Complete Fertilization Failure Research Articles

The Immediate Partial Removal of Cumulus-Oocyte Complexes: A Refined Approach for Rapid Observation of In Vitro Fertilization.

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients continue to experience fertilization disorders, leading to low fertilization rates and the production of nonviable embryos. Short-term fertilization involves the early removal of granulosa cells to observe the extrusion of the second polar body, enabling the assessment of fertilization and early remedial measures to address low fertilization rates and complete fertilization failure. However, the observation of short-term fertilization in IVF is impeded by challenges such as excessively large unprocessed clusters of cumulus-oocyte complexes and adhesion between eggs, necessitating complex external procedures for granulosa cell removal, and prolonged observation duration. To tackle this issue, this study proposes a method of immediate partial removal of granulosa cells post egg retrieval. This approach streamlines subsequent stages of short-term fertilization and traditional fertilization monitoring, reduces the time needed for oocyte extrusion, minimizes the likelihood of external environmental impacts on the oocytes, and decreases the risk of missing the critical fertilization observation window. Consequently, the study yields novel clinical evidence aimed at enhancing the operational efficiency of embryonic laboratories in IVF.

Assisted oocyte activation significantly improves zygote formation, cleavage, and implantation rates in patients with a history of fertilization failures.

Fertilization check performed at the 18th hour following classic in vitro fertilization procedure (IVF) or intracytoplasmic sperm injection (ICSI) is a critical stage in assisted reproduction. The success of the treatment is significantly reliant on the quantity of zygotes exhibiting two pronuclei. Consequently, low fertilization rates or complete fertilization failure are highly undesirable outcomes for both patients and reproductive specialists. Applying additional calcium ionophore for oocyte activation subsequent to ICSI may offer benefits and potentially enhance treatment outcomes, particularly for patients who have experienced low or absent fertilization rates (FR) in previous treatment cycles. The aim of the study is to evaluate the efficacy of Ca2+ ionophore application for oocyte activation. A retrospective analysis of 924 oocytes obtained from 120 patients who underwent ICSI cycles with a history of low or no fertilization as a result of previous unsuccessful treatment rounds. The next ART cycle followed with additional oocyte Ca2+ ionophore activation applied in 57 of the cases in order to optimize the treatment process (Group 1), and 63 patients were included and their outcomes followed as a control group (Group 2).We conducted a comparative analysis of results in both groups. The study's primary outcomes encompassed fertilization, cleavage embryo quality, blastocyst rate, and established clinical pregnancies. At day 1fertilization check we had 274/386 zygotes (71%FR) in group 1 and 132/410 in group 2 (32.2%FR), (P<0.0001). Twenty-two (34.9%) cycles in group 2 resulted in total fertilization failure (TFF). At the cleavage stage top-quality embryos from group 1 were significantly higher (P = 0.0021) in comparison to group 2. Forty-eight embryo transfers (ET) were performed in group 1 resulting in 41.67% clinical pregnancies versus 33 ET and only 4 pregnancies (12.12%) for group 2 (P = 0.0044). The results confirm the appropriateness of assisted oocyte activation as an additional method in cases of previous fertilization failure cycles.

P-060 When is it the sperm’s fault? The incidence of sperm-related poor fertilization after ICSI in 13,859 matched oocyte donation cycles

Abstract Study question What is the true incidence and impact of male factor-related poor fertilization in ICSI cycles when analyzed using matched pairs of high quality donor oocytes? Summary answer In ICSI cycles with donor oocytes, male factor-related poor fertilization occurs in 3.3% of cases and contributes to 86.7% of all poor (&amp;lt;30%) fertilization outcomes. What is known already Opting for oocyte donation is a complex decision for couples, involving significant financial and long-term implications. This decision is typically prompted by presumed female infertility as male fertility diagnostics remain limited. Although ICSI is a highly effective treatment, cases of poor or complete fertilization failure can still occur, even when sperm parameters appear normal. This suggests the presence of unidentified male-related factors. However, the precise incidence of such factors remains poorly understood. This study aims to address this gap, offering a unique perspective, by employing a matched oocyte donation model to investigate the influence of sperm-related factors on fertilization outcomes. Study design, size, duration This retrospective cohort study analyzed 14,928 oocyte donation ICSI cycles, derived from 7,519 ovarian stimulations (OS) of 2,963 unique oocyte donors. The data were from a single center from January 2015 to December 2022. Sibling oocytes from the same OS were utilized for at least two different recipients (n = 13,859), facilitating comparisons under varying paternal conditions. Participants/materials, setting, methods Cases from the same oocyte lot with poor (&amp;lt;30%) and good (&amp;gt;65%) fertilization rates were matched to isolate sperm-related factors influencing fertilization success. Paired t-tests were used to compare outcomes. Ordinary least squares regression was used to isolate variables associated with poor fertilization, such as oocyte status (fresh versus vitrified), sperm origin (partner versus donor) and the presence of severe male factor (defined as a concentration of &amp;lt; 1 million/mL and/or &amp;lt;1% progressive motility). Main results and the role of chance The mean fertilization rate was 73.0% across all cycles analyzed. The incidence of poor fertilization (≤30%) was found to be 3.8% across the entire cohort (532 out of 13,859 cycles). Of these, 86.7% (461/532) cases using the same oocyte lot could be matched to cases with high fertilization rates (&amp;gt;65%) in other recipient cycles (n = 1,441), indicating that poor fertilization was male-related. We observed comparable numbers of inseminated oocytes between matched cases with high and low fertilization (7.23 ± 1.32 and 7.42 ± 1.42, respectively, p = 0.011), while the mean number of fertilized oocytes (1.44 ± 0.77 versus 6.12 ± 1.36, p = &amp;lt;0.001) and mean fertilization rates (20% versus 83%, p &amp;lt; 0.001) differed significantly. Notably, oocyte vitrification and the presence of a severe male factor negatively affected fertilization outcomes (coeff. -0.0898, p &amp;lt; 0.001 and coeff. -0.1500, p &amp;lt; 0.001, respectively). Nevertheless, the majority of poor fertilization cases (92.7%) occurred in the absence of these factors, underscoring the limitations of conventional semen diagnosis tests as predictors of poor fertilization. Across the entire cohort male factor alone accounted for 3.3% (461/13,859) of cycles with &amp;lt;30% fertilization rates. Furthermore, sperm-related factors were the primary cause of poor fertilization outcomes in oocyte donation cycles, accounting for 86.7% of cases. Limitations, reasons for caution Certain variables were not analyzed due to the retrospective nature and extended timeframe of the study. The matched oocyte model may overlook certain non-male factor-related instances of poor fertilization, such as procedural or technical issues. Wider implications of the findings This extensive analysis emphasizes the clinical relevance of male factor in fertilization outcomes, highlighting the need for improved semen diagnosis. It also indicates the importance of considering male-related factors in treatment decisions and shifting the focus from female-centric to more balanced fertility evaluations. Trial registration number Not applicable

P-067 A physiological approach for oocyte activation when using testicular spermatozoa

Abstract Study question Can poor fertilization with testicular spermatozoa be rescued by oocyte activation? We propose a more physiological approach to the standard method. Summary answer Couples who utilized testicular spermatozoa benefitted from assisted oocyte activation (AOA), particularly when a more physiological approach such as recombinant human phospholipase-C-zeta (rhPLCζ) was utilized. What is known already ICSI overcomes sperm acrosomal dysfunction and other inherent defects of the male gamete. However, partial or complete fertilization failure may still occur, often attributable to the absence of PLCζ. These cases are often treated by calcium ionophore, a very popular method that is considered quite safe, however it still raises concerns because it induces intracellular calcium oscillations in a very aspecific manner. Testicular spermatozoa are particularly at risk of suboptimal fertilization; therefore, the availability of a more physiological method would be welcome. Study design, size, duration Over a period of 12 months, 21 couples utilizing testicular spermatozoa for ICSI, that resulted in poor fertilization, were identified and treated in subsequent ICSI cycles with assisted oocyte activation (AOA), either by exposure to ionomycin or co-injection of rhPLCζ at the time of ICSI. Embryology and clinical outcomes were recorded and compared between the two AOA methods as well as the couples’ historical and subsequent cycles. Participants/materials, setting, methods Couples with a history of poor/absent fertilization after undergoing ICSI using testicular spermatozoa were counseled for AOA in subsequent cycles (IRB 0712009553). For consenting couples, AOA was performed either by exposing post-ICSI oocytes to 50mM of ionomycin (Ionomycin-AOA), or by co-injecting spermatozoa with 0.4pL of rhPLCζ (5mg/mL) during ICSI (rhPLCζ-AOA). Fertilization, embryo development, and clinical outcomes were compared between the couples’ historical and AOA cycles. Main results and the role of chance We included 21 couples (maternal age, 35.1±4 yrs; paternal age, 36.8±7 yrs). Female partners all had negative infertility workups that consisted of a comprehensive review of their medical history as well as a targeted physical examination and tests assessing ovarian reserve, ovulatory function, and absence of uterine and tubule structural abnormalities. The couples underwent 53 historical ICSI cycles using poor testicular spermatozoa (concentration, 0.04±0.1x106/ml, 0.5±1% motility) that yielded a 22.5% (94/417) fertilization and only an 11.5% (3/26) clinical pregnancy rate. All couples subsequently underwent ICSI-AOA cycles. Fourteen couples (maternal age, 35.5±4 yrs; paternal age, 37.6±6 yrs) who were treated with Ionomycin-AOA obtained a higher fertilization of 44.4% (99/223) (P&amp;lt;0.00001), which resulted in a clinical pregnancy rate of 26.3% (5/19). Thus far, four couples have delivered 1 boy and 3 girls, with no major or minor congenital malformations. The remaining 7 couples (maternal age, 35.8±4 yrs; paternal age, 37.9±7 yrs) underwent subsequent ICSI cycles with rhPLCζ-AOA, which yielded a remarkably higher fertilization of 50.0% (50/134) (P&amp;lt;0.00001). These couples went on to achieve a clinical pregnancy rate of 27.3% (3/11), resulting in the delivery of 1 girl and 2 boys. Initial follow-up has confirmed normal neonatal development. Limitations, reasons for caution The effectiveness of AOA in ICSI cycles with testicular spermatozoa is very encouraging, particularly when a more physiological method is utilized. Nonetheless, these findings need to be validated in a larger cohort, confirming successful outcome and children health. Wider implications of the findings ICSI utilizing surgically retrieved spermatozoa, particularly testicular, are plagued by suboptimal fertilization, and using AOA may represent a way to normalize the generation of conceptuses. Moreover, a broader adoption of AOA would certainly benefit from the use of a recombinant cytosolic factor normally present in the sperm perinuclear theca. Trial registration number n/a

P-225 Implementing a more physiological method to trigger oocyte activation in cases of extremely poor fertilization with ICSI

Abstract Study question Can we utilize a more physiological compound to induce oocyte activation in couples with history of complete/partial fertilization failure with ICSI? Summary answer Recombinant human phospholipase-C-zeta (rhPLCζ) successfully activated oocytes with same efficiency as ionomycin, consistently normalized fertilization and enhanced clinical outcomes. What is known already While ICSI overcomes most aspects of male gamete dysfunction, partial or complete fertilization failure may seldomly occur mostly due absence of sperm-specific protein, known as PLCζ, which is required to trigger oocyte activation. Assisted oocyte activation (AOA), most often performed with an antibiotic (ionomycin), activates oocytes by triggering calcium release from intracellular storage. This agent induces indiscriminate permeabilization of ooplasmic membrane-bound organelles. This action has been considered nonspecific and may have possible deleterious effects on subsequent embryo development. Therefore, we propose a physiological alternative that employs a recombinant form of PLCζ, the native protein residing in the sperm perinuclear theca. Study design, size, duration In the past 12 months, couples with poor ICSI fertilization were included. PLCζ were assessed to confirm sperm-related oocyte activation deficiency with a normal threshold of ≥ 30%. In a preliminary comparison, sibling oocytes were equally allocated to AOA by ionomycin or to rhPLCζ at time of ICSI. Fertilization, embryo development, and clinical outcomes between the two cohorts. Subsequently, we describe the clinical outcomes of couples treated solely by rhPLCζ. Participants/materials, setting, methods Thirty couples, confirmed with low level of PLCζ in the ejaculates, were offered AOA with their subsequent ICSI cycles (IRB#0712009553). Conventional AOA was performed by exposing post-ICSI oocytes to 50 µM ionomycin. The rhPLCζ-AOA method was performed by co-injecting spermatozoa with 0.4pL of a rhPLCζ (5 µg/µL) during ICSI. Chi-square tests were utilized to compare rhPLCζ-AOA versus ionomycin-AOA and rhPLCζ-AOA versus history cycles. Main results and the role of chance In the preliminary study, 9 patients were included (maternal age: 35.7 ± 6, paternal age: 36.7 ± 6). PLCζ positivity was 11.7±3.6% confirming a sperm-related oocyte activation deficiency. A total of 123 oocytes were retrieved in 9 cycles, with maturity of 78.0% (96/123). The 96 MII oocytes were allocated to be treated with ionomycin-AOA (n = 58) or rhPLCζ-AOA (n = 38). While conventional ionomycin-AOA yielded a fertilization rate at 55.2% (32/58), rhPLCζ-AOA resulted in 63.1% (24/38). Both compounds generated embryos at similar rates and one pregnancy was achieve in each cohort. On the bases of such comparable outcomes, 21 couples had 40 previous cycles that generated a fertilization rate of 18.3% (60/327), cleavage rate of 51.7% (31/60), Following the replacement of 23 embryos in 14 transfer cycles, these cycles yielded 2 clinical pregnancies, but all resulted in pregnancy loss. In the subsequent cycles with rhPLCζ-AOA (n = 32), an enhanced fertilization rate was achieved at 41.1% (116/280, P&amp;lt;0.00001). The treatment cycle yielded a higher cleavage rate at 80.2% (93/116, P&amp;lt;0.0001), and after replacing 25 embryos in 17 embryo transfer cycles, yielded 6 pregnancies and 5 of them resulted in delivered or are ongoing. Limitations, reasons for caution We demonstrated that physiological rhPLCζ-AOA achieved an excelled fertilization and clinical outcomes comparing to the traditional ionomycin method. Although further observation is needed to confirm its safety, this approach represents a more physiological method capable of achieving fertilization, higher embryo cleavage and ultimately better pregnancy outcomes. Wider implications of the findings While ICSI has gained large popularity, poor fertilization may occasionally occur. The availability of an assisted oocyte activation that utilize a native compound capable of restoring fertilization capability of the male gamete is appealing and holds great promises. Trial registration number N/A

Identification of a new mutation in the ACTL9 gene in men with unexplained infertility.

Infertility is defined as the failure to achieve pregnancy after one year of unprotected intercourse within a marital relationship. Approximately 10%-15% of couples worldwide experience infertility issues, with nearly half of these cases attributed to male factors. Among men with unexplained infertility, genetic mutations have been identified as a potential cause. Studies have indicated that mutations affecting the function of the protein encoded by the ACTL9 gene may play a role in male infertility. The purpose of this research was to identify mutations in the ACTL9 gene associated with male infertility in a sample of 40 infertile men with unknown causes. Genomic DNA extraction and PCR amplification were carried out on samples from each individual. The genetic material was then analyzed using Sanger sequencing, followed by bioinformatics and segregation analysis to determine the potential effects of the observed variations. A novel genetic variant, c.376G>A (p.Glu126Lys), was identified in an infertile male individual, representing a previously unreported finding that was validated through segregation analyses. This specific variant induces a change from glutamate to lysine at the amino acid level by replacing the nucleotide G with A in the genomic DNA sequence, consequently impacting the secondary structure and function of the protein. The conclusive analysis of the procedure indicated that this alteration has the potential to interfere with the process of fertilization, ultimately resulting in the complete failure of fertilization (TFF) and causing male infertility.

Sperm acrosin activity may be a useful factor in choosing between ICSI and IVF for infertile male patients.

The clinical applications of acrosin activity are limited. We analyzed 61 578 male partners in infertile couples who visited the outpatient department of the Reproductive and Genetic Hospital of CITIC-Xiangya (Changsha, China) between August 2014 and December 2019 to determine the reference ranges and thresholds for acrosin activity in infertile Chinese men; to determine whether correlations exist between acrosin activity and age, sperm concentration, sperm morphology, or sperm motility; and to evaluate whether acrosin activity could serve as an effective prognostic indicator for choosing between in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in the clinic. The cut-off value for the normal reference range of acrosin activity for male partners in infertile couples was 24.78 µIU per 10 6 sperm. There was no significant association between acrosin activity and age, sperm concentration, semen volume, total sperm count, progressive motility, or total motile spermatozoa. A weak positive correlation was found between acrosin activity and normal sperm morphology. There was a statistically significant difference in abnormal acrosome morphology between the group with high acrosin activity (>24.78 µIU per 10 6 sperm) and the group with low acrosin activity (<24.78 µIU per 10 6 sperm). The group with a low IVF fertilization rate had a high index of abnormal acrosomal morphology at 21.2%, while the group with a high IVF fertilization rate had a low index of 0.2%. At an acrosin activity of <24.78 µIU per 10 6 sperm, in one cycle of the same patient, the fertilization rate, normal fertilization rate, and good-quality embryo rate for ICSI were significantly higher than those for IVF. Therefore, the most promising application of acrosin activity could be in the selection of ICSI over IVF for infertile male patients with complete fertilization failure or a low fertilization rate.

Transition nuclear protein 1 as a novel biomarker in patients with fertilization failure.

Although intracytoplasmic sperm injection (ICSI) is a way to deal with in vitro fertilization failure, 3% of couples still experience repeated fertilization failure after attempted ICSI, despite having sperm within normal parameters. These patients are a challenging group whose sperm cannot fertilize the egg during ICSI. Unfortunately, no test can predict the risk of fertilization failure. Phospholipase C zeta (PLCζ) and transition nuclear proteins (TNPs) are essential factors for chromatin packaging during sperm maturation. This study aimed to assess PLCζ1 and TNP1 expression in the sperm of patients with fertilization failure and the correlations among the DNA fragmentation index, PLCζ1 and TNP1 gene and protein expression, and the risk of fertilization failure. In this study, 12 infertile couples with low fertilization rates (<25%) and complete failure of fertilization in their prior ICSI cycles despite normal sperm parameters were chosen as the case group. Fifteen individuals who underwent ICSI for the first time served as the control group. After sperm analysis and DNA fragmentation assays, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to compare the gene and protein expression of PLCζ and TNP1 in both groups. DNA fragmentation was significantly higher in the fertilization failure group. The qRT-PCR and Western blot results demonstrated significantly lower PLCζ and TNP1 gene and protein expression in these patients than in controls. The present study showed that fertilization failure in normozoospermic men was probably due to deficient DNA packaging and expression of TNP1.

Assisted Oocyte Activation following Intracytoplasmic Sperm Injection: A Sensible Option for Infertile Couples with Severe Teratozoospermia.

The intracytoplasmic sperm injection (ICSI) has significantly improved male factor infertility treatment; however, complete fertilization failure still occurs in 1-5% of ICSI treatment cycles mainly due to oocyte activation failure. It is estimated that around 40-70% of oocyte activation failure is associated with sperm factors after ICSI. Assisted oocyte activation (AOA) as an effective approach to avoid total fertilization failure (TFF) has been proposed following ICSI. In the literature, several procedures have been described to overcome failed oocyte activation. These include mechanical, electrical, or chemical stimuli initiating artificial Ca2+ rises in the cytoplasm of oocytes. AOA in couples with previous failed fertilization and those with globozoospermia has resulted in varying degrees of success. The aim of this review is to examine the available literature on AOA in teratozoospermic men undergoing ICSI-AOA and determine whether the ICSI-AOA should be considered as an adjunct fertility procedure for these patients.

O-310 Browsing the Male Genome to Unravel Its Reproductive Potential

Abstract Study question Can whole genome profiling of sperm DNA be used to identify aspects of ART failure and predict embryo developmental competence? Summary answer Whole genome profiling of sperm DNA identifies germline gene mutations associated with reproductive failure and helps characterize subtle male factor infertility. What is known already A routine semen analysis provides limited information on the characteristics of the male gamete and is unable to predict spermatozoa performance in ART. Therefore, ancillary tests may be used to further assess the male gamete’s reproductive potential. Whole exome sequencing (WES) of the male genome carried out on somatic cells has proven to be a powerful technique capable of identifying the genetic roots of infertility. Here, we aim to preferentially detect germline mutations by exclusively sequencing spermatozoal DNA to identify genes related to the different underlying etiologies of reproductive failure. Study design, size, duration Over a 6-year period, 31 consenting couples with negative female infertility workups and normal semen parameters were included in this study. These couples were divided according to whether they reported a successful pregnancy with ART (fertile; n = 10) or not (infertile; n = 21). Sperm aneuploidy assessment by copy number variant (CNV) analysis with WES were carried out on ejaculated spermatozoa. Gene mutation profiles were enlisted and compared between the two patient cohorts to identify genes involved. Participants/materials, setting, methods DNA was extracted and amplified from at least 500 spermatozoa (DNA concentration, 760±486 ng/ul; quality, 1.7±0.1 nm) for CNV analyses by WES. Mutations corresponding to the CNV were then annotated and assessed using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when their read depth was &amp;gt;1.5 or &amp;lt; 0.5 times the median read depth in the control, respectively. Main results and the role of chance All couples (n = 31) (maternal age, 37.6±3yrs; paternal age, 39.7±5yrs) had adequate semen parameters (concentration, 59.2±30x106/mL, 44.8±18% motility, normal morphology) and normal peripheral karyotypes. The fertile cohort (n = 10) underwent 12 ICSI cycles, achieving an 82.6% (57/69) fertilization rate with 10/12 (83.3%) cycles resulting in live births. The infertile cohort (n = 21) underwent 25 ICSI cycles, achieving a 68.4% (91/133) fertilization rate and 6/14 (42.9%) clinical pregnancies, all resulting in pregnancy loss. CNV analysis indicated lower sperm aneuploidy in the fertile (4.0% vs. 8.4%) cohort (P &amp;lt; 0.00001). In both cohorts, mutations associated with sperm–egg fusion (ADAM3A) and acrosomal development (SPACA1, SPATA16) were identified, justifying ICSI utilization. The infertile cohort included complete fertilization failure, poor early embryo development, implantation failure, or pregnancy loss. Couples with complete fertilization failure (n = 4) had gene deletions (PLCZ1, PIWIL1, ADAM15) indicating sperm-related oocyte-activating deficiency. Those with poor early embryo development (n = 5) had mutations essential for centrosome integrity (HAUS1) and spindle/microtubular stabilization (KIF4A, XRN1). Couples who failed to achieve pregnancy (n = 7) had mutations commonly implicated in embryonic implantation (IL9R) and microtubule/centrosomal integrity (MAP1S). Those with pregnancy losses (n = 5) displayed mutations related to trophoblast development (NLRP7), cell cycle regulation (MARK4, TRIP13, DAB2IP, KIF1C), and a gene linked to recurrent miscarriage (TP53). Limitations, reasons for caution Using WES, we were able to identify germline mutations that appear to be involved in various aspects of human reproduction. These findings are new and should be validated in a larger study population. Moreover, although we attempted to control for maternal age, we still cannot exclude confounding female factors. Wider implications of the findings Evaluating the sperm genome can help identify elusive genetic factors associated with reproductive competence and help guide treatment options for couples unable to conceive who undergo ART. Therefore, screening spermatozoal DNA may serve as an additional tool in precision medicine to identify and treat subtle male factor infertility. Trial registration number N/A

O-044 ICSI in the lab: from vintage to AI

Abstract With the advent of in vitro fertilization (IVF) by Patrick Steptoe and Bob Edwards in the late 70s, the first conception outside of the human body resulted in the birth of Louise Brown. Although a terrific success, limitations of IVF surfaced, represented by the unexpected complete fertilization failure with suboptimal or dysfunctional spermatozoa. This prompted curiosity toward individual spermatozoa for a deeper understanding of its role aimed at enhancing the interaction between complementary gametes. Techniques were designed to manipulate the oocytes, such as stripping, partially digesting, or cracking the zona pellucida (ZP). These methods were palliative solutions to overcome fertilization failure and were often plagued by polyspermy. As a result, more direct approaches were implemented to overcome the ZP, such as subzonal injection (SUZI) that although more consistent, was still unable to overcome the shortcomings of dysfunctional spermatozoa. This laid the foundation for the utilization of ICSI that, whilst attempted by some investigators, became popular when Gianpiero Palermo serendipitously inserted one spermatozoon into the ooplasm during SUZI. Consistent fertilization then followed by injecting a cohort of oocytes by ICSI in SUZI cycles, and replacement of these embryos led to 4 pregnancies described in the first clinical ICSI report. To minimize oocyte damage, the procedure was further refined by inducing a deep invagination of the oolemma toward the 9 o’clock position, granting higher chances of post-injection survival. What set apart ICSI from other forms of ART was that any sperm sample, regardless of quality/quantity, would yield fertilization. Indeed, ICSI is the sole insemination method used with epididymal and testicular spermatozoa and has therefore revolutionized fertility treatment of azoospermic men. Indeed, even immotile testicular spermatozoa can still fertilize and yield successful pregnancies, albeit at a lower rate than their motile counterpart. Also, for these semen sources, aggressive sperm immobilization was introduced to enhance sperm membrane permeabilization and grant optimal fertilization results. Furthermore, ICSI has transformed the field of reproductive medicine by assisting other reproductive techniques, such as testing embryos for single gene defects to reduce the occurence of sperm DNA contamination, or overcoming the cryostress-induced changes of the ZP during cryopreservation allowing the oocyte to be fertilized at a higher rate. Oocyte cryopreservation now empowers women in their reproductive age to ordain their childbearing future. ICSI has proven to be the ultimate technique to overcome male infertility and has broadened its indication by yielding consistent fertilization and successful pregnancies in most circumstances, ensuring that men have the chance of fathering their own progeny. To date, ICSI is applied in several countries, and in some, is performed as the preferred/sole insemination method contributing to the birth of millions of babies worldwide. Thus far, no concerning differences have been seen in the health of ICSI versus standard IVF offspring, or even naturally conceived. In fact, it has been currently established that young adults of both genders born through ICSI retain their reproductive health. Despite its growing popularity, ICSI does not always succeed but still provides an invaluable platform to deepen our knowledge of gamete biology and helps to investigate/overcome some of the most severe and persistent forms of infertility. For example, combined with assisted gamete treatment, ICSI allows couples plagued by sperm-bound oocyte-activation-deficiency to achieve pregnancy. The need to increase access and curtail costs of reproductive care has led to the testing of automation in ART. This is also occurring with ICSI and to date, different automated modules have been proposed for oocyte denudation, sperm tracking, and robotic ICSI. Concurrently, there has been an interest in experimenting with artificial intelligence in the IVF laboratory to minimize human shortcomings and ensure that the best spermatozoon is chosen.

Infertility and Intracytoplasmic Sperm Injection (ICSI)

Infertility is generally defined as a Time to pregnancy (TTP) of longer than 12 months among couples who engage in unprotected intercourse in the fertile days of the menstrual cycle, any specific threshold is arbitrary. The prevalence of infertility differs greatly from one country to another, being 15% globally, &gt;30% in some developing countries, and 17-28% in industrialized countries. The term ‘implantation failure’ can be used to describe both patients who have never shown quantifiable signs of implantation such as increased levels of hCG, and those who have increased hCG production without later ultrasound evidence of a gestational sac. Intracytoplasmic sperm injection (ICSI) is a form of assisted reproductive technology in which a single sperm is injected into the cytoplasm of an egg in order to fertilize it. Complete fertilization failure following ICSI is an uncommon occurrence (1–3 percent), although it does occur even when spermatozoa appear to be normal. Furthermore, in some individuals, low to moderate fertilization (30 percent) has been reported in repeated ICSI cycles. Fertilization failure with ICSI is not the same as it is with traditional IVF technique. 60–90 percent of oocytes which showed a fertilization failure in traditional IVF are devoid of sperm nuclei, assuming that sperm ejection or penetration failure is the most common reason for the failure in fertilization process.

O-120 Unravelling reproductive failure by scrutinizing the male germline code

Abstract Study question Can whole exome sequencing (WES) of spermatozoal DNA provide insight into understanding the different steps that lead to inability of a couple to reproduce? Summary answer The identification of germline mutations can clarify different aspects of reproductive failure in couples with unexplained infertility. What is known already The limitation of a routine semen analysis in evaluating sperm characteristics, even according to the most stringent criteria, lies in its inability to provide substantial information on spermatozoa performance in ART. As a result, ancillary tests are being used to further assess the male gamete’s reproductive potential. More recently, WES of the male genome, carried out on somatic cells, has become a powerful technique that can potentially shed light on the genetic causes of infertility. Here, we aim to preferentially detect germline mutations by sequencing spermatozoal DNA to pinpoint genes related to different underlying etiologies for reproductive failure. Study design, size, duration In a 5-year period, 25 couples subdivided according to their ICSI outcomes were included in this study. Sperm aneuploidy assessment by fluorescent in situ hybridization (FISH) and copy number variant (CNV) analysis by WES were carried out on ejaculated specimens from consenting male partners. Following CNV analysis, gene mutation profiles were compared between the fertile (n = 10) and infertile cohorts (n = 15), as well as in relation to the reasons for reproductive failure. Participants/materials, setting, methods FISH was performed on at least 1,000 sperm cells with a threshold of &amp;gt; 1.6%. DNA was extracted and amplified from at least 500 spermatozoa (DNA concentration, 605±137 ng/ul; quality, 1.7±0.1 nm) for CNV analysis by WES. Mutations corresponding to the CNV were annotated and assessed using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when their read depth was &amp;gt;1.5 or &amp;lt; 0.5 times the median read depth in the control. Main results and the role of chance Couples (n = 25) (maternal age, 38.6±3yrs; paternal age, 39.7±5yrs) had normal somatic karyotypes with normal semen parameters (59.2±30x106/mL concentration, 44.8±18% motility) by WHO standards. The fertile (n = 10) cohort underwent 12 ICSI cycles, achieving an 82.6% (57/69) fertilization rate and 10/12 (83.3%) term pregnancies. The infertile cohort (n = 15) underwent 21 ICSI cycles, achieving a 66% (62/84) fertilization rate and 5/17 (29.4%) clinical pregnancies, all resulting in pregnancy loss. Sperm aneuploidy was consistently higher in the infertile (8.4%) versus fertile (4.0%) cohort (P&amp;lt;0.00001), as observed by FISH and DNA sequencing. For both cohorts, WES detected deletions responsible for sperm–egg fusion (ADAM3A) and acrosomal development (SPACA, SPATA), explaining the necessity for ICSI in these couples. The infertile cohort was characterized by 4 reasons for cycle failure: complete fertilization failure, poor embryo development, implantation failure, and pregnancy loss. Couples with complete fertilization failure (n = 4) had deletions (PLCZ1, PIWIL1) indicating a sperm-related oocyte-activating deficiency. Those with poor embryo development (n = 5) had mutations (HAUS1, KIF4A, XRN1) essential for centrosome integrity and spindle/microtubular stabilization. Couples who did not achieve pregnancy (n = 7) had a mutation (IL9R) in common related to cytokine constituents in the implantation pathway. Those with pregnancy losses (n = 5) had mutations (NLRP7, TP53) on post-implantation genes. Limitations, reasons for caution Several germline mutations, related to the different reasons for these couples’ reproductive failure, were identified. Although intriguing, these findings are still new and need to be validated in a larger study population. In addition, while maternal age was controlled for, we cannot definitively exclude other confounding female factors. Wider implications of the findings Additional screening methods for infertile couples, particularly those with unexplained infertility, can be used to clarify elusive factors underlying their reproductive ability. A genetic screening of spermatozoal DNA may therefore be considered a potential tool in precision medicine for the treatment of subtle male factor infertility. Trial registration number n/a

O-090 Correcting a PLCζ mutation in the human germ line to overcome hereditary infertility

Abstract Study question Can clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing result in the correction of a single base pair substitution that causes male infertility? Summary answer CRISPR/Cas9 administration during intracytoplasmic sperm injection (ICSI) leads to correction attempts of mutant phospholipase C zeta (PLCζ), howeverc loss-of-heterozygosity (LOH). What is known already Failed fertilization after ICSI can be caused by mutations in the sperm-related oocyte factor PLCζ which can be overcome by assisted oocyte activation (AOA). In this way, children may inherit the infertility-causing mutation. Mutation transmission can be overcome through CRISPR/Cas9 delivery during ICSI. In previous studies using CRISPR/Cas9 in the human germline for mutation correction, loss-of-heterozygosity (LOH, loss of the allele of one of the parents) was observed. Two different explanations were given, namely partial or complete paternal chromosomal loss or the correction of the mutation by using the maternal wild-type allele instead of the exogeneous supplied repair template. Study design, size, duration We injected a gRNA-Cas9 protein complex to target the PLCζ mutant allele, a repair template harboring the desired nucleotide substitution and an additional synonymous variant to track template usage, together with patient’s sperm. To overcome fertilization failure, AOA was applied during ICSI. After a culture period of maximal 6 days the embryos were collected. At day 3, some embryos were dissociated in individual blastomeres. The extracted DNA was analyzed through different genetic sequencing techniques. Participants/materials, setting, methods Donated sperm of a patient experiencing complete fertilization failure after routine ICSI, harboring a heterozygous base pair substitution in PLCZ1 (c.136-1G&amp;gt;C), was utilized. Sperm was injected in donated in vitro matured oocytes or in vivo matured oocytes containing clusters of smooth endoplasmic reticulum. Next-generation sequencing was used to assess correction potential. Short tandem repeat (STR) and single nucleotide polymorphism (SNP) assays were used to determine whether the sperm contained the mutation and to evaluate LOH. Main results and the role of chance CRISPR/Cas9 injections had no significant impact (p &amp;gt; 0.05) on embryonic development. Due to the heterozygous nature of the mutation, 47% (27/58) of the embryos originated from mutated sperm injection. The CRISPR components showed a high specificity with absence of insertions/deletions in 97% of the embryos originating from wild-type sperm (n = 31). Embryos originating from mutant sperm (n = 27) fall into three categories:(1) 22% showed the untargeted mutant allele, (2) 52% showed additional mutagenesis and (3) 26% showed the wild-type allele, which could be explained by correction. Mosaicism, defined as various editing events, was present in 17% (1), 21% (2) and 71% (3) of the embryos. The low occurrence of the synonymous variant, incorporated in the repair template, suggests that the template is not used during correction attempts. In only 29% (2/7) and 14% (1/7) of the ‘corrected embryos’, respectively long (&amp;gt;18Mb) or medium width LOH (4Mb) was observed through STR analysis. SNP analysis in closer proximity showed in 71% (5/7) of the embryos LOH, even in the absence of LOH through STR, suggesting also the occurrence of short width LOH. These results will be studied in more detail before definitive conclusions can be made. Chromosomal LOH will be studied by ddRADseq. Limitations, reasons for caution The occurrence of mosaicism and LOH might complicate the use of traditional CRISPR/Cas9 in human embryos and should be studied in detail to draw definite conclusions on its potential future use. To this end, genomic data have been produced from both individual blastomeres and whole-embryos which will be further analyzed. Wider implications of the findings Our findings demonstrate caution to use CRISPR/Cas9 to correct mutations in the germ line. They seem to contradict other reports that show predominant lack of mosaicism and presence of long width LOH. A deeper evaluation will be undertaken to define the length and type of LOH in this study. Trial registration number Not Applicable

Karyotypic abnormalities and Y chromosome microdeletions: How do these impact in vitro fertilization outcomes, and how common are they in the modern in vitro fertilization practice?

ObjectiveTo examine the outcomes of in vitro fertilization with intracytoplasmic sperm injection (IVF-ICSI) in couples in whom the male partner has a karyotypic abnormality or Y chromosome microdeletion (YCM).DesignRetrospective cohort.SettingSingle infertility center.Patient(s)Couples treated with IVF-ICSI from January 2014 to April 2019 with male factor infertility, sperm concentration of <5 × 106 sperm/mL, and results for karyotype and/or YCM panel.Intervention(s)In vitro fertilization with intracytoplasmic sperm injection.Main Outcome Measure(s)In couples in whom the male partner had a karyotypic abnormality or YCM: live birth rate/ongoing pregnancy rate, lack of partner sperm for fertilization, complete fertilization failure, cycle cancellation, and no embryos for transfer. The prevalence of karyotypic abnormalities and YCMs in the IVF population was calculated.Result(s)The live birth rate/ongoing pregnancy rate for those using partner sperm was 51.4% per transfer. However, 8.5% of cycles that intended to use partner sperm and 22.2% of cycles that intended to use surgically extracted partner sperm had no sperm available. Of cycles that created embryos with partner sperm, 12.5% had no embryo to transfer. The prevalence of karyotypic abnormalities was similar to previous reports (6.0%), while that of YCMs was lower (4.4%). Azoospermia factor a and b mutations were not represented in this population.Conclusion(s)It is reasonable to attempt IVF-ICSI with partner sperm in patients with genetic causes of male infertility. Patients should be counseled regarding the possibility of no sperm being available from the male partner, poor/failed fertilization, and genetic implications for potential offspring. Contingency plans, including IVF with donor sperm backup or oocyte cryopreservation, need to be made for these scenarios.

Identification of factors related to fertilization failure in in vitro fertilization-embryo transfer.

To investigate the possible factors relevant to fertilization failure in in vitro fertilization-embryo transfer (IVF-ET). The medical records of 4 205 infertile patients undergoing IVF-ET treatment at the Reproductive Medicine Center, Xiangya Hospital, Central South University from January 2016 to December 2017 were collected. The patients were divided into a complete fertilization failure group, a low fertilization rate group, and a control group based on fertilization rate. We examined the associations among the 3 groups in terms of female age, duration of infertility, duration of stimulation, gonadotropin (Gn) dosage, follicle-stimulating hormone (FSH) dosage, and total number of retrieved oocytes. According to theincidence factors, the patients were divided into a single female factor group, a single male factor group and a unisex factor group, and the correlation analysis of incidence factor among the 3 groups was performed. The patients were divided into a primary infertility and a secondary infertility in accordance with the type of infertility. We analyzed the correlation of infertility type among the three groups. Risk factors for complete fertilization failure and low fertilization rate in IVF-ET were obtained by stepwise multiple linear regression analysis. Primary infertility, long infertility duration, total number of retrieved oocytes, and unisex factor were associated with completefertilization failure and low fertilization rate in IVF-ET (P<0.05), but female age, duration of stimulation, FSH dosage as well as Gn dosage were not correlated with complete fertilization failure and low fertilization rate in IVF-ET (P>0.05). Stepwise multiple linear regression analysis showed that the incidence factor, type of infertility, and infertility duration were independent influential factors for complete fertilization failure and low fertilization rate. Complete fertilization failure and low fertilization rate in IVF-ET are related to duration of infertility, total number of retrieved oocytes, cause of onset, and type of infertility, but they are not relevant to female age, duration of stimulation, and Gn and FSH dosage.

Identification and treatment of men with phospholipase Cζ–defective spermatozoa

To identify and treat the gamete responsible for complete fertilization failure with intracytoplasmic sperm injection (ICSI) using a newly proposed assisted gamete treatment (AGT). Prospective cohort study. Center for reproductive medicine. One-hundred and fourteen couples with an adequate number of spermatozoa for ICSI and a fertilization rate of ≤10%, after controlling for maternal age. Couples with an oocyte-related oocyte activation deficiency (OAD) underwent a subsequent cycle with a modified superovulation protocol; couples with sperm-related OAD had an additional genetic and epigenetic assessment to identify mutations and expression levels of the corresponding genes. Treatment cycle outcome for couples undergoing ICSI with either a modified superovulation protocol or AGT compared with their historical cycle. A total of 114 couples matched the inclusion criteria, representing approximately 1.3% of the total ICSI cycles performed at our center, with age-matched controls. Fifty-two couples were confirmed negative for sperm-related OAD by the phospholipase Cζ (PLCζ) assay, indicating oocyte-related factors in their failed fertilization cycles. Couples were treated by one of two AGT protocols, AGT-initial or AGT-revised, in a subsequent attempt that was compared with their historical cycle. Subsequent ICSI cycles with a tailored superovulation protocol yielded significantly higher fertilization (59.0% vs. 2.1%) and clinical pregnancy (28.6% vs. 0) rates. In 24 couples (mean ± standard deviation: maternal age, 35.6 ± 5 years; paternal age, 39.8 ± 6 years) sperm-related OAD was confirmed; in four men, a deletion on the PLCZ1 gene was identified. Additional mutations were also identified of genes supporting spermiogenesis and embryo development (PIWIL1, BSX, NLRP5) and gene deletions confirming a complete absence of the subacrosomal perinuclear theca (PICK1, SPATA16, DPY19L). Subsequent AGT treatment provided higher fertilization (42.1%) and clinical pregnancy (36% vs. 0%) rates for couples with a history of impaired (9.1%) fertilization. A comparison of the two AGT protocols, AGT-initial or AGT-revised, revealed that the latter yielded even more favorable fertilization (37.6% vs. 45.9%) and clinical pregnancy (21.1% vs. 83.3%) rates. In couples with an oocyte-related OAD, tailoring the superovulation protocol resulted in successful fertilization, term pregnancies, and deliveries. In couples with a sperm-related OAD as determined by PLCζ assay, mouse oocyte activation test, and the assessment of gene mutations and function, AGT was successful. The AGT-revised protocol yielded an even higher fertilization rate than the AGT-initial protocol, resulting in the birth of healthy offspring in all couples who achieved a clinical pregnancy.

WEE2 Mutations as a Cause of Complete Fertilization Failure (CFF) When it is the Female Factor Responsible for CFF- A Short Communication

WEE2 Mutations as a Cause of Complete Fertilization Failure (CFF) When it is the Female Factor Responsible for CFF- A Short Communication

Development of ICSI.

The first conception outside of the human body that led to the birth of Louise Brown was a tremendous accomplishment, which opened the door to the utilization of assisted reproductive techniques globally. This brought the understanding that accomplishing life in a dish required several steps, the most obvious being the timing and characteristics of fertilization. It soon became obvious in the 1980s that the most disappointing phenomenon was unexpected and complete fertilization failure. Among the approaches that were attempted to treat male factor infertility, ICSI surfaced as the technique that brought the ratio of the gametes to 1:1 and was also able to grant consistent fertilization and a higher pregnancy rate. ICSI has now been implemented for a quarter of a century, proving itself as the ultimate technique utilizing ejaculated spermatozoa independent of the semen parameters and is the sole insemination method to be used with surgically retrieved spermatozoa. There are currently various indications for ICSI that are widely adopted, rendering it the most popular insemination method worldwide. The reliability of ICSI ensures its employment in upcoming techniques involving in vitro spermatogenesis and neogametogenesis.

Fertilization failure and gamete health: Is there a link?

Fertilization is a hallmark event of sexual reproduction marked by the fusion of male and female gamete to form zygote. It is a highly complex, yet a robust process that is intricately regulated by various signalling molecules. A healthy fertilization is determined by the quality of zygote which is contingent on the health of egg and sperm. The relationship between infertility and gametic health can be reciprocal. On one hand gametogenesis has to be dynamic and unremitting to sustain the reproductive health, while on the other hand it has to be error free for proper embryonic development. Complex cellular interactions make gametogenesis highly vulnerable to extrinsic as well as intrinsic intrusions. Molecular disparities during these phases may result in complete fertilization failure. Present review provides an overview of the regulation of gametogenesis, determinants of healthy gamete, players at fertilization window and what may go wrong during the development of zygote to embryo leading to implantation failure. We have outlined different 'windows' of vulnerability during gametogenesis supported by evidences affecting the fertility potential of both the partners.