Timing of Testicular Biopsy in Relation to Oocyte Retrieval and the Outcomes of Intracytoplasmic Sperm Injection.

PURPOSE: We evaluate microscopic (micro) testicular sperm extraction (TESE) timing relative to oocyte retrieval on intracytoplasmic sperm injection outcome. MATERIALS AND METHODS: Couples with nonobstructive azoospermia who underwent intracytoplasmic sperm injection with freshly retrieved spermatozoa were analyzed based on whether micro-TESE was performed at least 1 day prior to oocyte retrieval (TESE-day-before group) or on the day of oocyte retrieval (TESE-day-of group). Embryology and clinical outcomes were compared. RESULTS: The percentage of patients who underwent a successful testicular sperm retrieval was significantly lower in the TESE-day-before cohort (62%) than in the TESE-day-of cohort (69%; odds ratio [OR] 1.4, 95% CI [1.1, 1.7], P < .001). The fertilization rate was also found to be significantly lower in the TESE-day-before group (45%) than in the TESE-day-of group (53%; OR 1.4, 95% CI [1.2, 1.7], P = .01). Although the association between the cleavage rate and TESE timing was not statistically significant, the implantation rate was found to be significantly higher in the day-before cohort (28%) than in the day-of cohort (22%; OR 0.7, 95% CI [0.6, 0.9], P = .01). Nevertheless, it was found that the clinical pregnancy and delivery rates were not statistically significantly associated with the TESE timing. CONCLUSIONS: Although sperm retrieval and fertilization rates were lower in the TESE-day-before cohort, the 2 cohorts showed comparable embryologic and clinical outcomes. Micro-TESE can be performed before oocyte harvesting to provide physicians ample time to decide between cancelling oocyte retrieval or retrieving oocytes for cryopreservation.

Genetic profiling of azoospermic men to identify the etiology and predict reproductive potential.

PURPOSE: To identify germline mutations related to azoospermia etiology and reproductive potential of surgically retrieved spermatozoa, and to investigate the feasibility of predicting seminiferous tubule function of nonobstructive azoospermic men by transcriptomic profiling of ejaculates. MATERIALS AND METHODS: Sperm specimens were obtained from 30 men (38.4 ± 6 years) undergoing epididymal sperm aspiration for obstructive azoospermia (OA, n = 19) acquired by vasectomy, or testicular biopsy for nonobstructive azoospermia (NOA, n = 11). To evaluate for a correlation with azoospermia etiology, DNAseq was performed on surgically retrieved spermatozoa, and cell-free RNAseq on seminal fluid (n = 23) was performed to predict spermatogenesis in the seminiferous tubule. RESULTS: Overall, surgically retrieved sperm aneuploidy rates were 1.7% and 1.8% among OA and NOA cohorts, respectively. OA men carried housekeeping-related gene mutations, while NOA men displayed mutations on genes involved in crucial spermiogenic functions (AP1S2, AP1G2, APOE). We categorized couples within each cohort according to ICSI clinical outcomes to investigate genetic causes that may affect reproductive potential. All OA-fertile men (n = 9) carried mutations in ZNF749 (sperm production), whereas OA-infertile men (n = 10) harbored mutations in PRB1, which is essential for DNA replication. NOA-fertile men (n = 8) carried mutations in MPIG6B (stem cell lineage differentiation), whereas NOA-infertile individuals (n = 3) harbored mutations in genes involved in spermato/spermio-genesis (ADAM29, SPATA31E1, MAK, POLG, IFT43, ATG9B) and early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). Transcriptomic assessment of cell-free RNAs in seminal fluid from NOA men allowed the prediction of residual spermatogenic foci. CONCLUSIONS: Sperm genome profiling provides invaluable information on azoospermia etiology and identifies gene-related mechanistic links to reproductive performance. Moreover, RNAseq assessment of seminal fluid from NOA men can help predict sperm retrieval during testicular biopsies.

A novel approach toward the generation of oocytes by direct diploid cell haploidization.

Oocyte-mediated somatic cell haploidization is a process in which a diploid cell halves its chromosomal content by segregating its homologue within the ooplasm. Replacing the donor oocyte nucleus with a patient's female diploid somatic nucleus can generate patient-genotyped oocytes. Insemination of these resulting constructs enables their activation and induces a reductive meiotic division, haploidizing the diploid female donor cell that can subsequently support syngamy with the male genome and create a zygote. So far, experimental data for this method have been limited and have not consistently proven the generation of chromosomally normal embryos. Overall, we achieved reconstruction of murine oocytes with a micromanipulation survival rate of 56.5%, and a correct haploidization and fertilization rate of 31.2%, resulting in a 12.7% blastocyst rate. Time-lapse analysis revealed that reconstructed embryos underwent a timely polar body extrusion and pronuclear appearance followed by a satisfactory embryonic cleavage, comparable with the control. Whole genome sequencing of the analyzed embryos indicated that 27.3% (6/22) were properly diploid. Our findings suggest that diploid cell haploidization may be a feasible technique for creating functional gametes in mammals.

A comparison between the Felix™ electrophoretic system of sperm isolation and conventional density gradient centrifugation: a multicentre analysis.

PURPOSE: Developing optimized techniques for the isolation of human spermatozoa possessing low levels of DNA damage is an important objective for the ART industry. The purpose of this study was to compare a novel electrophoretic system (Felix™) of sperm isolation with a conventional method involving density gradient centrifugation (DGC). METHODS: Five international ART Centres in Australia, India, Sweden, the USA, and China have collaborated in order to compare the quality of the sperm populations isolated by Felix™ and DGC in terms of processing time, sperm concentration, motility, vitality, and DNA integrity as assessed by 3 methods: SCSA, Halo, and TUNEL. RESULTS: Across all centers, 112 comparisons were performed. Although significant differences were noted between centers in terms of the quality of the semen samples subjected for analysis, overall, both methods were equally capable of isolating populations of spermatozoa exhibiting high levels of vitality and progressive motility. The absolute numbers of spermatozoa recovered were significantly (p < 0.001) lower with the Felix™ device although sperm quality was higher with 4/5 centers reporting a significant improvement in DNA integrity relative to DGC (p < 0.01-p < 0.001). In practical terms, the Felix™ device featured a standardized 6 min preparation time whereas clinical DGC protocols varied from center to center but generally took around 40 min to complete. CONCLUSIONS: The Felix™ device is a positive technical development capable of isolating suspensions of highly motile spermatozoa exhibiting low levels of DNA damage in a fraction of the time taken by conventional procedures such as DGC.

Single-center thorough evaluation and targeted treatment of globozoospermic men.

PURPOSE: To 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. METHODS: We 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). RESULTS: Ultrastructural 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. CONCLUSIONS: Sperm bioassays and genomic studies can be used to characterize this gamete's capacity to support embryonic development and to tailor treatments maximizing reproductive outcome.

Thoughts on the popularity of ICSI.

PURPOSE: Intracytoplasmic sperm injection (ICSI) is the most widely utilized assisted reproductive technique (ART) worldwide. In this feature, we review the early assisted fertilization attempts that eventually led to the development of ICSI, and discuss its current utilization in cases of male and non-male factor infertility. METHODS: We researched the literature related to the development, indications, and current use of ICSI, such as sperm structural abnormalities, male genetic indications, surgically retrieved sperm, high sperm chromatin fragmentation, oocyte dysmorphism, and preimplantation genetic testing (PGT). We also describe the potential future applications of ICSI. RESULTS: This review summarizes the early micromanipulation techniques that led to the inception of ICSI. We also explore its current indications, including non-male factor infertility, where its use is more controversial. Finally, we consider the benefits of future advancements in reproductive biology that may incorporate ICSI, such as in vitro spermatogenesis, neogametogenesis, and heritable genome editing. CONCLUSION: The versatility, consistency, and reliability of ICSI have made it the most prevalently utilized ART procedure worldwide.

Sperm Genomic Integrity by TUNEL Varies throughout the Male Genital Tract.

PURPOSE: We assessed sperm chromatin fragmentation at different levels of the male genital tract. MATERIALS AND METHODS: Ejaculated specimens from consenting male partners were screened for sperm chromatin fragmentation by TUNEL (terminal deoxynucleotidyl deoxyuridine triphosphate nick end labeling). Men with intracytoplasmic sperm injection failure and high ejaculated sperm chromatin fragmentation underwent surgery to retrieve spermatozoa from different levels of the male genital tract, which were then reassessed for sperm chromatin fragmentation. Approximately 500 or more spermatozoa were assessed per patient with a 15% threshold. Intracytoplasmic sperm injection results of cycles using spermatozoa from different levels of the male genital tract were compared. RESULTS: Topographical assessment of the male genital tract showed a mean ± SD of 20.4% ± 10% sperm chromatin fragmentation in the vas deferens, 15.8% ± 8% in the epididymis and 11.4% ± 6% in the testis. All values were lower than in ejaculated controls (mean 32.9% ± 20%, p <0.05). A total of 25 couples who underwent intracytoplasmic sperm injection with surgically retrieved spermatozoa had lower sperm chromatin fragmentation (p <0.001), and higher implantation, clinical pregnancy and delivery rates (p <0.01). A total of 45 couples with a history of intracytoplasmic sperm injection failure with ejaculate performed elsewhere were treated solely with surgically retrieved spermatozoa at our center. Compared to historical cycles, surgically retrieved spermatozoa had a lower fertilization rate (65%, p <0.05) but enhanced rates of implantation (19.1%), clinical pregnancy (40.0%) and delivery (34.3%) (each p <0.01). CONCLUSIONS: To our knowledge we report for the first time that sperm chromatin fragmentation increases progressively from the testicle to the epididymis and the vas deferens, and is highest in the ejaculate. Men with high ejaculated sperm chromatin fragmentation can benefit from using surgically retrieved sperm for in vitro fertilization and/or intracytoplasmic sperm injection.

A treatment approach for couples with disrupted sperm DNA integrity and recurrent ART failure.

OBJECTIVE: To test a novel method to select spermatozoa with high chromatin integrity. DESIGN: Specimens with high sperm chromatin fragmentation (SCF) were selected by density gradient selection (DGS) and microfluidic sperm sorting (MSS). SETTING: Academic medical center. PATIENT(S): Ejaculates from consenting men were processed by DGS/MSS. Couples underwent ICSI cycles with spermatozoa processed by DGS/MSS. Clinical outcomes were evaluated after embryo transfer. INTERVENTION(S): SCF was measured by TUNEL. ICSI with spermatozoa selected by DGS and MSS was performed. MAIN OUTCOME MEASURE(S): Fertilization, embryo implantation, and pregnancy outcomes were compared between DGS and MSS. RESULT(S): A total of 23 men had an average SCF of 20.7 ± 10%. After DGS and MSS, the SCF was 12.5 ± 5% and 1.8 ± 1%, respectively. In couples who underwent ICSI, the average SCF was 28.8 ± 9%, which fell to 21.0 ± 9% after DGS and 1.3 ± 0.7% after MSS. Four couples underwent 11 ICSI cycles with DGS and achieved one (25%) pregnancy that resulted in pregnancy loss. In four subsequent ICSI cycles with MSS, an ongoing clinical pregnancy rate of 50% was achieved. Five additional couples underwent 12 cycles of ICSI with DGS. After preimplantation genetic testing for aneuploidy, 30.3% of the embryos were euploid. One pregnancy was achieved, resulting in pregnancy loss. With MSS, 31.5% of the embryos were euploid and 4 couples obtained a pregnancy. Finally, sixteen couples underwent 20 ICSI cycles solely with MSS at our center. Of these couples, 8 had failed 13 ICSI cycles with DGS elsewhere. These couples achieved an overall implantation of 34.5% (10/29) and a pregnancy rate of 58.8% (10/17). CONCLUSION(S): Microfluidic selection yielded spermatozoa with optimal genomic integrity and improved chances of obtaining a euploid conceptus.

Faster sperm selected by rheotaxis leads to superior early embryonic development in vitro.

To understand the impact of sperm speed as they swim against the flow on fertilization rates, we created conditions similar to the female reproductive tract (FRT) on a microfluidic platform for sperm selection. Selected sperm were evaluated based on early development of fertilized embryos. Bovine and human spermatozoa were selected at various fluid flow rates within the device. We found that the speed of bovine spermatozoa increases as the flow rate increases and that the amount of DNA fragmentation index is lowered by increasing the flow rate. Bovine spermatozoa selected by our platform at low (150 µL h-1, shear rate 3 s-1), medium (250 µL h-1, shear rate 5 s-1), and high flow rates (350 µL h-1, shear rate 7 s-1) were used for fertilization and compared to sperm sorted by centrifugation. The samples collected at the highest flow rate resulted in the formation of 23% more blastocysts compared to the control. While selecting for higher quality sperm by increasing the flow rate does result in lower sperm yield, quality improvement and yield may be balanced by better embryonic development.

Profiling the male germline genome to unravel its reproductive potential.

OBJECTIVE: To identify specific germline mutations related to sperm reproductive competence, in couples with unexplained infertility. DESIGN: In this retrospective study, couples were divided according to whether they had successful intracytoplasmic sperm injection outcomes (fertile) or not (infertile). Ancillary sperm function tests were performed on ejaculates, and whole exome sequencing was performed on spermatozoal DNA. Sperm aneuploidy and gene mutation profiles were compared between the 2 cohorts as well as according to the specific reasons for reproductive failure. SETTING: Center for reproductive medicine at a major academic medical center. PATIENT(S): Thirty-one couples with negative infertility workups and normal semen parameters. INTERVENTION(S): Couples with mutations on fertilization- or embryo development-related genes were subsequently treated by assisted gamete treatment or microfluidics, respectively. MAIN OUTCOME MEASURE(S): Intracytoplasmic sperm injection cycle outcomes including fertilization, clinical pregnancy, and delivery rates. RESULT(S): Sperm aneuploidy was lower in the fertile group (4.0% vs. 8.4%). Spermatozoa from both cohorts displayed mutations associated with sperm-egg fusion (ADAM3A) and acrosomal development (SPACA1), regardless of reproductive outcome. The infertile cohort was then categorized according to the reasons for reproductive failure: absent fertilization, poor early embryo development, implantation failure, or pregnancy loss. Spermatozoa from the fertilization failure subgroup (n = 4) had negligible PLCζ presence (10% ± 9%) and gene mutations (PLCZ1, PIWIL1, ADAM15) indicating a sperm-related oocyte-activating deficiency. These couples were successfully treated by assisted gamete treatment in their subsequent cycles. Spermatozoa from the poor early embryo development subgroup (n = 5) had abnormal centrosomes (45.9% ± 5%), and displayed mutations impacting centrosome integrity (HAUS1) and spindle/microtubular stabilization (KIF4A, XRN1). Microfluidic sperm processing subsequently yielded a term pregnancy. Spermatozoa from the implantation failure subgroup (n = 7) also had abnormal centrosomes (53.1% ± 13%) and carried mutations affecting embryonic implantation (IL9R) and microtubule and centrosomal integrity (MAP1S, SUPT5H, PLK4), whereas those from the pregnancy loss subgroup (n = 5) displayed mutations on genes involved in trophoblast development (NLRP7), cell cycle regulation (MARK4, TRIP13, DAB2IP, KIF1C), and recurrent miscarriage (TP53). CONCLUSION(S): By assessing the sperm genome, we identified specific germline mutations related to various reproductive processes. This information may clarify elusive factors underlying reproductive competence and enhance treatment for couples with unexplained infertility.

A non-randomized clinical trial to determine the safety and efficacy of a novel sperm sex selection technique.

The desire to have offspring of a specific sex has a long history but has been particularly present with the appearance of assisted reproduction. However, embryo selection raises ethical concerns. Thus, several techniques to select sex-specific spermatozoa have been proposed but carry limitations. There are many variations of each technique, and some are time consuming and costly. Concerns about effectiveness and safety have also rendered many of them unappealing. Therefore, we propose a novel sperm sex selection technique (SST) that appears to be consistently safe and effective. A single-center, non-randomized clinical trial was designed. We included 1,317 couples, who were assigned to one of two groups: ICSI/PGTA or ICSI/PGTA+GS. Ejaculates from male partners of couples in the ICSI/PGTA+GS group (n = 105) were processed using SST to enrich spermatozoa for their desired sex. Standard sperm processing was carried out for couples undergoing PGT-A solely for aneuploidy (n = 1,212), comprising the ICSI/PGTA control group. To validate the efficacy of our technique, we performed an analysis on spermatozoa pre- and post-selection, followed by an assessment of the proportion of the conceptuses' sex to confirm clinical reliability. We also followed up on ICSI clinical outcomes and child/newborn health to establish the safety of our method. Our main outcome measures included the proportion of spermatozoa and embryos enriched for female and male sex, as well as embryo euploidy rates and ICSI clinical outcomes. These outcomes were compared between the two groups. For the ICSI/PGTA group (n = 1,212) (maternal age, 37.0±4yrs; paternal age, 39.1±6yrs), with ejaculated spermatozoa processed in the standard fashion, 2,303 ICSI cycles (1.2±1) yielded an 81.0% (14,375/17,737) fertilization. PGT-A results indicated a euploidy rate of 73.1% (n = 3,718) for female and 72.4% (n = 3,054) for male embryos. These couples achieved a 76.4% (699/915) implantation and 65.2% (597/915) clinical pregnancy rate, with 551 deliveries (48.5% female, 51.5% male). All 105 men in the ICSI/PGTA+GS group had sperm specimens with an equal sex distribution at baseline. Of them, 59 (paternal age, 40.9±6yrs) who desired female offspring obtained an 81.6% enrichment after SST. They underwent 73 ICSI cycles with their partners (maternal age, 37.9±4yrs), achieving a 77.3% (583/754) fertilization. This resulted in 79.1% (231/292) female embryos that generated a 79.3% (23/29) implantation rate, with 16 singleton deliveries of the desired female sex without major or minor congenital malformations. Forty-six couples (maternal age, 37.3±4yrs; paternal age, 40.7±6yrs) desiring male offspring obtained an 80.8% sperm sex enrichment. They underwent 50 ICSI cycles, achieving a 75.4% (462/613) fertilization and equivalent proportion of male embryos (223/280, 79.6%). Their implantation was 90.5% (19/21), with 13 singleton deliveries of healthy male offspring. Furthermore, 78.8% (182/231) of female and 66.4% (148/223) of male embryos from the ICSI/PGTA+GS cohort were euploid. These euploid rates were comparable to those from the ICSI/PGTA group. In couples undergoing ICSI with PGT-A, SST consistently enriched spermatozoa, resulting in a higher proportion of embryos and thus offspring of the desired sex. Moreover, SST did not impair the fertilization or embryo developmental competence of spermatozoa, nor did it affect offspring health. Trial registration: Clinicaltrials.gov NCT05500573.

Male haploid cells through direct spherification.

OBJECTIVE: To obtain de novo male gametes capable of inducing full preimplantation blastocyst development using a novel three-dimensional (3D) culture system. DESIGN: Mouse embryonic stem cells (mESCs) were spherified by plunging in sodium alginate followed by calcium chloride, delineating a 3D environment that simulates the seminiferous tubule. As a control, mESCs cultured on two-dimensional plates were used. Plates and spheres containing mESCs from both methods were exposed to Activin-A, bFGF, and KSR followed by exposure to BMP4, LIF, SCF, and EGF to promote differentiation into male germ-like cells. MAIN OUTCOME MEASURES: Cells were assessed for VASA, DAZL, and BOULE on days 3 and 10. Cells were later injected into activated oocytes and monitored using time-lapse imaging on days 15, 22, 29, and 36. Control conceptuses generated using mature epididymal spermatozoa were also monitored via time-lapse imaging. RESULTS: On day 3, cells differentiated on plates expressed VASA at 1% and DAZL at 29%. In spheres, VASA was expressed at a rate of 15% and DAZL at a rate of 45% (P<.001). On day 10, cells differentiated on plates had VASA expression of 7%, DAZL of 23%, and BOULE of only 0.5%. Cells differentiated into spheres expressed VASA at a rate of 20%, DAZL at 43%, and BOULE at 10% (P<.001). Subsequent differentiation in spheres on day 3 exhibited a DAZL (expressed in spermatogonia) expression of 43% and a VASA (further spermatogenesis progression) expression of 15%. On day 10, DAZL and VASA expressions were reassessed and increased to 45% and 18%, respectively. BOULE, a marker expressed solely in postmeiotic spermatocytes, was expressed at 8%, whereas acrosin was expressed in spermatids at 2%. On day 15, VASA expression plateaued at 17%, BOULE peaked at 10%, and acrosin reached 5%. On day 22, expression of VASA increased to 19%, BOULE decreased to 8%, and acrosin peaked at 7%. On day 29, VASA expression peaked at 20%, BOULE dropped to 2%, and acrosin remained stable at 7%. On day 36, VASA expression remained at 13%, whereas BOULE and acrosin expression decreased to 0% and 1%, respectively. The control cohort attained 88.4% fertilization and 76.9% blastocyst rates. De novo gametes achieved fertilization rates of 35.0%, 61.1%, 81.8%, and 50.0% on days 15, 22, 29, and 36, respectively. Neogametes-generated blastocyst rates were 5.0%, 16.7%, 36.4%, and 8.3% for days 15, 22, 29, and 36, respectively. CONCLUSION: Our novel 3D differentiation model can generate functional gametes and is aimed at obviating the need for allogeneic/xenogeneic transplantation. The decreased overall marker expression and the reduced blastocyst development indicated that intrasphere germ cell differentiation correlated with the length of mouse spermatogenesis at approximately 30 days. Future experiments will be conducted to confirm the reproducibility of our findings and the eventual generation of offspring.

Can a sperm selection technique improve embryo ploidy?

BACKGROUND: Spermatozoa with the highest motility retain a superior genomic integrity, and elevated sperm chromatin fragmentation (SCF) has been linked to a lower ability of the conceptus to develop and implant. Therefore, the utilization of a sperm selection method, such as microfluidic sperm selection (MFSS), is capable of reducing the SCF by yielding the most motile fraction of spermatozoa with the highest embryo developmental competence. What remains unclear, however, is the causal mechanism that links SCF to an impaired embryo development. OBJECTIVES: To identify a relationship between SCF and an unexpectedly high proportion of embryo aneuploidy, while addressing treatment options. MATERIALS AND METHODS: We identified couples with a high incidence of embryo aneuploidy in a previous intracytoplasmic sperm injection (ICSI) cycle with pre-implantation genetic testing for aneuploidy (PGT-A), utilizing spermatozoa selected by density gradient (DG). Terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) and neutral Comet assays were carried out on the semen specimens to assess total SCF and double-stranded DNA (dsDNA) fragmentation, respectively. These couples underwent subsequent ICSI/PGT-A cycles with MFSS. Total SCF and dsDNA fragmentation were compared between the two sperm selection methods. Embryo aneuploidy, implantation, clinical pregnancy, delivery, and pregnancy loss rates were compared between the couples' historical DG and subsequent MFSS cycles. RESULTS: In 57 couples undergoing 71 ICSI/PGT-A cycles, where DG sperm selection was carried out, a high incidence of aneuploid embryos (74.7%) resulted in poor implantation and no viable pregnancies. Testing for SCF, inclusive of dsDNA breaks, evidenced a SCF of 26.2% and dsDNA break of 3.6% in the raw specimen, that decreased to 18.0% (p < 0.001) and 3.1%, respectively, in the DG processed specimen. Following MFSS, total SCF and dsDNA fragmentation decreased to 1.9% and 0.3%, respectively (p < 0.001). The embryo euploidy rate remarkable improved from 25.3% in the DG cycles to 42.9% in the MFSS cycles (p < 0.001). The 6.7% implantation rate in the DG cycles increased to 65.5% in the MFSS cycles (p < 0.001). Similarly, the clinical pregnancy rate rose from 10.5% (DG) to 64.6% (MFSS), resulting in a 62.5% delivery rate (p < 0.001). DISCUSSION AND CONCLUSIONS: In couples with a relatively young female partner with a negative infertility workup, and a male partner with semen parameters adequate for ICSI, presenting with a high rate of embryo aneuploidy, an additional subtle male factor component may be the culprit. Thus, it is crucial to assess the SCF and test for the dsDNA breaks, which can eventually contribute to embryo chromosomal abnormalities. Given the inverse relationship between SCF and motility, a selection of the most motile gamete by MFSS enhanced the proportion of spermatozoa with an intact genome, contributing to the generation of more euploid embryos that are capable of implanting and yielding increased term pregnancies.

Assessing male gamete genome integrity to ameliorate poor assisted reproductive technology clinical outcome.

OBJECTIVE: To assess the role of evaluating sperm chromatin fragmentation (SCF) as a tool to guide treatment in couples who achieved unexpectedly poor clinical outcomes after intracytoplasmic sperm injection (ICSI). DESIGN: We identified couples with an unexpectedly suboptimal clinical outcome after ICSI who were then screened for SCF. Consequently, the same couples were counseled to undergo a subsequent ICSI cycle using either ejaculates processed by microfluidic sperm selection (MFSS) or spermatozoa retrieved from the testis, and clinical outcomes were compared between history and treatment cycles. To confirm the sole effect of a compromised male gamete, we compared the ICSI outcome in cycles where male gametes with abnormal SCF were used to inseminate autologous and donor oocytes. Finally, to eliminate an eventual confounding female factor component, we compared the clinical outcome of ICSI cycles using sibling donor oocytes injected with spermatozoa with normal or abnormal SCF. SETTING: Academic reproductive medicine center point of care. PATIENT(S): The patient population consisted of 76 couples with reproductively healthy and relatively young female partners and male partners with compromised semen parameters, but suitable for ICSI. In a subanalysis, we identified 67 couples with abnormal SCF who underwent ICSI cycle(s) with donor oocytes. Furthermore, we identified 29 couples, 12 with normal SCF and 17 with abnormal, uncorrected SCF, and 7 couples with abnormal, corrected SCF vs. a control, who used sibling donor oocytes for their ICSI cycle(s). INTERVENTION(S): For couples who resulted in surprisingly low clinical outcomes after ICSI, despite semen parameters adequate for ICSI and a normal female infertility evaluation, a SCF assessment was performed on the semen specimen using the terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labeling (TUNEL) assay. The couples then underwent a subsequent ICSI cycle with spermatozoa processed by MFSS or surgically retrieved. Moreover, cycles with donor oocytes were used to confirm the sole contribution of the male gamete. MAIN OUTCOME MEASURE(S): Clinical outcomes, such as fertilization, embryo implantation, clinical pregnancy, delivery, and pregnancy loss rates were compared between history and treatment cycle(s) using ejaculated spermatozoa selected by MFSS or from a testicular biopsy, taking into consideration the level of SCF. In a subanalysis, we reported the clinical outcomes of 67 patients who used donor oocytes and compared them with cycles where their own oocytes were used. Furthermore, we compared the ICSI clinical outcomes between cycles using sibling donor oocytes injected with low or high SCF with or without sperm intervention aimed at correcting, or alleviating the degree of SCF. RESULT(S): In a total of 168 cycles, 76 couples had in a prior cycle a 67.1% fertilization rate, and clinical pregnancy and pregnancy loss rates of 16.6% and 52.3%, respectively. After testing for SCF, the DNA fragmentation rate was 21.6%. This led to a subsequent ICSI cycle with MFSS or testicular sperm extraction, resulting in clinical pregnancy and delivery rates of 39.2%, and 37.3%, respectively. The embryo implantation rate increased to 23.5%, whereas the pregnancy loss rate decreased to 5% in the treatment cycle. This was particularly significant in the moderate SCF group, reaching embryo implantation, clinical pregnancy, and delivery rates of 24.3%, 40.4%, and 36.2%, respectively, and reducing the pregnancy loss rate to 10.5% in post-sperm treatment cycles. In 67 patients with high SCF who used donor oocytes, a significantly higher fertilization rate of 78.1% and embryo implantation rate of 29.1% were reported, compared with those in couples also with an elevated SCF who used their own. Interestingly, the clinical pregnancy and delivery rates only increased slightly from 28.0%-36.1% and from 23.7%-29.2%, respectively. To further control for a female factor, we observed couples who shared sibling donor oocytes, 17 with normal SCF and 12 with abnormal (uncorrected) SCF. Interestingly, the abnormal SCF group had impaired fertilization (69.3%), embryo implantation (15.0%), and delivery (15.4%) rates. For an additional 15 couples who split their donor oocytes, 8 had normal SCF, and although 7 couples originally had abnormal SCF, 4 used microfluidic processing, 2 used testicular spermatozoa, and 1 used donor spermatozoa to alleviate the degree of SCF, resulting in comparable clinical outcomes with the normal SCF group. CONCLUSION(S): A superimposed male factor component may explain the disappointing ICSI outcome in some couples despite reproductively healthy female partners. Therefore, it may be useful to screen couples for SCF to guide treatment options and maximize chances of a successful pregnancy. The improved, but suboptimal pregnancy and delivery outcomes observed in couples using donor oocytes confirmed the exclusive detrimental role that the male gamete exerted on embryo development despite the presence of putative oocyte repair mechanisms.

Assisted gamete treatment to pinpoint acquired meiotic maturity and overcome oocyte activation deficiency contributed by both gametes.

Objective: To treat couples with total fertilization failure (TFF) based on a combined oocyte- and sperm-related oocyte activation deficiency by optimizing oocyte response to chemical activation with calcium ionophore. Design: Case report. Setting: Tertiary Hospital. Patients: Two couples with a history of TFF after intracytoplasmic sperm injection intracytoplasmic sperm injection (ICSI). Interventions: To overcome oocyte-related oocyte activation deficiency (OAD), extended in vivo/in vitro oocyte maturation was performed to enhance ooplasmic maturity; to address sperm-related OAD, assisted gamete treatment (AGT) was performed to trigger oocyte activation. Main outcome measures: Treatment cycle outcomes for the 2 couples undergoing ICSI with extended oocyte maturation (EOM) and AGT. Results: We identified 2 couples with TFF after ICSI because of a combined factor of OAD confirmed by phospholipase C zeta expression and genomic assessment. Initial AGT treatment alone failed to enhance fertilization, suggesting superimposed oocyte dysmaturity prohibiting oocytes from responding to chemical stimuli. To address this complex form of OAD, in couple 1, 27 oocytes out of 34 retrieved presented normal metaphase II spindles after EOM; ICSI with AGT yielded a fertilization rate of 63.0% (17/27). All 17 zygotes were cryopreserved initially. Two embryos were thawed and transferred, yielding a monochorionic diamniotic twin pregnancy. Couple 2 underwent 3 ICSI cycles with EOM and AGT; 91.4% (32/35) of oocytes displayed normal metaphase II spindle and achieved an overall fertilization rate of 43.8% (14/32). A total of 12 blastocysts were cryopreserved. A single 46XY blastocyst was thawed and transferred, resulting in a singleton pregnancy. Conclusions: Our study has demonstrated the usefulness of EOM by targeting spindle presence to enhance chemical responses to AGT.

Successful cryptozoospermia management with multiple semen specimen collection.

OBJECTIVE: To determine the prevalence of sperm suitable for intracytoplasmic sperm injection (ICSI) in fresh ejaculated semen samples provided by men scheduled for a microdissection testicular sperm extraction (mTESE) procedure. Secondary objectives included an evaluation of the effect of a short abstinence period on semen quality and ICSI outcomes for men with cryptozoospermia. DESIGN: Retrospective cohort study. SETTING: Academic medical center. PATIENTS: All men were scheduled to undergo a mTESE procedure by a single, high-volume surgeon at an academic center from September 1, 2015, to May 1, 2021. INTERVENTION: Presence of sperm suitable for ICSI in the ejaculate on the day of scheduled mTESE. MAIN OUTCOME MEASURES: Prevalence of sperm suitable for ICSI in the ejaculate among previously diagnosed men with azoospermia. Secondary outcomes included changes in semen parameters, clinical pregnancy rate, and live birth rate. RESULTS: Of 727 planned mTESE procedures, 69 (9.5%) were canceled because sperm suitable for ICSI were identified in a fresh ejaculated sample produced on the day of scheduled surgery (typically one day before oocyte retrieval). Overall, 50 men (50/727, 6.9%) used these rare, ejaculated sperm for ICSI. Semen samples obtained with <24 hours of abstinence were more likely to have better motility than the sample initially provided on the day of the planned mTESE. The live birth rate per ICSI attempt using these rare, ejaculated sperm was 36% (19/53). CONCLUSION: Providing a fresh ejaculated semen sample on the day of mTESE allows nearly 10% of men with azoospermia to avoid surgery with satisfactory ICSI outcomes. Providing multiple ejaculated samples over a short period of time does not adversely affect sperm concentration and may enhance sperm motility in men with cryptozoospermia.

Sperm centriolar factors and genetic defects that can predict pregnancy.

The human sperm centrosome, comprising the two morphologically distinct centrioles and associated pericentriolar materials, plays a crucial role in fertilization and early embryonic development after fertilization. Once inside the oocyte, the sperm centrosome serves as a microtubule-organizing center, orchestrating mitotic spindle formation, chromosome segregation, and syngamy. Abnormalities of the sperm centrosome can lead to abnormal embryonic development and embryonic chromosomal instability, and are associated with pregnancy loss. Recent research has shed light on the molecular composition, regulation, and function of this vital organelle. Understanding the intricacies of the sperm centrosome is crucial for elucidating the mechanisms underlying successful fertilization and early embryonic development, as well as addressing infertility and developmental disorders associated with centrosomal defects.

Non-contact ultrasound oocyte denudation.

Cumulus removal (CR) is a central prerequisite step for many protocols involved in the assisted reproductive technology (ART) such as intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing (PGT). The most prevalent CR technique is based upon laborious manual pipetting, which suffers from inter-operator variability and therefore a lack of standardization. Automating CR procedures would alleviate many of these challenges, improving the odds of a successful ART or PGT outcome. In this study, a chip-scale ultrasonic device consisting of four interdigitated transducers (IDT) on a lithium niobate substrate has been engineered to deliver megahertz (MHz) range ultrasound to perform denudation. The acoustic streaming and acoustic radiation force agitate COCs inside a microwell placed on top of the LiNbO3 substrate to remove the cumulus cells from the oocytes. This paper demonstrates the capability and safety of the denudation procedure utilizing surface acoustic wave (SAW), achieving automation of this delicate manual procedure and paving the steps toward improved and standardized oocyte manipulation.

Haploidy in somatic cells is induced by mature oocytes in mice.

Haploidy is naturally observed in gametes; however, attempts of experimentally inducing haploidy in somatic cells have not been successful. Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0/G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos. Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.