Manipulation of Development by Nuclear Transfer

By Gianpiero D. Palermo, MD, PhD

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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.

The purpose of this study was 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.

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.

Single-center thorough evaluation and targeted treatment of globozoospermic men

S. Cheung, A. Parrella, D. Tavares, D. Keating, P. Xie, Z. Rosenwaks, G. Palermo.

In the current study, we aim to characterize, by specific biomarkers and nucleic acid sequencing, the structural and genomic sperm characteristics of PG and CG men in order to identify the best reproductive treatment. Specimens from these individuals were screened by multiple gamete functional assays to measure the presence of OASCF and ultrastructural features, and genomic assessments of histone content, chromatin integrity, and aneuploidy were also performed. Clinical outcomes were compared between the two globozoospermia forms and their respective treatment protocols.

Sperm Genomic Integrity by TUNEL Varies throughout the Male Genital Tract

P. Xie, D. Keating, A. Parrella, S. Cheung, Z. Rosenwaks, M. Goldstein, G. Palermo.

For this study, we assessed sperm chromatin fragmentation in men with intracytoplasmic sperm injection failure and high ejaculated sperm chromatin fragmentation who underwent surgery to retrieve spermatozoa from different levels of the male genital tract. In assessing intracytoplasmic sperm injection results of cycles using spermatozoa from different levels of the male, we found that men with high ejaculated sperm chromatin fragmentation can benefit from using surgically retrieved sperm for assisted reproductive procedures.

Genetic and epigenetic profiling of the infertile male

S. Cheung, A. Parrella, Z. Rosenwaks, G. Palermo.

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. 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.

Strictures of a microchannel impose fierce competition to select for highly motile sperm

M. Zaferani, G. Palermo, A. Abbaspourrad.

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.

Revisiting aneuploidy profile of surgically retrieved spermatozoa by whole exome sequencing molecular karyotype.

S. Cheung, P. Schlegel, Z. Rosenwaks, G. Palermo

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. Our result 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.

Histone variant H3.3-mediated chromatin remodeling is essential for paternal genome activation in mouse preimplantation embryos

Q. Kong, L. Banaszynski, F. Geng, X. Zhang, J. Zhang, H. Zhang, C. O’Neill. P. Yan, Z. Liu, K. Shido, G. Palermo, C. Allis, S. Rafii, Z. Rosenwaks, D. Wen.
Histone variant H3.3 in both male and female gametes is thought to be important for remodeling the paternal and maternal genomes for activation during both fertilization and embryogenesis. However, the underlying mechanisms remain poorly understood. Using our H3.3B-HA-tagged mouse model, engineered to report H3.3 expression in live animals and to distinguish different sources of H3.3 protein in embryos, we show here that sperm-derived H3.3 (sH3.3) is removed from the sperm genome shortly after fertilization and extruded from the zygotes via the second polar bodies (PBII). We also found that the maternal H3.3 (mH3.3) is incorporated into the paternal genome as early as 2 h postfertilization and is detectable in the paternal genome until morula stage. Knockdown of mH3.3 resulted in compromised embryonic development both of fertilized embryos and of androgenetic haploid embryos. Furthermore, we report that mH3.3 depletion in oocytes impairs both activation of the Oct4 pluripotency marker gene and global de novo transcription from the paternal genome important for early embryonic development. Our results suggest that H3.3-mediated paternal chromatin remodeling is essential for the development of preimplantation embryos and the activation of the paternal genome during embryogenesis.

Cap-Score™ prospectively predicts probability of pregnancy.

J. Schinfeld, F. Sharara, R. Morris, G. Palermo, Z. Rosenwaks, E. Seaman, S. Hirshberg, J. Cook, C. Cardona, G. Ostermeier, A. Travis
In this study, we prospectively tested whether the percentage of capacitated sperm determined by GM1 localization ("Cap-Score™") can predict male fertility with the outcome being clinical pregnancy within ≤3 IUI cycles. Cap-Score and semen analysis (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%). These data show that Cap-Score provides a practical, predictive assessment of male fertility, with applications in assisted reproduction and treatment of male infertility.

A Treatment algorithm for couples with unexplained infertility based on sperm chromatin assessment

C. O’Neill. A. Parrella, D. Keating, S. Cheung, Z. Rosenwaks, G. Palermo
In this study, A total of 354 couples with unexplained infertility and normal semen parameters underwent 1133 intrauterine insemination (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 sperm DNA fragmentation (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 intracytoplasmic sperm injection (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. 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.

Development of ICSI

C. O’Neill. S. Chow, Z. Rosenwaks, G. Palermo

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

A rationale for biopsying embryos reaching the morula stage on Day 6 in women undergoing preimplantation genetic testing for aneuploidy

M. Irani, N. Zaninovic, C. Canon, C. O’Neill, V. Gunnala, Q. Zhan, G. Palermo, D. Reichman, and Z. Rosenwaks

The study included 763 cycles in which 1260 morulae and 3014 blastocysts were biopsied. Women were divided into four age groups (<35, 35–37, 38–39 and ≥40 years): the prevalence of aneuploidy was consistently lower among blastocysts (40.3, 50.8, 56 and 78.3%, respectively) than among compacted morulae (68.7, 75.5, 88.9 and 98.1%, respectively) and cavitating morulae (57, 66.4, 81 and 91.6%, respectively) throughout the different age groups (P < 0.001). Of note, the majority of compacted morulae (98.1%) and cavitating morulae (91.6%) were aneuploid in women aged ≥40 years. Compacted and cavitating morulae had significantly higher rates of complex aneuploidy, which involves ≥3 chromosomes, compared with blastocysts (P < 0.001). Furthermore, euploid morulae were associated with a significantly lower IR (28.2 versus 54.6%; P = 0.002) and live birth rate (23.1 versus 55.0%; P = 0.001) compared to euploid blastocysts.
 

Optimizing the first-line fertility treatment

Mohamad Irani, Stephen Chow, Derek Keating, Simone Elder, Zev Rosenwaks & Gianpiero Palermo

The objective of this study was to identify sperm score thresholds to achieve satisfactory intrauterine insemination (IUI) success rates according to the response to stimulation with clomiphene citrate (CC). To minimize the confounding effect of female age, we included only CC/IUI cycles of women ≤35 years old. A total of 1,194 CC/IUI cycles were included. Semen volume, concentration, and motility influenced the clinical pregnancy rate (CPR). Normal morphology (≥4%) was associated with a comparable CPR with 3%, 2%, and 1% normal forms (15.6%, 16.1%, 18.1%, and 13.1%, respectively). A combination of the total number of motile spermatozoa in the ejaculate before semen preparation (TM) at a threshold ≥20 × 106 was associated with a CPR of 17.8% compared to 4.6% for a threshold <20 × 106 (p < .001). Interestingly, the TM threshold to achieve satisfactory outcomes was lower (10 × 106) in patients who had an optimal response to CC (≥2 dominant follicles with an endometrial thickness ≥7 mm) compared to 40 × 106 for those who had a suboptimal response (one dominant follicle with an endometrial thickness <7 mm). In conclusion, the response to superovulation with CC determines each patient’s TM threshold required for satisfactory outcomes. Couples whose TM is below the threshold may benefit from a superovulation with gonadotropins or in vitro fertilization.

In this investigation we assess the incidence of round cells (RCs) in semen samples in our infertile patient population and their significance on intracytoplasmic sperm injection (ICSI) cycle outcomes. We also evaluate the usefulness of RCs as indicators of bacterial infection and highlight the origin of this cell-type, as well as its role in the human ejaculate. In a prospective fashion, a total of 4,810 ejaculated samples were included in the study during a period of 24 months. RCs were characterized for white blood cell (WBC) components versus exfoliated germ cells by testing for multiple markers of ploidy as well as protamine assays. Cases displaying ≥ 2 x 106/ml RCs were screened for bacteria. Raw specimens containing RC were processed by peroxidase and other leukocyte assays, specific stains for protamines were used to identify spermiogenic stage, aneuploidy (FISH) assessment was carried out, and the presence of various Sertoli-cell cytoplasmic remnants was analyzed to identify and characterize immature germ cells. The effect of RC on clinical outcome was assessed in specimens used for ICSI. The average age of the men involved was 39.2 ± 7 years. Semen samples had a mean concentration of 40.7 ± 31 x 106/ml, motility of 42.6 ± 35%, and morphology of 2.3 ± 2%. RCs were identified in 261 specimens, representing a proportion of 5.4%. Men with RCs had comparable age but lower sperm concentration and morphology than the control group (P<0.001). The aneuploidy rate of 4.3% in RCs group was remarkably higher than the control group (2.3%; P<0.001). Sperm aneuploidy rate positively correlated with the number of RCs (P<0.001). Of 44 men, 17 of them in 18 cycles had up to 1.9 x 106/ml RCs without affecting fertilization and clinical pregnancy rates when compared to controls (n = 365 cycles). In 27 men undergoing 33 ICSI cycles with ≥ 2 x 106/ml RCs, the fertilization rate trended lower and the miscarriage rate was significantly increased (P = 0.05). There was lack of correlation between RC and bacteriological growth. Specific markers indicated that seminal RCs are mostly immature germ cells encased in the remnants of Sertoli cell cytoplasm. Moreover, their modest protamine content and their haploid status confirm that they are post-meiotic. Sequential observation in the same man showed that RC episodes were followed by an amelioration of semen parameters, and interestingly, the episodic occurrence of RCs often coincides with flu season peaks. Seminal RCs are not a marker of infectiousness but rather a transient indicator of spermatogenic insult that possibly occurs in most men following a mild and transient ailment such as the flu.

The World Health Organization declaimed that infertility is a major global public health issue of the last few decades. Infertility is commonly defined as the failure to conceive after 1 year of unprotected intercourse and is estimated to concern 72.4 million people worldwide with 40.5 million currently seeking medical care. The overall burden of subfertility/infertility is significant, is likely underestimated, and has not displayed any decrease over the last 20 years. Male factors are estimated to be involved, at least partially, in half of the cases. While the diagnosis, medical treatment, and psychosocial management of infertility have rapidly evolved over the past 4 decades, some difficulties still persist. Little is known about the physiopathology of altered sperm production, its genetic causes, or the genetic and epigenetic consequences for the gamete and the forthcoming conceptus. The information generated by conventional semen analysis has historically classified patients into categories lacking knowledge of causality and leaving conventional therapy as somewhat empirical. One of the reasons for this lack of fundamental understanding is the heterogeneity of causal factors as male infertility is a typical multifactorial disorder with a strong genetic basis and additional factors such as urogenital infections, immunological or endocrine diseases, attack from reactive oxygen species (ROS), or perturbations from endocrine disruptors. Since assisted reproduction technology (ART) is widely used to achieve conception with gametes produced by compromised spermatogenesis, there is a clear need to detail the molecular pathogenesis of male infertility to improve long-term risk assessment on a case-by-case basis. In this context, research on the male partner will shed a much-needed light on the physiopathology of male reproduction, will enhance patient management, and constitutes a prerequisite for the development of new therapeutic solutions.

This study investigates whether the timing of in-vivo and in-vitro maturation influences ooplasmic dysmaturity. This is a retrospective comparison of intracytoplasmic sperm injection (ICSI) cycles (index cycles) complicated by complete fertilization failure (CFF) to cycles with successful fertilization in the same patient. The cycle following the index cycle was modified intentionally to increase fertilization. The times between human chorionic gonadotrophin (HCG) trigger and oocyte retrieval, HCG trigger and removal of cumulus cells, and HCG trigger and sperm injection were recorded. Fifteen patients were included. Compared with successful fertilization cycles, index (CFF) cycles showed a shorter time interval between HCG trigger and oocyte retrieval (2029.0 ± 16 versus 2195.0 ± 10 min; P < 0.001), HCG trigger and removal of cumulus cells (2201.4 ± 15 versus 2309.0 ± 23 min; P < 0.001) and oocyte retrieval and removal of cumulus cells (114.0 ± 13 versus 171.8 ± 15 min; P < 0.001). The interval between HCG trigger and ICSI was comparable between groups. Findings reveal novel patterns in time intervals between HCG trigger, oocyte retrieval, removal of cumulus cells and ICSI. Thus, modulating time intervals between HCG trigger, oocyte retrieval, removal of cumulus cells and ICSI to grant fertilization seems feasible.

Objective. To investigate the outcomes of intracytoplasmic sperm injection (ICSI) cycles where sibling oocytes from a single donor were split between two recipients based on strict sperm morphology. Methods. Retrospective cohort study. All ICSI cycles had one donor's oocytes split between two recipients in a 1 : 1 ratio based on strict sperm morphology, that is, one male partner had morphology of 0% and the other had morphology of >1%. Fertilization, positive hCG, clinical pregnancy, spontaneous miscarriage, and live birth rates of the aforementioned groups were compared. Results. The baseline characteristics of the two groups (n = 103), including semen parameters of the male partners, were comparable. There was no difference in the fertilization rates when comparing the 0% group to the >1% group (78.7% versus 81.6%; P = 0.66). The overall positive hCG, clinical pregnancy, spontaneous miscarriage, and live birth rates for the 0% group were 61.2%, 49.5%, 10.7%, and 38.8%, respectively. The corresponding rates in the >1% group were positive hCG (63.1%), clinical pregnancy (55.3%), spontaneous miscarriage (7.77%), and live birth (46.6%). Conclusions. The fertilization and pregnancy outcomes of ICSI cycles for strict sperm morphology of 0% versus morphology of >1% are equivalent. These results can provide reassurance to couples undergoing ICSI for severe teratospermia.

To create a rapid, inexpensive, efficient, and reproducible real-time three-dimensional (3-D) analysis of viable spermatozoa. Previous studies have demonstrated that abnormal semen profiles are associated with a modest increase in the frequency of sperm chromosomal abnormalities, and that sperm with aberrations in the shape and contours of the head may be carriers of chromatinic defects. Although high-power magnification and enhanced video-generated magnification have been suggested, these techniques are inherently limited by the clarity of the image, the time required for the analysis, and the risk of variable head-positioning during imaging.

Intracytoplasmic sperm injection (ICSI) is the most effective assisted reproductive procedure enabling fertilization in severe forms of male factor indications and male gamete dysfunction. Reliability of ICSI has allowed the expansion of its application to other forms of infertility rendering it the most popular assisted reproduction technology (ART) insemination method worldwide. The concern related to the invasiveness of ICSI together with the arbitrary selection of the inseminating spermatozoon has induced the execution of studies to compare the performance of ICSI in non-male factor infertility with standard in vitro insemination approach. Not surprisingly, the outcome has evidenced that ICSI does not yield higher pregnancy rates than in vitro fertilization but functions invariably as a normalizer of fertilization mollifying the absent or low fertilization. The follow-up studies on ICSI children have evidenced that the procedure is safe and the slightly higher incidences of neonatal malformations or de novo gonosomal abnormalities are related to the genetics of the infertile couples. Furthermore, ICSI is accepted for some specific indications such as low number and poor morphology oocytes, thicker zona, excess polyspermia, PGD/PGS/PGT (preimplantation genetic diagnosis/preimplantation genetic screening/preimplantation genetic testing), discordant HCV/HIV (hepatitis C virus/human immunodeficiency virus) couples, in vitro maturation (IVM), and oocyte cryopreservation. Only the advent of new biomarkers in combination with routine semen analysis capable of identifying the fertilization competence of the spermatozoon can guide the reproductive physician toward the proper insemination method.