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The evident ability of the intracytoplasmic sperm injection (ICSI) procedure to achieve high fertilization and pregnancy rates regardless of semen characteristics has induced its application with spermatozoa surgically retrieved from azoospermic men. Here, ICSI outcome was analysed in 308 cases according to the cause of azoospermia; four additional cycles were with cases of necrozoospermia. All couples were genetically counselled and appropriately screened. Spermatozoa were retrieved by microsurgical epididymal aspiration or from testicular biopsies. Epididymal obstructions were considered congenital (n = 138) or acquired (n = 103), based on the aetiology. Testicular sperm cases were assessed according to the presence (n = 14) or absence (n = 53) of reproductive tract obstruction. The fertilization rate using fresh or cryopreserved epididymal spermatozoa was 72.4% of 911 eggs for acquired obstructions, and 73.1% of 1524 eggs for congenital cases; with clinical pregnancy rates of 48.5% (50/103) and 61.6% (85/138) respectively. Spermatozoa from testicular biopsies fertilized 57.0% of 533 eggs in non-obstructive cases compared to 80.5% of 118 eggs (P = 0.0001) in obstructive azoospermia. The clinical pregnancy rate was 49.1% (26/53) for non-obstructive cases and 57.1% (8/14) for testicular spermatozoa obtained in obstructive azoospermia, including three established with frozen-thawed testicular spermatozoa. In cases of obstructive azoospermia, fertilization and pregnancy rates with epididymal spermatozoa were higher than those achieved using spermatozoa obtained from the testes of men with non-obstructive azoospermia. [ABSTRACT FROM PUBLISHER]

Genetic abnormalities, including partial deletions of the Y-chromosome, are commonly detectable in men with non-obstructive azoospermia (NOA). NOA can be treated using testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI). Recent studies have shown that the presence of deletions involving the AZFc region do not appear to affect the chance of retrieving spermatozoa or have a significant impact on fertilization or pregnancy rates with ICSI. We investigated the effect of Y-chromosome partial deletions on the chance of sperm retrieval with TESE. Eighty attempts at sperm retrieval were performed using TESE on men who were previously evaluated for Y-chromosome partial deletions. Y-chromosome analysis was performed using a polymerase chain reaction (PCR)-based technique with 35 sequence-tagged-sites. Of the 80 men, nine (11%) had partial Y-chromosome deletions detected. Two azoospermic men with AZFc deletions had successful sperm retrieval, ICSI and a subsequent clinical pregnancy. Seven men had deletions involving the AZFb region (three men had isolated AZFb deletions, one had a AZFa, AZFb and AZFc deleted, and three had AZFb and AZFc deleted). None of the seven men had spermatozoa extracted by TESE, a result that is significantly different from the overall 64% (47/73) sperm retrieval rate achieved at our centre (P = 0.001). Two men with AZFb deletions had cells consistent with round spermatids identified and injected into oocytes without effecting any normal fertilizations. Although preliminary, these results suggest that the presence of an AZFb deletion is a significantly adverse prognostic finding for TESE. Men with AZFb deletions should be apprised of these results before attempting TESE-ICSI. Alternatives such as donor insemination or adoption should be considered or therapy delayed until improved results with round spermatid injections are likely. [ABSTRACT FROM PUBLISHER]

The impact of intracytoplasmic sperm injection (ICSI) on cryopreserved zygotes and embryos was evaluated by comparing embryo survival and implantation between embryos derived from ICSI and those derived from standard insemination procedures. The study included patients whose excess zygotes and embryos were cryopreserved between September 1993 and December 1994 and who subsequently underwent a frozen embryo transfer. Embryo survival, clinical pregnancy rates per transfer and pregnancy outcome were compared. Three hundred and thirty eight cryopreservation cycles, during which 1471 embryos were cryopreserved, were included in this study. Of those, 961 were derived from oocytes fertilized by insemination in vitro and 510 were derived from oocytes fertilized by ICSI. A total of 690 of the embryos (451 in the insemination group and 239 in the ICSI group) have since undergone a thaw cycle. The embryo survival rates were similar between the two groups (70.5 and 73.2%, insemination and ICSI respectively) and were not significantly affected by the stage at cryopreservation. There was no significant difference in pregnancy rates per transfer (31.8 and 32.3%), the preclinical pregnancy loss rate (16.7 and 23.8%), or the clinical miscarriage rate (16.7 and 23.8%) between the insemination and the ICSI groups respectively. It is concluded that ICSI does not have an adverse impact on the survival and successful implantation of cryopreserved and thawed embryos. [ABSTRACT FROM PUBLISHER]

With the introduction of intracytoplasmic sperm injection (ICSI), couples with severe male factor infertility have achieved fertilization and clinical pregnancy rates comparable to other in-vitro fertilization (IVF) patients. However, failure of fertilization still occurs in some patients despite the utilization of microsurgical sperm injection techniques. How such fertilization failure after ICSI might impact later ICSI treatment(s) is unknown. In this investigation, couples with complete fertilization failure after ICSI treated from August 1993 to August 1996 were identified (index cycle, n = 21). Additionally, fertilization data from any previous or subsequent infertility treatments were evaluated. Seven patients (33%) had at least one IVF treatment before the index cycle, although no deliveries occurred. Of patients with complete fertilization failure in the index cycle, 48% (n = 10) underwent at least one subsequent ICSI cycle which proceeded to oocyte retrieval. The remainder (n = 11) elected to discontinue treatment. Although six subsequent cycles were cancelled due to poor follicular response (< or = 2 mature oocytes), all patients electing to continue treatment eventually achieved a subsequent embryo transfer. The clinical pregnancy rate per transfer was 45.4% for this group; the delivery and ongoing pregnancy rate per transfer was 36.3%. Review of semen parameters, superovulation characteristics or other clinical parameters during the three study cycles (pre-index, index, and post-index) was not prognostic of fertilization success or reproductive outcomes in later treatments. Fertilization failure with ICSI therefore could not be predicted by prior cycle performance, although total immotility of spermatozoa at time of oocyte retrieval, total teratozoospermia, and low oocyte yield were common characteristics of couples experiencing complete fertilization failure with ICSI. These findings suggest that fertilization failure in one ICSI cycle does not preclude successful fertilization and delivery in a later ICSI treatment. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate any influence of maternal and/or paternal age on gamete characteristics and pregnancy outcomes in intracytoplasmic sperm injection (ICSI) cycles. In all, 821 consecutive ICSI cases were analysed retrospectively. While a significant linear decline in semen volume was detected, no significant differences in the concentration, motility or morphology of the spermatozoa were found with paternal ageing. A significant decline in the number of oocytes retrieved and the number of mature oocytes obtained was found with advancing maternal age. An increase in the occurrence of digyny was noted with parental ageing, while no difference in single or bipronuclear fertilization was found. Older women had a decreased incidence of single pronucleus formation and an increase in digyny, but no significant difference in the percentage of oocytes that underwent two-pronuclear fertilization was detected with regard to maternal ageing. Pregnancy outcomes were not influenced by the age of the male partner, while a strong negative correlation was found with maternal ageing. To better analyse male partner ageing as a factor affecting pregnancy outcome, we analysed a subgroup of patients with a female partner aged <35 years who underwent ICSI. No paternal influence on ICSI pregnancy outcome was found in this subgroup of patients. We conclude that the influence of pregnancy outcome after ICSI is related mostly to maternal and not paternal age. [ABSTRACT FROM PUBLISHER]

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.

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests J.A.M. has nothing to disclose. A.B. has nothing to disclose. C.K. has nothing to disclose. N.P. has nothing to disclose. P.X. has nothing to disclose. N.Z. has nothing to disclose. G.D.P. has nothing to disclose. Z.R. has nothing to disclose. P.N.S. has nothing to disclose., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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., (© 2022 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

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., Competing Interests: Declaration of interests P.X. has nothing to disclose. O.M.K. has nothing to disclose. S.C. has nothing to disclose. L.N. has nothing to disclose. D.F.A. has nothing to disclose. Z.R. has nothing to disclose. G.D.P. has nothing to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Cheung et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

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., (© 2022 The Authors.)

Purpose: Men who survive cancer as children or young adults may have severe spermatogenic impairment with azoospermia requiring surgical sperm retrieval and assisted reproductive technologies. We assessed treatment outcomes from a large series of cancer patients with prior radiation and/or chemotherapy., Materials and Methods: Men with nonobstructive azoospermia who underwent initial microsurgical testicular sperm extraction from 1995-2020 from a high-volume surgeon at a single institution were identified. Those with a history of malignancy treated by radiation therapy and/or chemotherapy were included. The primary outcome was successful sperm retrieval., Results: A total of 106 men were evaluated, of whom 57 received chemotherapy and radiation, 44 received only chemotherapy and 5 received only radiation. Sperm retrieval was successful in 39 of 106 (37%) men, with higher likelihood of retrieval in men who received only chemotherapy compared to men who received chemotherapy and radiation (61% vs 18%, p <0.001). None of the 18 patients who received chemotherapy with radiation to the pelvis had successful sperm retrieval, compared to 26% of patients who received chemotherapy with extra-pelvic radiation (p=0.02)., Conclusions: Chemotherapy and radiation for cancer may result in nonobstructive azoospermia that can be treated to allow fertility. However, pelvic radiation therapy is associated with the worst prognosis for successful treatment with microsurgical sperm retrieval and in vitro fertilization; we observed no cases of successful retrieval in men who received pelvic radiation therapy. These data are useful for pretreatment counseling, suggesting that men with prior radiation therapy may not be candidates for surgical sperm retrieval.

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., (© 2022. The Author(s).)

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., (© 2021. The Author(s).)

The aim of this article is to gather 9 thought leaders and their team members to present their ideas about the future of in vitro fertilization and the andrology laboratory. Although we have seen much progress and innovation in the laboratory over the years, there is still much to come, and this article looks at what these leaders think will be important in the future development of technology and processes in the laboratory., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The in vitro fertilization and andrology laboratories are at the center of assisted reproductive technologies and the place where technicians and embryologists manipulate gametes and preimplantation-stage embryos with the goal of achieving the best embryo for transfer. Through the years, these laboratories have seen developments in technique, technology, and testing. The goal of this Views and Interviews series is to bring together the thought leaders in the field and envision what the laboratories will look like in the next 10 years., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Despite the large number of babies born worldwide following intracytoplasmic sperm injection, concerns about the procedure's safety still exist owing to the use of suboptimal spermatozoa. Thus, follow-up of children conceived via intracytoplasmic sperm injection is highly recommended. We propose the use of parent-administered questionnaires to monitor the development of offspring conceived via intracytoplasmic sperm injection., Objective: This study aimed to determine whether male infertility treatment affects offspring development., Study Design: We compared obstetrical and neonatal outcomes and physical and psychological development of toddlers conceived via in vitro fertilization and intracytoplasmic sperm injection. Once newborns reached 3 years of age, participating patients were sent a set of parent-administered questionnaires, including the Ages and Stages Questionnaires; Prescreening Developmental Questionnaire 2; Peabody Developmental Motor Scales, Second Edition; Social Skills Rating System; Parenting Stress Index, Third Edition; and Child Behavior Checklist for Ages 2-3. Child development was measured by the Ages and Stages Questionnaires; Prescreening Developmental Questionnaire 2; and Peabody Developmental Motor Scales, Second Edition, questionnaires, whereas Social Skills Rating System; Parenting Stress Index, Third Edition; and Child Behavior Checklist for Ages 2-3 questionnaires were used to measure child behavior. The child's developmental or behavioral outcome was considered "abnormal" when he or she scored below average in ≥2 questionnaires from the respective category. We also conducted subanalyses to assess the effects of male genomic integrity, DNA fragmentation, chemical exposure, utilization of surgically retrieved spermatozoa, and extended embryo culture to determine the development of a child conceived via intracytoplasmic sperm injection., Results: A total of 12,306 couples met the inclusion criteria for this study; 1914 of 7433 patients (25.8%) who underwent intracytoplasmic sperm injection and 451 of 4873 patients (9.3%) who underwent in vitro fertilization returned the questionnaires. Our comparison of obstetrical outcomes between the 2 groups did not reveal any significant differences in the mode of delivery distribution, with most mothers having uncomplicated vaginal deliveries. Furthermore, gender distribution, gestational ages, and birthweights were also comparable between children conceived via intracytoplasmic sperm injection and in vitro fertilization. However, children conceived via in vitro fertilization displayed impaired developmental characteristics compared with the intracytoplasmic sperm injection-conceived cohort (adjusted odds ratio, 0.72; 95% confidence interval, 0.5-0.9; P=.0004). There was no difference in child behavior. Furthermore, 3 cases of autism were reported, 1 case from the in vitro fertilization group and 2 from the intracytoplasmic sperm injection group, all conceived from couples with an older male partner. Ages and Stages Questionnaires outcomes were also compared for the offspring conceived via in vitro fertilization and intracytoplasmic sperm injection by gender; however, no significant differences were observed. In addition, 5 separate subanalyses were then conducted exclusively for the intracytoplasmic sperm injection-conceived group. Levels of spermatogenic failure, DNA fragmentation, and chemical exposure did not significantly affect offspring development. Interestingly, although the length of embryo culture did not seem to influence child development, the abnormal behavior rate was significantly higher in children from the day 3 embryo transfer cohort (adjusted odds ratio, 0.4; 95% confidence interval, 0.05-0.34; P=.04). Children conceived via intracytoplasmic sperm injection from ejaculated spermatozoa displayed impaired developmental and behavioral characteristics compared with toddlers conceived from surgically retrieved specimens (adjusted odds ratio, 4.9; 95% confidence interval, 1.2-20.7; P=.05)., Conclusion: Most children conceived via intracytoplasmic sperm injection and in vitro fertilization are developing well without significant delays. Although the development of a child conceived via intracytoplasmic sperm injection was not affected by most of the variables assessed, those conceived from surgically retrieved spermatozoa were at a considerably lower risk of abnormal developmental and abnormal behavioral characteristics than offspring conceived from ejaculated specimens. However, given the small numbers of respondents available for many subgroups of interest, further studies of outcomes of children born from fathers with severe male factor infertility are warranted., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Objective: To identify and treat the gamete responsible for complete fertilization failure with intracytoplasmic sperm injection (ICSI) using a newly proposed assisted gamete treatment (AGT)., Design: Prospective cohort study., Setting: Center for reproductive medicine., Patient(s): One-hundred and fourteen couples with an adequate number of spermatozoa for ICSI and a fertilization rate of ≤10%, after controlling for maternal age., Intervention(s): Couples with an oocyte-related oocyte activation deficiency (OAD) underwent a subsequent cycle with a modified superovulation protocol; couples with sperm-related OAD had an additional genetic and epigenetic assessment to identify mutations and expression levels of the corresponding genes., Main Outcome Measure(s): Treatment cycle outcome for couples undergoing ICSI with either a modified superovulation protocol or AGT compared with their historical cycle., Result(s): A total of 114 couples matched the inclusion criteria, representing approximately 1.3% of the total ICSI cycles performed at our center, with age-matched controls. Fifty-two couples were confirmed negative for sperm-related OAD by the phospholipase Cζ (PLCζ) assay, indicating oocyte-related factors in their failed fertilization cycles. Couples were treated by one of two AGT protocols, AGT-initial or AGT-revised, in a subsequent attempt that was compared with their historical cycle. Subsequent ICSI cycles with a tailored superovulation protocol yielded significantly higher fertilization (59.0% vs. 2.1%) and clinical pregnancy (28.6% vs. 0) rates. In 24 couples (mean ± standard deviation: maternal age, 35.6 ± 5 years; paternal age, 39.8 ± 6 years) sperm-related OAD was confirmed; in four men, a deletion on the PLCZ1 gene was identified. Additional mutations were also identified of genes supporting spermiogenesis and embryo development (PIWIL1, BSX, NLRP5) and gene deletions confirming a complete absence of the subacrosomal perinuclear theca (PICK1, SPATA16, DPY19L). Subsequent AGT treatment provided higher fertilization (42.1%) and clinical pregnancy (36% vs. 0%) rates for couples with a history of impaired (9.1%) fertilization. A comparison of the two AGT protocols, AGT-initial or AGT-revised, revealed that the latter yielded even more favorable fertilization (37.6% vs. 45.9%) and clinical pregnancy (21.1% vs. 83.3%) rates., Conclusion(s): In couples with an oocyte-related OAD, tailoring the superovulation protocol resulted in successful fertilization, term pregnancies, and deliveries. In couples with a sperm-related OAD as determined by PLCζ assay, mouse oocyte activation test, and the assessment of gene mutations and function, AGT was successful. The AGT-revised protocol yielded an even higher fertilization rate than the AGT-initial protocol, resulting in the birth of healthy offspring in all couples who achieved a clinical pregnancy., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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.

Research Question: Ooplasmic maturity has been studied for some time, but remains poorly defined. This study aimed to evaluate metaphase II (MII) oocyte competence in terms of fertilization, embryo development and cycle outcomes, according to the oocyte maturity ratio., Design: Couples treated by intracytoplasmic sperm injection (ICSI) between 1993 and 2017 with female partners ≤35 years old were included. Cycles were divided into four groups according to proportion of MII oocytes at the time of retrieval: optimal (76-100%), adequate (51-75%), partial (26-50%) and minimal (1-25%)., Results: A total of 7672 ICSI cycles (optimal: 4838; adequate: 2252; partial: 518; minimal oocyte maturity: 64) were included, in which 95,667 MII oocytes were injected using ejaculated spermatozoa. The decreasing proportion of MII significantly reduced normal fertilization (two pronuclei) (78.9% to 71.3%; P < 0.0001) with a corresponding increase in digynic three-pronuclei that rose from 2.6% in the optimal group to 4.7% in the minimal group (P = 0.003). Implantation (33% to 17%; P < 0.0001), clinical pregnancy (63.6% to 37.5%; P < 0.0001) and live birth rates (49.2% to 26.6%; P < 0.0001) were affected by the decreasing proportion of MII oocytes., Conclusions: A high proportion of immature sibling oocytes in the retrieved cohort affects the fertilization rate and embryo developmental competence of MII inseminated oocytes, clinical pregnancy and live birth rates, suggesting that, in addition to nuclear maturity, ooplasmic and membrane maturity are required for developmental competence of MII oocytes. These findings may provide guidance toward ovarian stimulation protocols aimed at achieving a greater proportion of MII oocytes, leading to higher fertilization rates and better pregnancy outcomes., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Evaluation of reproductive quality of spermatozoa by standard semen analysis is often inadequate to predict ART outcome. Men may be prone to meiotic error and have higher proportion of spermatozoa with fragmented chromatin, capable of affecting the conceptus' health. In men with unexplained infertility, supplementary tests may be pivotal to gain insight into the paternal contribution to the zygotic genome. A total of 113 consenting men were included in the study, with an additional 5 donor specimens used as control. Among study participants, 87 were screened for sperm aneuploidy by fluorescent in situ hybridization (FISH) and ranked according to their increasing age. A total of 18 men were assessed by whole exome sequencing and categorized according to their reproductive outcome as either fertile or infertile. Another set of men (n = 13) had their gene expression analyzed by RNA-seq and were profiled according to their reproductive capacity. FISH revealed that the average aneuploidy rate was highest for men over-55 age group (9.6%), while men >55 had the highest average disomy for chromosomes 17(1.2%) and 18(1.3%). ART results for the entire cohort comprised 157 cycles, stratified by paternal age. The youngest age group (25-30 years) had a fertilization rate of 87.7% which decreased to 46.0% in the >55 age group. Clinical pregnancy rate was highest in the 25-30yr group (80.0%) while no pregnancies were attained in the >55 age groups. Pregnancy loss was characterized by a steadily increasing trend, highest in the 51-55 age group (50.0%). NGS was performed on a cohort of patients classified as having recurrent pregnancy loss. This cohort was classified as the infertile group (n = 10) and was compared to a control group (n = 8) consisting of patients successfully treated by ART. Eight couples in 17 ICSI cycles achieved a clinical pregnancy rate of 82.4% while 10 infertile couples treated in 21 cycles achieved a pregnancy rate of 23.8%, all resulting in pregnancy loss. DNA-sequencing on spermatozoa from these patients yielded overall aneuploidy of 4.0% for fertile and 8.6% for the infertile group (P<0.00001). In the infertile cohort, we identified 17 genes with the highest mutation rate, engaged in key roles of gametogenesis, fertilization and embryo development. RNA-seq was performed on patients (n = 13) with normal semen analyses. Five men unable to attain a pregnancy after ART were categorized as the infertile group, while 8 men who successfully sustained a pregnancy were established as the fertile control. Analysis resulted in 86 differentially expressed genes (P<0.001). Of them, 24 genes were overexpressed and 62 were under-expressed in the infertile cohort. DNA repair genes (APLF, CYB5R4, ERCC4 and TNRFSF21) and apoptosis-modulating genes (MORC1, PIWIL1 and ZFAND6) were remarkably under-expressed (P<0.001). Sperm aneuploidy assessment supported by information on gene mutations may indicate subtle dysfunctions of the spermatozoon. Furthermore, by querying noncoding RNA we may gather knowledge on embryo developmental competence of spermatozoa, providing crucial information on the etiology of unexplained infertility of the infertile male., Competing Interests: The authors have declared that no competing interests exist.

Investigating sperm locomotion in the presence of external fluid flow and geometries simulating the female reproductive tract can lead to a better understanding of sperm motion during fertilization. Using a microfluidic device featuring a stricture that simulates the fluid mechanical properties of narrow junctions inside the female reproductive tract, we documented the gate-like role played by the stricture in preventing sperm with motilities below a certain threshold from advancing through the stricture to the other side (i.e., fertilization site). All the slower sperm accumulate below (i.e., in front of) the stricture and swim in a butterfly-shaped path between the channel walls, thus maintaining the potential for penetrating the stricture and ultimately advancing toward the fertilization site. Accumulation below the stricture occurs in a hierarchical manner so that dense concentrations of sperm with higher velocities remain closer to the stricture, with more sparsely distributed arrays of lower-velocity sperm lagging behind.

Previous studies, including our own, have reported that spermatozoa isolated from the testis have remarkably higher occurrence of aneuploidy once isolated from azoospermic men. This notion, however, did not translate into a lower pregnancy rate nor a greater proportion of miscarriages. Indeed, ICSI offspring generated from surgically retrieved gametes did not suffer from increased karyotypic aneuploidy than children generated from ejaculated specimens. In recent years, aneuploidy assessments on a larger number of cells and utilizing more chromosome probes have reported a progressive decrease in chromosomal aberrations in spermatozoa directly retrieved from the seminiferous tubules. In light of the availability of more accurate molecular genetic techniques, we have decided to challenge the notion that sampling epididymal and testicular tissues yields spermatozoa with higher incidence of aneuploidy than those retrieved in the ejaculate. In a retrospective manner, we have carried out an analysis by FISH with 9 chromosome probes on at least 1000 cells from the ejaculates of 87 consenting men and the specimens of 6 azoospermic men, while spermatozoa of fertile donors were used as control. Aneuploidy by FISH yielded 0.9% for the donor control but rose in the study group to 3.6% in the ejaculated, 1.2% for the epididymal, and 1.1% for testicular spermatozoa. There were no differences in autosomal or gonosomal disomies, nor nullisomies. In this group, once the specimens of these men were used for ICSI, ejaculated spermatozoa yielded a 22% clinical pregnancy rate that resulted in 62.5% pregnancy loss. The surgically retrieved specimens yielded a 50% clinical pregnancy rate that progressed to term. To confirm our findings, in a prospective analysis, DNA sequencing was carried out on the ejaculates and surgical samples of 22 men with various spermatogenic characteristics. In this comparison, the findings were similar with actually a higher incidence of aneuploidy in the ejaculated spermatozoa (n = 16) compared to those surgically retrieved (n = 6) (P<0.0001). For this group, the clinical pregnancy rate for the ejaculated specimens was 47.2% with 29.4% pregnancy loss, while the surgically retrieved yielded a 50% clinical pregnancy rate, all progressing to term. A subsequent prospective combined assessment on ejaculated and surgically retrieved spermatozoa by FISH and NGS was performed on non-azoospermic men with high DNA fragmentation in their ejaculate. The assessment by FISH evidenced 2.8% chromosomal defects in the ejaculated and 1.2% in testicular biopsies while by NGS became 8.4% and 1.3% (P = 0.02), respectively. Interestingly, we evidenced a pregnancy rate of 0% with ejaculated while 100% with the testicular spermatozoa in this latter group. This indicates that improved techniques for assessing sperm aneuploidy on a wider number of cells disproves earlier reports and corroborates the safe utilization of testicular spermatozoa with a positive impact on chances of pregnancy., Competing Interests: The authors have declared that no competing interests exist.

Objectives: This work assessed sexual and neurobehavioral parameters after ovarian treatment with autologous PRP., Design: Questionnaire study., Material and Methods: Patients receiving ovarian PRP injection (n=80) due to low ovarian reserve and/or at least 1 prior failed IVF cycle were sampled. Pre- and post-treatment levels in self-reported daily energy, sleep quality, skin tone/hair thickness/nail growth, cognitive clarity, menstrual pattern, cervical mucus/vaginal lubrication, libido, sexual activity, ability to achieve orgasm, and overall sexual experience were measured., Results: Mean±SD age and baseline BMI among patients were 45.5±6yrs and 25±5.1kg/m2, respectively. Average weight loss after ovarian PRP was 1kg (p=0.056). After ovarian PRP, superior nail growth, skin tone, and hair thickness was observed by 46.3% of patients [95%CI=35%,57.8%]; the same ratio experienced increased "clarity of thinking" following the procedure. Irregular or absent menses affected 56.3% of patients at enrollment, and menses returned or cyclicity improved in 24.4% after treatment [95%CI=12.9%,39.5%]. Increased post-treatment vaginal lubrication/cervical mucus production was reported by 51.3% of women [95%CI=39.8%, 62.6%] accompanied by increased libido in 55% [95%CI=43.5%,66.2%]. More frequent sexual activity after ovarian PRP was noted from 46.3% of subjects [95%CI=35%, 57.8%] coinciding with a 45% improvement in overall sexual experience before vs. after ovarian PRP [95%CI=33.9%, 56.5%]., Conclusion: This investigation is the first to document responses across neurobehavioral and metabolic parameters after ovarian PRP. Injection of PRP-derived growth factors directly into ovarian tissue seems to enable a local signaling milieu favoring development of hormonally active ovarian elements, thus "re-potentiating" low or absent reserve.

Objective: To design a reproductive treatment algorithm based on the sperm DNA fragmentation (SDF) for couples with unexplained infertility following a poor intrauterine insemination (IUI) outcome., Design: Couples that failed IUI with no apparent reproductive issue in both partners were allocated to diverse reproductive treatments on the basis of SDF., Setting: Reproductive medical center in an academic setting., Patient(s): Over 4 years, couples with an unexpected poor IUI outcome and no apparent female or male partner reproductive issues were recruited., Intervention(s): IUI, IVF, and ICSI were performed in the standard fashion following sperm SDF assays., Main Outcomes Measure(s): Fertilization rate, implantation rate, pregnancy characteristics, and delivery rates., Result(s): A total of 354 couples with unexplained infertility and normal semen parameters underwent 1133 IUI cycles. Clinical pregnancy rate (CPR) with IUI at our center in an age-matched cohort is 23.9% while the study cohort had 1.8%. Following SDF assessment, couples with failed IUI attempts but normal SDF (SCSA 9.8 ± 4.6%; TUNEL 11.8 ± 6.2%) underwent IVF with a CPR of 12.7%; those with abnormal SDF underwent ICSI with ejaculated spermatozoa, resulting in a CPR of 18.7%. This group included couples with normal SDF that had failed IVF. Couples with abnormal SDF that failed ICSI with ejaculated spermatozoa achieved a CPR of 31.0% with surgically retrieved spermatozoa., Conclusion(s): Couples with unexplained infertility that present with unexpectedly poor IUI outcomes can be funneled into a treatment algorithm guided by the integrity of the sperm genome for higher chances of pregnancy using an alternate method of insemination.

Platelets modulate clinically relevant yet incompletely understood tissue regeneration processes, and platelet rich plasma (PRP) has been previously used with some success in various non-reproductive medical contexts. Here, we extended PRP application to ovarian tissue with a view to document impact on ovarian reserve among women attending for infertility treatment. PRP was freshly isolated from patients (n= 4) with diminished ovarian reserve as determined by at least one prior IVF cycle canceled for poor follicular recruitment response or estimated by serum AMH and/or FSH, no menses for ≥1 year. Immediately following substrate isolation and activation with calcium gluconate, approximately 5 mL of autologous PRP was injected into each ovary under direct transvaginal sonogram guidance. For each study subject, AMH, FSH, and serum estradiol data were recorded at two-week intervals post-PRP and compared to baseline (pre-PRP) values. In this pilot group, mean (±SD) patient age was 42 ± 4 years with infertility duration reported as 60 ± 25 months. Following this protocol of intraovarian PRP administration, increases in serum AMH (p = .17), decreases in FSH (p < .01), or both, were observed in all cases, sufficient to permit retrieval of 5.3 ± 1.3 MII oocytes. IVF occurred 78 ± 22 (range =  59-110) days after activated PRP injection, and results appeared independent of patient age, infertility duration, baseline platelet concentration or pretreatment antral follicle count. Each patient had at least one blastocyst suitable for cryopreservation. While autologous PRP has been successfully applied therapeutically to various tissues to accelerate healing and wound repair, this is the first description of direct injection of activated PRP into the human ovary of poor prognosis IVF patients. Evidence of improved ovarian function was noted in all who received intraovarian PRP, possibly as early as two months after treatment. Additional research is needed to clarify (and enhance) which PRP components are responsible for altered ovarian function, and to identify predictive characteristics for patients most likely to benefit from this intervention.

Semen analysis (SA) poorly predicts male fertility, because it does not assess sperm fertilizing ability. The percentage of capacitated sperm determined by G M1 localization ("Cap-Score™"), differs between cohorts of fertile and potentially infertile men, and retrospectively, between men conceiving or failing to conceive by intrauterine insemination (IUI). Here, we prospectively tested whether Cap-Score can predict male fertility with the outcome being clinical pregnancy within ≤3 IUI cycles. Cap-Score and SA were performed (n = 208) with outcomes initially available for 91 men. Men were predicted to have either low (n = 47) or high (n = 44) chance of generating pregnancy using previously-defined Cap-Score reference ranges. Absolute and cumulative pregnancy rates were reduced in men predicted to have low pregnancy rates versus high ([absolute: 10.6% vs. 29.5%; p = 0.04]; [cumulative: 4.3% vs. 18.2%, 9.9% vs. 29.1%, and 14.0% vs. 32.8% for cycles 1-3; n = 91, 64, and 41; p = 0.02]). Only Cap-Score, not male/female age or SA results, differed significantly between outcome groups. Logistic regression evaluated Cap-Score and SA results relative to the probability of generating pregnancy (PGP) for men who were successful in, or completed, three IUI cycles (n = 57). Cap-Score was significantly related to PGP (p = 0.01). The model fit was then tested with 67 additional patients (n = 124; five clinics); the equation changed minimally, but fit improved (p < 0.001; margin of error: 4%). The Akaike Information Criterion found the best model used Cap-Score as the only predictor. These data show that Cap-Score provides a practical, predictive assessment of male fertility, with applications in assisted reproduction and treatment of male infertility., (© 2018 The Authors. Molecular Reproduction and Development Published by Wiley Periodicals, Inc.)