients at their first IVF attempt (female age ≤38 years, anti-Müllerian hormone concentrations >1.5 ng/ml and/or FSH ≤10 mIU/ml). Induction of multiple follicular growth was based on a fixed protocol consisting of clomiphene citrate (100 mg/day) from day 3 to 7 of the menstrual cycle and 150 IU of recombinant FSH on days 5, 7 and 9. In case of low follicular recruitment (fewer than four follicles), the cycle was cancelled. The IVF Lite programme was considered complete after a live birth delivery or up to three embryo transfers within 12 months. The primary outcome was the cumulative live birth rate (cLBR) per couples that completed the programme.RESULTS:A total of 369 patients completed the IVF Lite programme, with 239 live births; 132 patients delivered after one embryo transfer (35.8%), 70 after a second embryo transfer (cLBR 54.7%), and 37 after a third attempt (cLBR 64.8%). No cases of ovarian hyperstimulation syndrome or clinical complications occurred. Spontaneous dropout rate from the programme was 4.5%. The cLBR per intention to treat was 46.8%.CONCLUSIONS:The IVF Lite programme proved to be effective and safe in good prognosis patients with a good response to clomiphene citrate stimulation. It was well tolerated and implied low gonadotrophin consumption. Two-thirds of the patients achieved a live birth at the completion of the programme.

Research question: Is the efficacy of imported vitrified oocyte donation affected by the cryobank of origin? Design: Longitudinal cohort study, including 249 completed oocyte warming cycles from 200 recipients (January 2016-July 2020). No severe male factor was included. Vitrified oocytes were provided by three Spanish cryobanks. Primary outcome was cumulative live birth delivery rate (CLBR) per completed oocyte warming cycle. Results: After warming 1535 oocytes, 1244 survived (81.0%) and 945 fertilized (76.0%); embryo utilization rate was 65.3%. The overall CLBR per completed cycle was 47.0% but was lower in cryobank 1 (31.2%) versus cryobank 2 (56.0%, P = 0.0010) and cryobank 3 (50.8%, P = 0.0241). Multivariate logistic regression analysis identified survival of four or more oocytes as the strongest predictor for delivery (P = 0.0282). Only 202 out of 249 oocyte warming cycles had four or more survived oocytes in a proportion that was significantly lower in cryobank 1 versus cryobank 2 (70.1% versus 89.0%, P = 0.0020); comparison with cryobank 3 (81.0%) was not significant. In the 202 oocyte warming cycles, CLBR in cryobank 1 (370%) was lower versus cryobank 2 (58.8%, P = 0.0115) and cryobank 3 (60.8%, P = 0.0019), suggesting a reduced viability in oocytes from cryobank 1 that survived warming. Conclusions: Differences were found in the efficacy of imported vitrified oocytes in relation to the cryobank of origin. Each centre needs to evaluate the results internally when starting a collaboration with an oocyte cryobank to establish the necessary measures to maximize treatment efficacy.

Abstract Study question Do RIF patients have the preimplantation genetic testing for aneuploidy (PGT-A) overcome their infertility condition? Summary answer PGT-A positively impact on implantation rate in RIF patients What is known already The most common definition of RIF is failure to achieve a pregnancy after three consecutive transfers of good quality embryos. This term possibly represents a heterogeneous category of infertile couples as the causes of repeated failures can be diverse. Especially intriguing is the case of patients with an age lower than 39 years for which the oocyte quality is expected not to be compromised by the well known age effect on female fertility. The chromosome analysis of the resulting embryos has been proposed as a valid method to improve implantation in the great majority of RIF patients Study design, size, duration This retrospective study included 49 patients with at least three previous consecutive implantation failures, which underwent PGT-A from January 2016 to April 2020. Both partners had a normal karyotype. Only patients with a female age below 39 years were included, who presented with a normal uterine cavity. Couples with a severe male factor were excluded. Single frozen blastocysts were transferred according to chromosomal results Participants/materials, setting, methods Maternal age was 35.5 ± 3.1 years. All blastocysts were vitrified after trophectoderm biopsy. Whole genome amplification and array comparative genomic hybridization were performed on biopsies. Only euploid embryos were transferred. The primary outcome was the live-birth delivery rate after the first transfer Main results and the role of chance Before starting a PGT-A cycle, these patients underwent 213 embryo transfers with 251 embryos replaced. A total of 264 blastocysts were analyzed, 140 of which were aneuploid (53%). Monosomy or trisomy was reported in 67 of the diagnosed samples (67/140, 48%) whereas the remaining 73 carried complex aneuploidies (73/140, 52%). The remaining 124 blastocysts (47%) were diagnosed as euploid. All patients performed an embryo transfer resulting in 28 clinical pregnancies (57%). There were 5 spontaneous abortions and the live-birth delivery rate per patient was 47% Limitations, reasons for caution This study suffers from the weakness related to retrospectivity. In addition, as euploid embryos are still cryopreserved, the delivery rate could change at completion of the cycles Wider implications of the findings: A RIF condition can be attributed, at least in a good proportion of cases, to the generation of high percentages of aneuploid embryos. In this case, the transfer of euploid blastocysts has high chances to classify this category of RIF patients has having an embryonic cause of infertilit. Trial registration number Not applicable

Benralizumab and mepolizumab are new therapies for severe eosinophilic asthma. Theyare both humanized IgG antibodies, targeting the IL‐5 receptor and IL‐5, respectively, suppressing the corresponding pathways. No specific biomarkers have been proposed to evaluate treatment response to benralizumab or mepolizumab. The aim of this proteomic study was to compare serum protein profiles of patients with severe eosinophilic asthma before and after anti‐IL5 or anti‐IL5R therapies. Proteomic analysis highlighted 22 differently abundant spots. Among the proteins identified, CAYP1, A1AT and A2M expression was significantly modified in both groups of patients after therapies while ceruloplasmin showed a significant modification in the group of benralizumab treatment. These differentially expressed proteins could be potential biomarkers of response to mepolizumab and benralizumab treatments and need further evaluation.

The relationship between varicocele and fertility has always been a matter of debate because of the absence of predictive clinical indicators or molecular markers able to define the severity of this disease. Even though accumulated evidence demonstrated that the endocannabinoid system (ECS) plays a central role in male reproductive biology, particularly in the testicular compartment, to date no data point to a role for ECS in the etiopathogenesis of varicocele. Therefore, the present research has been designed to investigate the relationship between testicular ECS gene expression and fertility, using a validated animal model of experimental varicocele (VAR), taking advantage of traditional statistical approaches and artificial neural network (ANN). Experimental induction of VAR led to a clear reduction of spermatogenesis in left testes ranging from a mild (Johnsen score 7: 21%) to a severe (Johnsen score 4: 58%) damage of the germinal epithelium. However, the mean number of new-borns recorded after two sequential matings was quite variable and independent of the Johnsen score. While the gene expression of biosynthetic and degrading enzymes of AEA (NAPE-PLD and FAAH, respectively) and of 2-AG (DAGLα and MAGL, respectively), as well as their binding cannabinoid receptors (CB1 and CB2), did not change between testes and among groups, a significant downregulation of vanilloid (TRPV1) expression was recorded in left testes of VAR rats and positively correlated with animal fertility. Interestingly, an ANN trained by inserting the left and right testicular ECS gene expression profiles (inputs) was able to predict varicocele impact on male fertility in terms of mean number of new-borns delivered (outputs), with a very high accuracy (average prediction error of 1%). The present study provides unprecedented information on testicular ECS gene expression patterns during varicocele, by developing a freely available predictive ANN model that may open new perspectives in the diagnosis of varicocele-associated infertility.

ABHD2 is a serine hydrolase that belongs to the subgroup of the α,β-hydrolase fold-containing proteins, which is involved in virus propagation, immune response, and fertilization. Chemical tools to selectively modulate the activity of ABHD2 in an acute setting are highly desired to investigate its biological role, but are currently lacking. Here, we report a library-versus-library screening using activity-based protein profiling (ABPP) to evaluate in parallel the selectivity and activity of a focused lipase inhibitor library against ABHD2 and a panel of closely related ABHD proteins. This screen resulted in the rapid identification of novel inhibitors for ABHD2. The selectivity of the inhibitor was further investigated in native mouse testis proteome by competitive ABPP, revealing a highly restricted off-target profile. The progesterone-induced acrosome reaction was reduced in a dose-dependent manner by the newly identified inhibitor, which provides further support for the key-role of ABHD2 in the P4-stimulated acrosome reaction. On this basis, the ABHD2 inhibitor is an excellent starting point for further optimization of ABHD2 inhibitors that can modulate sperm fertility and may lead to novel contraceptives.