Subsequent ICSI Research Articles

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<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<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-072 Fresh testicular sperm seems to yield more fertilization abnormalities and early pregnancy loss than frozen testicular sperm

Abstract Study question How do ICSI outcomes using fresh testicular sperm, compare to those using frozen samples cryopreserved from the previous fresh testicular sperm extraction (TESE)? Summary answer Although fresh testicular sperm led to a better fertilization and pregnancy rate,it yielded more fertilization abnormalities and miscarriages than frozen sperm for the same couples. What is known already Testicular biopsies can be performed in cases of severe male infertility to search for spermatozoa. In the current era of advanced reproductive techniques, ICSI in association with TESE may be an effective treatment for patients with azoospermia. TESE could be performed the day of oocyte pick-up, or instead, it may be accomplished previously, and in this case TESE spermatozoa are cryopreserved for the future ICSI cycles. However, when reviewing the literature, not a lot of papers tried to compare the ICSI outcomes between fresh and frozen testicular sperm in the same couples. Study design, size, duration This retrospective study included the clinical and embryological data of pairs of fresh and frozen sperm injection cycles from couples after TESE between 2010 and 2023. The first ICSI was performed with fresh spermatozoa retrieved from TESE. The subsequent ICSI cycles used frozen spermatozoa from the first TESE. Participants/materials, setting, methods All infertile couples that underwent ICSI+TESE with testicular sperm cryopreservation in the study period were identified (110 couples). Of whom, 34 couples who underwent 40 subsequent ICSI cycle using frozen spermatozoa from the previous TESE were included. ICSI cycles were divided in: Group 1:ICSI with TESE and fresh testicular sperm injection Group 2:ICSI with frozen testicular sperm injection ICSI outcomes were compared for: fertilization/fertilization abnormalities rate, embryo quality, pregnancy and miscarriages rate, and live birth rate. Main results and the role of chance The average age of men who underwent ICSI was 42 years, while that of women was 33 years. On average, the couples had their ICSI with frozen sperm within 1.7 years after the TESE. There were no significant differences in women’s clinical parameters, the type of ovarian stimulation used nor its primary outcome between the two ICSI attempts. Although the primary outcome of ICSI showed a significantly higher fertilization rate for fresh testicular sperm compared to frozen sperm (64.4% vs 44.8%; p = 0.003), there was a tendency for more fertilization abnormalities when fresh testicular sperm was used (8.3% vs 6.2%, p = 0.4). Despite a better quality of cleavage embryos in ICSI with fresh testicular sperm (43.9% vs 39.2%, p = 0.6) and a higher pregnancy rate for the same group (21.9% vs 15.9%, p = 0.52), the miscarriage rate was twice as high when compared to the miscarriage rate of ICSI with frozen testicular sperm (6.7% vs 3.4%, p = 0.8), indicating that cryopreservation of testicular sperm could play a role in selecting better sperm for micro-injection. As for the live birth rate, ICSI with frozen sperm led to not statistically inferior rates when compared with ICSI with fresh sperm in the same couples (6.7% vs 10%, p = 0.6). Limitations, reasons for caution Due to our inclusion criteria, the study population may seem to be too reduced to reach significant results. To gain more informative insights, we recommend combining results from different centers and referring to the etiology of azoospermia that led to the TESE procedure. Wider implications of the findings Genetic testing and chromosomal aberration detection on testicular spermatozoa before and after cryopreservation could be proposed to compare the proportion of defected spermatozoa in both types of samples. Trial registration number not applicable

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<0.00001). The treatment cycle yielded a higher cleavage rate at 80.2% (93/116, P<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

P-176 A novel electrophoretic sperm isolation system achieves equivalent ICSI outcomes to the combined density gradient centrifugation and swim-up method in a significantly shorter processing time

Abstract Study question Does an electrophoretically sperm isolation method, the Felix™ system, have clinical or operational advantages over the combined density gradient centrifugation and swim-up (DGCS) method? Summary answer The Felix™ system produces results that are comparable to the DGCS method, however, Felix™ has the ability to isolate spermatozoa more efficiently. What is known already The isolation of healthy spermatozoa to improve ART outcomes has received enormous attention in the literature recently. Although swim-up and density gradient centrifugation are the most common sperm separation methods, the optimal technique is still unclear. Recently, good sperm selection approaches using various non-centrifugal devices have emerged. Previous reports have shown that a quick and simple sperm separation device, the Felix™ system, can isolate high-quality spermatozoa and improve the results of ART. The Felix™ system combines electrophoresis and separation membranes to collect negatively charged mature normal sperm. Study design, size, duration A prospective oocyte sibling study was conducted between March 2023 and December 2023. The study involved infertile couples (n = 45) with a semen volume of 2.3 mL or more, a motile sperm concentration of 2 million/mL or more, maternal age under 43 years, and at least four mature oocytes at the time of oocyte retrieval. Ejaculated semen was prepared separately using DGCS and Felix™ on the day of oocyte retrieval and subsequent ICSI results were analyzed. Participants/materials, setting, methods In the DGCS group, semen was layered on a 45/90% of sperm grad and centrifuged for 15 minutes, followed by a 30 min swim-up. Subsequently, 0.3 mL of supernatant was collected. In the Felix™ group, 0.3 mL of sperm was collected following six minutes of membrane hydration and electrophoresis. Sperm concentration, total motility, progressive motility and DNA fragmentation (SDFR) were measured. Fertilization, blastocyst development, good-quality blastocyst development, and embryo utilisation rates were also assessed. Main results and the role of chance The average female age at the time of oocyte retrieval was 36.5±4.0 years, and the average male age was 38.1±5.9 years. Of 401 oocytes, 200 and 201 oocytes underwent ICSI using sperm prepared by DGCS and Felix™, respectively. The average processing time of Felix™ was significantly shorter than that of DGCS (54.0±4.3 vs. 7.4±0.2 minutes, p < 0.001, respectively). No significant difference was observed between the DGCS and Felix™ groups for sperm concentration (p = 0.49), motility (p = 0.05), progressive motility (p = 0.34), and SDFR (p = 0.09). Furthermore, there were no significant differences between the two groups with regards to fertilization rate (81.0% vs. 80.6%, p = 0.92), blastocyst development rate (52.9% vs. 58.4%, p = 0.60), good-quality blastocyst development rate (26.1% vs. 35.7%, p = 0.06), and embryo utilisation rate (54.3% vs 58.0%, p = 0.50). Limitations, reasons for caution Although the DGCS method was used as a comparison group, it is necessary to compare the Felix™ system with DGC alone, swim-up alone, or other sperm isolation methods. Larger, more powered studies are required to determine the impact on pregnancy and live birth outcomes. Wider implications of the findings The Felix™ system is a viable option for sperm isolation. While DGCS requires complicated procedures and time-consuming process including centrifugation and incubation; Felix™ requires six minutes only. The Felix™ system reduces processing times significantly and therefore can improve laboratory operations and workflow. Trial registration number Not Applicable

#305 : Preparation of Epididymal/Testicular Spermatozoa; Technical Aspects and Its Impact on Success

Background and Aims: The introduction of ICSI 30 years ago paved the way for epididymal/testicular spermatozoa to be used to fertilise oocytes in couples undergoing ART. Currently, spermatozoa is recovered from the epididymis or testis using varying approaches, including micro-surgery and fine-needle aspiration, mostly in men with obstructive and nonobstructive azoospermia. The samples delivered to the laboratory for processing are of varying quality with regards to amount of material/tissue, presence of spermatozoa, and other cells. The success of processing the tissue can have a direct impact on the outcome of the subsequent ICSI and our aim for this review was to identify ways to improve the preparation techniques for epididymal/testicular samples. Ethics approval was obtained from WSLHD HREC Ref 5583QA. Methods: The review included 527 ICSI cycles for 311 patients (2016-2022) where epididymal/testicular spermatozoa had been used. Approaches for sample preparation included mini-gradient centrifugation and removal of red blood cells using erythrocyte lysis buffer (ELB). In cases with severe oligoasthenozoospermia (105 cycles, 79 patients), a phosphodiesterase inhibitor (PI) was added to induce motility. Results: Employing mini-gradient centrifugation or adding ELB did not result in lost spermatozoa for any cases, but vastly reduced sample debris allowing for easier identification of spermatozoa. Fertilisation rate after ICSI was 57.9±1.2%. Motile spermatozoa were found and injected in all 105 samples after addition of PI and the fertilisation rate after ICSI was 55.3±2.7. To date cycles with PI have resulted in 26 live births with no abnormalities and all infants were of appropriate weight for gestational age. Conclusions: Employing precise preparation techniques for epididymal/testicular samples is paramount to allow for timely identification of motile spermatozoa for ICSI and assure optimal overall outcome. Sharing and discussing successful approaches with the wider embryology community allows for discussion and refining of techniques which can benefit all.

The Prevalence of Bacterial Vaginosis among Infertile Iraqi Women and Subsequent ICSI outcome

The vaginal microbial makeup has a substantial impact on women's health. The typical vaginal microbial profile in healthy women of reproductive age comprises aerobic, facultatively anaerobic, as well as obligately anaerobic organisms. However, the optimal vaginal microbiota is dominated by lactobacilli and is thought to defend against pathogenic microbial colonization and infection by producing lactic acid, antimicrobial metabolites, and lowering immune system activation. Recent research has come up with the term "molecular bacterial vaginosis (BV)" to describe non-optimal vaginal microbiota that is low in Lactobacillus species (spp.) and has a lot of facultative and absolute anaerobic bacteria specifically Gardnerella vaginalis. In the context of IVF, BV has been linked to infertility. The study comprised forty-six women to explore the vaginal and follicular microbiota of asymptomatic infertile Iraqi women starting an ICSI trial. At the time of ova pick-up, a high vaginal swab and follicular fluid were acquired from each instance for DNA extraction to test for the presence of microorganisms by the use of a real-time PCR special commercial kit. In conclusion, molecular technologies have offered a more thorough view into the vaginal microbiota, which may explain why vaginal dysbiosis shifts during the ICSI-ET treatment. Even though the majority of women have no clinically visible infection, the fact is that they have an aberrant vaginal micro-ecology profile. Nevertheless, isolating microorganisms from follicular fluid did not reduce the likelihood of fertilization or the pregnancy rate during ICSI cycles.

Enzymatic tissue processing after testicular biopsy in non-obstructive azoospermia enhances sperm retrieval.

What is the added value of enzymatic processing of testicular biopsies on testicular sperm retrieval (SR) rates for patients with non-obstructive azoospermia (NOA)? In addition to mechanical mincing, enzymatic digestion increased SR rates in testicular biopsies of NOA patients. Many studies focus on the surgical approach to optimize recovery of testicular sperm in NOA, and in spite of that, controversy still exists about whether the type of surgery makes any difference as long as multiple biopsies are taken. Few studies, however, focus on the role of the IVF laboratory and the benefit of additional lab procedures, e.g. enzymatic digestion, in order to optimize SR rates. This retrospective single-center cohort study included all patients who underwent their first testicular sperm extraction (TESE) by open multiple-biopsy method between January 2004 and July 2022. Only patients with a normal karyotype, absence of Y-q deletions and a diagnosis of NOA based on histology were included. The primary outcome was SR rate after mincing and/or enzymes. The secondary outcome was cumulative live birth (CLB) after ICSI with fresh TESE and subsequent ICSI cycles with frozen TESE. Multiple biopsies were obtained from the testis, unilaterally or bilaterally, on the day of oocyte retrieval. Upon mechanical mincing, biopsies were investigated for 30 min; when no or insufficient numbers of spermatozoa were observed, enzymatic treatment was performed using Collagenase type IV. Multivariable regression analysis was performed to predict CLB per TESE by adjusting for the following confounding factors: male FSH level, female age, and requirement of enzymatic digestion to find sperm. We included 118 patients, of whom 72 (61.0%) had successful SR eventually. Spermatozoa were retrieved after mechanical mincing for 28 patients (23.7%; 28/118) or after additional enzymatic digestion for another 44 patients (37.2%; 44/118). Thus, of the 90 patients requiring enzymatic digestion, sperm were retrieved for 44 (48.9%). Male characteristics were not different between patients with SR after mincing or enzymatic digestion, in regard to mean age (34.5 vs 34.5 years), testis volume (10.2 vs 10.6 ml), FSH (17.8 vs 16.9 IU/l), cryptorchidism (21.4 vs 34.1%), varicocele (3.6 vs 4.6%), or histological diagnosis (Sertoli-cell only 53.6 vs 47.7%, maturation arrest 21.4 vs 38.6%, sclerosis/atrophy 25.0 vs 13.6%).Of the 72 patients with sperm available for ICSI, 23/72 (31.9%) achieved a live birth (LB) after the injection with fresh testicular sperm (and fresh or frozen embryo transfers). Of the remaining 49 patients without LB, 34 (69.4%) had supernumerary testicular sperm frozen. Of these 34 patients, 19 (55.9%) continued ICSI with frozen testicular sperm, and 9/19 (47.4%) had achieved an LB after ICSI with frozen testicular sperm. Thus, the total CLB was 32/118 (27.1%) per TESE or 32/72 (44.4%) per TESE with sperm retrieved.Of the female characteristics (couples with sperm available), only female age (30.3 vs 32.7 years; P = 0.042) was significantly lower in the group with a LB, compared to those without.The CLB with testicular sperm obtained after enzymatic digestion was 31.8% (14/44), while the CLB with sperm obtained after mincing alone was 64.3% (18/28). Multivariable logistic regression analysis showed that when enzymatic digestion was required, it was associated with a significant decrease in CLB per TESE (OR: 0.23 (0.08-0.7); P = 0.01). Limitations of the study are related to the retrospective design. However, the selection of only patients with NOA, and specific characteristics (normal karyotype and absence Y-q deletion) and having their first TESE, strengthens our findings. Enzymatic processing increases the SR rate from testicular biopsies of NOA patients compared to mechanical mincing only, demonstrating the importance of an appropriate laboratory protocol. However, NOA patients should be counseled that when sperm have been found after enzymatic digestion, their chances to father a genetically own child may be lower compared to those not requiring enzymatic digestion. None reported. N/A.

P-003 Obstructive azoospermia; MESA should be employed as the method for sperm retrieval, not TESE

Abstract Study question TESE is widely used for obstructive azoospermia (OA) as a surgical method for sperm retrieval, but is it benefiting the patient? Summary answer Since MESA-ICSI has a very good fertilization rate, clinical pregnancy rate and delivery rate, MESA should be employed for the OA subjects, not TESE. What is known already TESE and micro-TESE are technically simple, and widely used as a sperm retrieval surgery because they do not require microsurgical skills. Some review articles have shown no significant differences in the rates of cleavage, good-quality embryos, implantation, clinical pregnancy between ICSI with epididymal sperm or testicular sperm. Clinical usefulness of MESA is still controversial according to previous reports. Study design, size, duration We studied 110 patients diagnosed with OA and treated with MESA at the Asada Ladies Clinic between April 2004 and December 2021. Participants/materials, setting, methods The MESA was performed using a micropipette with a micropuncture technique under operative microscope. When no sperm were present or motility was not observed, additional punctures to the epididymal tubule were performed. Aspirated samples were transferred into modified human tubal fluid and sent to the in vitro fertilization (IVF) laboratory for cryopreservation. Main results and the role of chance Motile sperm were recovered in all cases (110 cases). Of these, ICSI using frozen thawed sperm was performed in 101 cases. The rate of normal fertilization rate was 76%. Of the 399 embryo transfer (ET) cycles, 168 had a clinical pregnancy (41% per ET). Of the 101 patients who underwent ART, 94 (93% per case) had clinical pregnancies resulting in 90 (89.1%) deliveries. It should be emphasized that since MESA does not involve incision of the testes, there are fewer postoperative peritoneal irritation symptoms and no concerns about postoperative testicular atrophy or low testosterone levels. Some review articles showed no significant differences in the rates of cleavage, good-quality embryo, implantation, clinical pregnancy between ICSI with epididymal sperm or testicular sperm. Although clinical usefulness of MESA is still controversial, if the ART results of MESA-ICSI and TESE-ICSI are the same, MESA should be performed as it is less invasive on the patient and reduces the burden on the embryologist who have to process the TESE tissue. Limitations, reasons for caution Some authors have reported that since MESA specimens contain DNA fragmentated sperm when compared with specimen of TESE, subsequent ICSI results in poorer fertilization and pregnancy rates. We did not evaluate sperm DNA fragmentation. Wider implications of the findings A large quantity of uncontaminated sperm can be retrieved using MESA which is less invasive for the patient, and there is no need for special processing before cryopreservation. Additionally, it can reduce the laboratory workload. Trial registration number not applicable

P-211 “Survival rates after vitrification of oocytes obtained after luteal phase vs follicular phase ovarian stimulation”

Abstract Study question Do the oocytes retrieved after luteal phase ovarian stimulation (OS) have the same survival rate as those retrieved after follicular phase (OS)? Summary answer Oocyte survival rate after vitrification is similar in oocytes retrieved in a follicular phase OS compared to those in luteal phase. What is known already Oocytes retrieved in luteal phase have recently captured scientific interest as dual stimulation is gaining popularity. Oocytes obtained in luteal phase OS added to those obtained in follicular phase shorten time to pregnancy per intention to treat in poor responders. Embryos obtained with these oocytes show similar ploidy rates, as well as clinical outcomes. Study design, size, duration We analyzed data from a randomized, non-inferiority Duo Stim Preimplantation Genetic Testing for aneuploidies (PGT-A) trial performed from 2017 to 2021 in women >38 years old. Only cycles with at least one oocyte retrieved in both follicular and luteal were included. We compared survival rates from oocytes obtained in after follicular phase OS (n = 154 oocytes) versus luteal phase OS (n = 201 oocytes) within the same patient. Participants/materials, setting, methods A subtotal of 38 patients were included who underwent 2, 3, or 4 OS cycles to accumulate oocytes for subsequent ICSI with PGT-A. After retrieval and decumulation, oocyte vitrification was performed with Kitazato vitrification protocol (Kitazato, Japan) and Cryotop device, same as subsequent thawing. Main results and the role of chance Mean of age in the selected patients was 39,7 years. In total, 195 cumulus-oocyte complexes were retrieved after follicular phase OS vs 245 in luteal phase. From this group of oocytes, 145/195 were mature (78.97%) vs 201/245 (82.04%), respectively. No statistically significant differences were observed in survival rate whether oocytes came from a follicular phase OS 81.16 % (125/154) versus luteal phase 87.06% (175/201) (p = 0.3312). Our results confirm that survival rate of oocyte obtained in a luteal phase OS is comparable to conventional follicular phase OS, validating oocyte accumulation strategy in a Duo Stim protocol. Limitations, reasons for caution The limited sample size was due to the nature of the randomized, controlled trial. Our results need to be replicated in larger series for validation. Wider implications of the findings Luteal phase OS oocytes showed similar survival rates after thawing than conventional OS cycles, opening a new venue for oocyte vitrification in oncological patients or poor responders accumulating oocytes. This approach might be of interest as well in patients or countries where ethical/legal limitations may limit embryo accumulation. Trial registration number not applicable

P-260 A more physiological approach to support oocyte activation in cases with fertilization failure

Abstract Study question Can we use a physiological compound to trigger oocyte activation in couples undergoing ICSI with history of fertilization failure due to sperm activating factor deficiency? Summary answer In a pilot sibling oocyte study, oocyte activation using recombinant human phospholipase C-zeta (rhPLCζ) yielded a comparable fertilization compared to conventional method using ionomycin. What is known already ICSI overcomes most forms of male gamete defects, however partial or total fertilization failure still occurs regardless of adequate semen parameters and optimal oocyte maturity. This has been attributed to the lack of a cytosolic factor identified as PLCζ. To address this, various approaches of assisted oocyte activation have been proposed, including gamete exposure to chemical or electrical stimuli. The most popular approach remains to be ionomycin. Despite its apparent safety, this stimulus is unnatural and may be aggressive on the oocytes. Therefore, we propose a more physiological approach by utilizing a recombinant form of this specific enzyme. Study design, size, duration In the past 2 months, couples with poor fertilization at their initial ICSI cycles were identified and counseled for assisted oocyte activation (AOA) in the subsequent cycle after confirmation of a PLCζ deficiency. Oocytes were separated into 2 equal cohorts activated either by ionomycin or co-injection with recombinant human rhPLCζ during ICSI. Fertilization rates between ionomycin-activated and rhPLCζ-activated cohorts were compared, as well as with the couples’ history cycles. Participants/materials, setting, methods Six couples with suboptimal fertilization outcomes consented for the study (IRB 0712009553) and were offered AOA with their subsequent ICSI cycles. Conventional AOA was carried out by exposing post-ICSI oocytes to 50 mM ionomycin. The rhPLCζ had a confirmed >95% purity. Our physiological rhPLCζ-activation method was performed by co-injecting spermatozoa with 0.4pL of a rhPLCζ (5mg/mL in glycerol) during ICSI. Main results and the role of chance Couples (n = 6, female age: 36.2 ± 6yrs, male age: 38.0 ± 7yrs) had negative infertility workups with normal semen parameters (3.3 ± 3 mL volume, 42.1 ± 35 x106/mL concentration, 30.8 ± 23% motility, 2.0 ± 1% normal morphology). They underwent 7 unsuccessful ICSI cycles with 20.0% (12/60) fertilization rate and yielded a 91.7% cleavage rate, of those, 3 were transferred at cleavage stage but did not yield clinical pregnancy. A total of 2 blastocysts were obtained but were all aneuploid. In their subsequent treatment cycles, overall AGT achieved a significantly improved fertilization rate of 51.6% (32/62) (P<0.001). We then compared the efficiency between the two AGT methods in equally distributed sibling oocyte cohort. Although mathematically insignificant, we observed a trend that rhPLCζ-activation outperforms ionomycin-activation in total activation (67.9% vs 50.0%) as well as fertilization (57.1% vs 47.0%). The new rhPLCζ-activation method yielded a comparable embryo cleavage rate at 87.5% compared to the conventional method at 81.3%. Although fresh embryo transfer at cleavage stage did not yield pregnancy rate yet, rhPLCζ-activated cohort had one euploid blastocyst while the ionomycin-activated cohort did not yield euploid blastocyst. Limitations, reasons for caution While we observed that a more physiological rhPLCζ-activation yields a comparable fertilization compared to the conventional ionomycin method, the study is preliminary, and a confirmatory clinical outcome is not yet available. Wider implications of the findings This is the first attempt to use recombinant sperm-specific protein to mimic physiological oocyte activation for patient treatment. If this data is confirmed in a larger series, this may represent a way to normalize fertilization in cases of sperm activating factor deficiency even in different sources of spermatozoa. Trial registration number N/A

MP30-03 MOTILITY ASSESSMENT OF SPERM RETRIEVED FROM MICRO-TESTICULAR SPERM EXTRACTION COMPARING FROZEN-THAW AND FRESH SPERM AT A SINGLE CENTER

You have accessJournal of UrologyCME1 Apr 2023MP30-03 MOTILITY ASSESSMENT OF SPERM RETRIEVED FROM MICRO-TESTICULAR SPERM EXTRACTION COMPARING FROZEN-THAW AND FRESH SPERM AT A SINGLE CENTER Raneen Sawaid Kaiyal, Darren Bryk, Shinnosuke Kuroda, Johnathan Doolittle, Neel V. Parekh, Edmund Sabanegh, Nina Desai, Scott Lundy, and Sarah C. Vij Raneen Sawaid KaiyalRaneen Sawaid Kaiyal More articles by this author , Darren BrykDarren Bryk More articles by this author , Shinnosuke KurodaShinnosuke Kuroda More articles by this author , Johnathan DoolittleJohnathan Doolittle More articles by this author , Neel V. ParekhNeel V. Parekh More articles by this author , Edmund SabaneghEdmund Sabanegh More articles by this author , Nina DesaiNina Desai More articles by this author , Scott LundyScott Lundy More articles by this author , and Sarah C. VijSarah C. Vij More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003258.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Microsurgical testicular sperm extraction (m-TESE) is the gold standard treatment for male patients with Non-Obstructive Azoospermia (NOA). The published success rates of this procedure are typically reported as sperm retrieval rates (SRR), which is estimated to be 50%. However, since motile or twitching sperm is preferentially selected for ICSI and testicular sperm retrieved is often non-motile or of poor morphology it leads to complex decisions for embryologists. SRR does not accurately capture the success of this procedure as measured by pregnancy and live birth outcomes. Our primary objective is to assess the motility of sperm retrieved from m-TESE at the time of fresh m-TESE (same day as egg retrieval) compared to the motility of sperm post thaw from frozen m-TESE cases (sperm is retrieved ahead of egg retrieval with subsequent cryopreservation) . The secondary outcomes include pregnancy and live birth rates. METHODS: We retrospectively reviewed our electronic records of m-TESE procedures performed between 2007-2021.Patients with obstructive azoospermia and cryptozoospermia were excluded. We reviewed all cases with retrieved sperm and recorded motility assessment for those cases. Sperm motility was classified as motile sperm, twitching sperm, or tail sway (sperm with distinctly flexible tail). We compared the motility and ICSI outcomes between fresh m-TESE and frozen-thaw m-TESE. RESULTS: 129 m-TESE surgeries were performed on 114 patients with NOA. 31 m-TESEs were performed fresh and 98 were performed ahead of time with sample freezing for subsequent ICSI cycle. SRR from “fresh” cases was 58%, while SSR from frozen-thaw cycles was 44.8% (p=0.200). Motile sperm (Motile sperm, Twitching sperm and Tail Sway) was obtained in 58% of “fresh” cases. Motile sperm was found for “frozen” mTESE in 37% of cases when examined on day of surgery and 19.3% of cases on post-thaw. Pregnancy and live birth rate were 55.5% and 27.7% respectively for “fresh” cases and 27% and 21.6% respectively for frozen-thaw cases (p-value <0.05 and 0.6140). CONCLUSIONS: Patients scheduled for frozen-thaw ICSI cycle had lower rates of obtaining motile sperm for ICSI, pregnancy and live births compared to patients undergoing for fresh m-TESE at one institution. These results help inform our counseling and treatment strategy for patients with non-obstructive azoospermia. Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e391 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Raneen Sawaid Kaiyal More articles by this author Darren Bryk More articles by this author Shinnosuke Kuroda More articles by this author Johnathan Doolittle More articles by this author Neel V. Parekh More articles by this author Edmund Sabanegh More articles by this author Nina Desai More articles by this author Scott Lundy More articles by this author Sarah C. Vij More articles by this author Expand All Advertisement PDF downloadLoading ...

Exclusive Sertoli Cell Syndrome (ESCS) is a rare condition that has male infertility as its main consequence. It is one of the most serious forms of non-obstructive azoospermia, with a poor reproductive prognosis

Abstract Exclusive Sertoli Cell Syndrome (ESCS) is a rare condition that has male infertility as its main consequence. It is one of the most serious forms of non-obstructive azoospermia, with a poor reproductive prognosis. In some cases, however, such as the type II of the syndrome, sperm can be recovered through testicular puncture and subsequent ICSI, with a 13% success rate. This article aims to report the case of an azoospermic 35-year-old patient, with no other significant changes in complementary exams. 35 years old man with history of primary infertility, he was married since 17 years with normal sexual activity and and average built. Hematologic examination and biochemistry lab data were within normal limits. LH and total testosterone values were also within normal range, together with slightly elevated FSH value (119.88mUI/ml. There was azoospermia in the first semen analysis. Repeated semen analysis, 3 months later, revealed azoospermia. The karyotype of the patient is 46, XY with no Y chromosome micro deletions. Scrotal Color Doppler ultrasound and MRI examinations revealed that both testicles were in the left hemiscrotum. The lower testis had 53×26×43 mm dimensions and the posterosuperior located testis had 27×16×25 mm dimensions. Total abdominal ultrasonic examination revealed no developmental anomaly of urinary system. Family history of the patient revealed that mother and father were cousins no family history of infertility, the picture suggestive a case of sertoli only cell syndrome.

DEVELOPMENT OF A MACHINE LEARNING APPLICATION FOR INTRAOPERATIVE OBJECT DETECTION OF POSITIVE SEMINIFEROUS TUBULES IN MICRODISSECTION TESTICULAR SPERM EXTRACTION FOR NONOBSTRUCTIVE AZOOSPERMIA

Approximately 1 in 100 men in the general population have azoospermia, and 80% of them have nonobstructive azoospermia (NOA). For couples with NOA who wish for a baby, the only effective treatment options are sperm retrieval by microdissection TESE (micro-TESE) and subsequent ICSI. However, the current sperm recovery rate is generally only 30-40%. Favorable seminiferous tubules that contain sperm are generally white and thicker in diameter than other tubules, and the success of sperm retrieval is largely dependent on the surgeon's attentiveness and observational ability.

Is There an Increased Incidence of Aneuploid Embryos in Cases of Obstructive and Non-Obstructive Azoospermia?

Background: Current evidence supports that males with low spermatozoa concentrations, as well as obstructive azoospermia (OA) or non-obstructive azoospermia (NOA), present with high rates of aneuploid spermatozoa. This issue could potentially lead to an increased number of aneuploid embryos in subsequent ICSI cycles using surgically retrieved spermatozoa, however, azoospermia is not routinely considered to be a clinical indication for pre-implantation genetic testing for aneuploidies (PGT-A). Aim: To identify the incidence of aneuploid embryos in PGT-A cycles that used testicular spermatozoa extracted through surgical sperm retrieval (SSR). In addition, whether aneuploidy rates differed between embryos fertilised with SSR compared with ejaculated spermatozoa. Method: A retrospective study of OA and NOA patients who underwent SSR between March 2017 and September 2020 and had PGT-A cycles. Next-generation sequencing was employed to detect embryo aneuploidies. Data were compared and analysed using a chi-square test with PGT-A cycles using ejaculated spermatozoa within the same time-period. Results: A total of 57 males underwent SSR, leading to 68 cycles that included the use of PGT-A. A total of 152 embryos with conclusive results identified there to be 57.2% euploid embryos (87/152) and 38.2% aneuploid embryos (58/152) in the SSR group. In comparison, the ejaculated spermatozoa group resulted in 59.2% (2115/3677) euploid embryos and 35.7% (1312/3677) aneuploid embryos. The incidence of aneuploid embryos (38.2%) in the SSR group was not statistically significant [Odds ratio: 1.11 (0.79-1.56) p=0.53]. Importantly, there were no differences for mean maternal age for both study groups (SSR female partners: 36.7 years; ejaculated spermatozoa female partners: 37.1 years; p&gt;0.1). Conclusion: The current outcomes indicate there is not an increased incidence of aneuploid embryos in PGT-A cycles of men who underwent to SSR compared with men using ejaculated spermatozoa.

P-112 Utilizing surgical sperm retrieval to improve clinical outcomes for men with high sperm chromatin fragmentation in their ejaculates

Abstract Study question In men with elevated sperm chromatin fragmentation (SCF), can surgical sperm retrieval from the proximal area of the male genital tract improve clinical outcomes? Summary answer Spermatozoa retrieved from the epididymis or testes demonstrate higher genomic integrity and are associated with enhanced embryo implantation, clinical pregnancy, and delivery rates. What is known already Sperm genomic integrity is a key factor in achieving a successful pregnancy. During spermiogenesis, alteration of DNA topology involving nuclease and ligase mechanisms is required for supercoiling and DNA compaction. If a proper DNA repair mechanism fails during this intricate process, spermatozoa with DNA fragmentation should be picked up by the epididymis and phagocytized. When this mechanism fails, along with the presence of superimposed reactive oxygen species in the male genital tract, spermatozoa with DNA fragmentation may reach the ejaculates. We have previously demonstrated that retrieving spermatozoa directly from the epididymis and testes yields gametes with higher conserved genomic integrity. Study design, size, duration In a nine-year timespan, we identified over 1,000 men with high SCF in their ejaculates; 144 patients consented to undergo surgical retrieval of spermatozoa. In a preliminary assessment, their clinical outcomes were compared to those from men with normal SCF (n = 539). Of these men, 53 consented to undergo subsequent ICSI cycles with their female partners using surgically retrieved spermatozoa from the epididymis (n = 15) or testes (n = 38). Fertilization and clinical outcomes were compared. Participants/materials, setting, methods Ejaculated, epididymal, and testicular specimens were screened by terminal deoxynucleotidyl dUTP nick-end labeling (TUNEL) to assess SCF using a commercially available kit. A total of 500 spermatozoa/sample were screened with a normal threshold of 15%. Surgical samples from the epididymis and testes were cryopreserved in multiple vials for subsequent ICSI cycles. Main results and the role of chance In the preliminary assessment, ICSI outcomes utilizing ejaculated spermatozoa were compared between men with normal (n = 539, 9.3±3%) and abnormal (n = 144, 28.2±13%) SCF. Although fertilization did not differ, implantation (24.1% vs. 7.8%) and clinical pregnancy rates (CPR, 35.8% vs. 14.6%) were significantly impaired (P &amp;lt; 0.001) in men with elevated SCF. SCF assessment on surgically retrieved spermatozoa revealed a decreasing trend from 28.2±13% in the ejaculate to 16.6±9.2% in the epididymis and 10.1±5.7% in the testes. A total of 53 men underwent 79 subsequent ICSI cycles using surgically retrieved spermatozoa. Compared to their historical cycles using ejaculates (n = 65), ICSI with surgically retrieved sperm substantially improved implantation (18.8% vs. 2.1%; P &amp;lt; 0.001), CPR (36.5% vs. 5.3%; P &amp;lt; 0.0001), and ongoing/delivery rates (33.8% vs. 4.1%; P &amp;lt; 0.0001). Among these couples, 15 underwent 21 ICSI cycles utilizing epididymal spermatozoa with a mean SCF of 16.6±9%. Implantation (26.5% vs. 3.7%; P &amp;lt; 0.05), CPR (68.4% vs. 4.2%; P &amp;lt; 0.0001), and ongoing/delivery rates (57.1% vs. 4.2%; P &amp;lt; 0.0001) were significantly improved compared to the historical cycles. The remaining 38 patients underwent ICSI with testicular spermatozoa with an average SCF of 10.1±6%. Implantation (15.0% vs. 2.6%; P &amp;lt; 0.01), CPR (25.5% vs. 6.1%; P &amp;lt; 0.01), and ongoing/delivery rates (23.6% vs. 3.0%; P &amp;lt; 0.05) were higher when compared to historical cycles. Limitations, reasons for caution Although surgically retrieved spermatozoa can be used to enhance clinical outcomes in couples with high SCF, epididymal spermatozoa yielded higher pregnancy rates despite the higher level of SCF in these specimens compared to testicular spermatozoa. This can be explained by the corrective action of the ICSI procedure itself. Wider implications of the findings Our study demonstrated that sperm DNA integrity progressively increases through the journey of the male genital tract. In couples with a compromised sperm genome, surgically retrieved spermatozoa can be beneficial. Therefore, assessing SCF might be used a routine tool to evaluate the male gamete. Trial registration number N/A

A SPERM SELECTION TECHNIQUE TO IMPROVE EMBRYO PLOIDY

To assess the role of an enhanced sperm selection method in mitigating paternal contributions to embryo aneuploidy. Over the last 4 years, 57 couples underwent ICSI with sperm selected by density gradient centrifugation (DGC), resulting in few frozen embryo transfers (FETs) due to consistent embryo aneuploidy following preimplantation genetic testing for aneuploidy (PGT-A). These men consented to sperm chromatin fragmentation (SCF) assessment, inclusive of double-stranded DNA breaks (dsDNA) in their raw semen, as well as post-DGC and post-microfluidic sperm selection (MFSS). These couples underwent subsequent ICSI cycles with MFSS. Outcomes of cycles processed by DGC and MFSS were analyzed and compared. SCF was assessed by terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) on ≥500 spermatozoa per patient, with a normal threshold of ≤15%. A neutral Comet assay was used to assess dsDNA on ≥200 spermatozoa, utilizing a modified in-house protocol and a normal threshold of ≤3%. A total of 57 men had the following semen parameters: concentration of 40.0±32x106/mL, 37.1±11% motility, and 2.2±1% normal morphology. After selection by DGC or MFSS, the concentrations were 3.3±3.4 and 8.0±13x106/mL, with 58.0±29% and 96.9±9% motility, respectively (P<0.0001). The SCF decreased from 21±14% in raw specimens to 18±6% following DGC and to 1.9±1% following MFSS (P<0.001). The dsDNA fell from 3.6±2% in raw specimens to 3.1±1% after DGC and to 0.3±0.2% after MFSS (P<0.001). These men (40.9±6 years) underwent DGC selection for 71 ICSI cycles with their female partners (36.5±5 years), achieving a fertilization rate of 58.4% (403/690) and a blastocyst euploidy rate of 28.5% (47/165). Only 19 FET cycles were performed, with a 6.7% implantation rate (2/30) and two clinical pregnancies resulting in miscarriage. Subsequently, these men had their specimens selected by MFSS in 71 ICSI cycles, resulting in a higher fertilization rate of 75.9% (647/852; P<0.0001) and a much improved (P<0.0001) blastocyst euploidy rate of 48.9% by PGT-A (192/389). In 48 FET cycles, 51 embryos were replaced with an increased implantation rate of 60.8% (31/51; P<0.0001), a CPR of 64.6% (31/48; P<0.001), and an ongoing/delivery rate of 62.5% (30/48; P<0.0001). With its dsDNA component, SCF tangibly contributes to embryo structural chromosomal abnormalities. An enhanced spermatozoa selection method for ICSI appears to remarkably increase the proportion of euploid blastocysts with consequent successful clinical outcomes.

Single-center thorough evaluation and targeted treatment of globozoospermic men

PurposeTo characterize, by specific biomarkers and nucleic acid sequencing, the structural and genomic sperm characteristics of partial (PG) and complete globozoospermic (CG) men in order to identify the best reproductive treatment.MethodsWe assessed spermatozoa from 14 consenting men ultrastructurally, as well as for histone content, sperm chromatin integrity, and sperm aneuploidy. Additional genomic, transcriptomic, and proteomic evaluations were carried out to further characterize the CG cohort. The presence of oocyte-activating sperm cytosolic factor (OASCF) was measured by a phospholipase C zeta (PLCζ) immunofluorescence assay. Couples were treated in subsequent cycles either by conventional ICSI or by ICSI with assisted gamete treatment (AGT) using calcium ionophore (Ionomycin, 19657, Sigma-Aldrich, Saint Louis, MO, USA).ResultsUltrastructural assessment confirmed complete acrosome deficiency in all spermatozoa from CG men. Histone content, sperm chromatin integrity, and sperm aneuploidy did not differ significantly between the PG (n = 4) and CG (n = 10) cohorts. PLCζ assessment indicated a positive presence of OASCF in 4 PG couples, who underwent subsequent ICSI cycles that yielded a 36.1% (43/119) fertilization with a 50% (2/4) clinical pregnancy and delivery rate. PLCζ assessment failed to detect OASCF for 8 CG patients who underwent 9 subsequent ICSI cycles with AGT, yielding a remarkable improvement of fertilization (39/97; 40.2%) (P = 0.00001). Embryo implantation (6/21; 28.6%) and clinical pregnancies (5/7; 71.4%) were also enhanced, resulting in 4 deliveries. Gene mutations (DPY19L2, SPATA16, PICK1) were identified in spermatozoa from CG patients. Additionally, CG patients unable to sustain a term pregnancy had gene mutations involved in zygote development (NLRP5) and postnatal development (BSX). CG patients who successfully sustained a pregnancy had a mutation (PIWIL1) related to sperm phenotype. PLCZ1 was both mutated and underexpressed in these CG patients, regardless of reproductive outcome.ConclusionsSperm bioassays and genomic studies can be used to characterize this gamete’s capacity to support embryonic development and to tailor treatments maximizing reproductive outcome.

Sertoli Cells Only Syndrome - Case Report

Exclusive Sertoli Cell Syndrome (ESCS) is a rare condition that has male infertility as its main consequence. It is one of the most serious forms of non-obstructive azoospermia, with a poor reproductive prognosis. In some cases, however, such as the type II of the syndrome, sperm can be recovered through testicular puncture and subsequent ICSI, with a 13% success rate. This article aims to report the case of an azoospermic 35-year-old patient, with no other significant changes in complementary exams. After percutaneous puncture of the epididymis and biopsy with no sperm, we diagnosed ESCS, and indicated IVF with donor semen.

ICSI OUTCOMES USING SPERMATOZOA WITH OPTIMAL GENOMIC INTEGRITY

To test whether selecting spermatozoa with an intact genome enhances implantation and pregnancy rates in couples undergoing ICSI. During the past 3 years, semen specimens from consenting men with prior ICSI failure were screened for sperm chromatin fragmentation (SCF) and double-stranded DNA breaks (DSBs). These tests were performed on raw specimens that were processed by density gradient centrifugation (DGC) and microfluidic sperm selection (MFSS). Couples underwent subsequent ICSI cycles with MFSS processing in order to select spermatozoa with the highest genomic integrity. Embryological and pregnancy outcomes were recorded in the study groups and compared to the historical cycles. Consenting men underwent standard semen analysis according to WHO 2010 criteria. DGC and MFSS were used to select spermatozoa with the highest portion of progressively motile spermatozoa. SCF was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) on at least 500 spermatozoa utilizing a threshold of ≤15%. DSBs were measured by a neutral Comet assay using a modified in-house protocol which assessed at least 200 spermatozoa per patient considering a normal threshold of ≤3%. ICSI was performed in the standard fashion. A total of 63 men with an average age of 41±10 years had the following average semen parameters: concentration of 26±31 x 106/mL, 33±14 motility, and 2±1% morphology. After DGC and MFSS, the sperm concentration was 18±23 and 8±13 x106/mL, with 55±33% and 97±9% motility (P<0.0001), respectively. The morphology remained 2±1% after DGC, and improved to 3.2±1% after MFSS (P<0.001). The SCF decreased from 24±8% in the raw samples to 19±10% following DGC, and fell to 1.7±1% after MFSS processing (P<0.0001). DSB rates were 4.1±1% in the raw samples, 3.3±2% after DGC, and only 0.3±0.1% after MFSS (P<0.0001). Men (n=39; 42±7 years) who underwent ICSI with their female partners (38±3 years) had an average SCF and DSB rate in their raw sample of 23±9% and 3.9±1.5%, respectively, which fell to 19±2% and 3.2±3% after DCG selection, and dropped to only 1.4±1% and 0.2±0.1% after MFSS (P < 0.0001). These couples underwent 25 reference ICSI cycles with DGC sperm selection and achieved a fertilization rate of 60.5% (98/162). The implantation rate was only 2.1% (1/47) with a clinical pregnancy rate of 5.0% (1/20), resulting in a pregnancy loss. These couples subsequently underwent 48 ICSI cycles with MFSS and achieved a fertilization rate of 73.1% (333/455; P<0.01). The implantation rate rose to 27.3% (26/95; P<0.001) and the clinical pregnancy rate increased to 53.3% (24/45; P<0.001), resulting in 46.6% (21/45) (P<0.0001) of deliveries/ongoing pregnancies.The pregnancy loss rate was 12.5% (3/24). According to our study, SCF is strongly correlated with sperm motility. MFSS is the only device capable of yielding the highest portion of progressively motile spermatozoa that therefore have the least amount of DNA damage. Spermatozoa with an intact genome are associated with improved embryonic development and implantation rates in couples undergoing ICSI.

A NOVEL METHOD TO ATTENUATE EMBRYO ANEUPLOIDY DUE TO PATERNAL INHERITANCE

To test the efficacy of a sperm-selection method to generate genetically healthy embryos in couples with recurrent embryo aneuploidy. Consenting men with high sperm DNA fragmentation (SCF) in their spermatozoa who were treated at our center during the last three years were included. Spermatozoa were selected by density gradient centrifugation (DGC) for ICSI cycles, which frequently yielded aneuploid embryos, resulting in no embryo transfer or failed implantation. In subsequent ICSI cycles, their spermatozoa were processed by microfluidics sperm selection (MFSS). SCF, including the assessment of double-stranded DNA breaks (DSBs), was performed before and after each selection method. SCF, embryo aneuploidy, and clinical outcomes were assessed and compared. A total of 29 men underwent semen analysis according to WHO 2010 criteria. SCF was carried out by TUNEL on at least 500 spermatozoa with an established threshold of 15%. DSBs were measured by a neutral Comet test using a modified in-house protocol. A threshold of ≤3% was considered normal, with at least 200 spermatozoa assessed per patient. Clinical outcomes were compared between ICSI cycles performed with the DGC and MFSS methods. Preimplantation genetic testing for aneuploidy (PGT-A) was carried out on the resulting blastocysts. Euploid embryos were replaced into the uterine cavity, and implantation and clinical pregnancy rates (CPR) were recorded. A total of 29 men (40±7 years) had the following semen parameters: a concentration of 38.7 ± 39 x106/mL, 31 ± 17% motility, and 2.1 ± 1% morphology. After DGC and MFSS, the sperm concentration was 18±29 and 7.3±11 x106/mL, with 55.2±30% and 98.3±2% motility, respectively (P<0.0001). The average SCF decreased from 22±9% in the raw samples to 16±3% following DGC, and dropped to 1.8±1% after MFSS processing (P<0.0001). The DSB rate was 3.8±2% in the raw samples, 2.8±1% after DGC, and only 0.3±0.1% after MFSS. These couples (female partner, 37.2±5 years) underwent 42 ICSI cycles with DGC-selected spermatozoa and achieved a fertilization rate of 61.8% (254/411) with 23.8% (26/109) of the embryos identified as euploid. The replacement of these euploid embryos yielded an implantation rate of only 4.3% (1/23) and a CPR of 8.3% (1/12), with 8.3% (1/12) resulting in a pregnancy loss. When these couples underwent subsequent ICSI cycles with MFSS, a fertilization rate of 80.2% (334/416) (P<0.00001) was achieved, with 48.9% (90/184) of the embryos identified as euploid (P<0.0001). A total of 29 embryos were replaced, resulting in an implantation rate of 65.5% (19/29; P<0.0001) and a CPR of 73% (19/26) (P<0.001), resulting in 5.2% (1/19) pregnancy loss and 69.2% (18/26) ongoing pregnancy or deliveries (P<0.0001). Spermatozoa with DNA damage, particularly double-stranded nicks/breaks, may impair the ability of the oocyte to repair the male genome. Because the male genome contributes to embryo aneuploidy, selecting spermatozoa with the highest motility and chromatin integrity may enhance the chances of obtaining a euploid embryo for transfer and boost embryo implantation.