Q.V. Neri

ICSI: where we have been and where we are going.

Notwithstanding the broad success of in vitro fertilization (IVF), a failure to achieve fertilization still plagues a substantial group of patients, with sperm abnormalities the main culprit. In the 1980s, several micromanipulation procedures were adopted from animal husbandry to facilitate gamete interaction, and this resulted in the development of intracytoplasmic sperm injection (ICSI), a procedure through which an oocyte can be fertilized independently of the morphology and/or motility of the single spermatozoon injected. The procedure was first used in cases of fertilization failure after standard IVF or when an inadequate number of sperm cells were available. The consistency of fertilization independent of the functional quality of the spermatozoon has extended the application of ICSI to immature spermatozoa retrieved surgically from the epididymis and testis. Moreover, the need to denude the oocyte has allowed assessment of the nuclear maturity of the oocyte. ICSI is also preferred in conjunction with preimplantation genetic diagnosis and recently has been used to treat HIV discordant couples, where there is a pressing need to minimize the exposure of the oocyte to a large number of spermatozoa. For all ages and with all the different sperm types used, fertilization after ICSI is at approximately 70 to 80% and it ensures a clinical pregnancy rate of up to 45%. These results have made ICSI a procedure comparable in popularity with IVF and have minimized the need for couples suffering from all forms of male infertility to resort to adoption or the use of donor sperm.

Effect of Treating Induced Mitochondrial Damage on Embryonic Development and Epigenesis

Abstract Germinal vesicle transplantation (GVT) has been proposed as a possible treatment to correct age-related oocyte aneuploidy caused by dysfunctional ooplasm. How healthy ooplasm regulates normal meiosis and subsequent development has yet to be elucidated, but impaired mitochondrial metabolism may be attributable to incomplete segregation of the oocyte chromosomes. In the present study, after ooplasmic mitochondrial damage by photoirradiating chloromethyl-X-rosamine, examination of the oocyte nucleis ability to survive after transfer into healthy ooplasts was performed. To assess their fertilizability and potential for development, GVT oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and transferred to foster mice. Condition of the offspring at birth was assessed, and epigenetic analysis was performed. Photosensitization consistently inhibited oocyte maturation. However, after GVT of photosensitized nuclei into healthy ooplasts, 67.2% were reconstituted, and 76.2% of these matured normally, with an overall rate of 51.2%, much higher than that (6.0%) in the mitochondrially injured oocytes. After ICSI, 65.8% (52/79) of GVT oocytes were fertilized normally, and 21.1% (11/52) eventually reached the blastocyst stage. The transfer of 132 two-cell GVT embryos into the oviducts of pseudopregnant females resulted in 17 apparently healthy live offspring. For some key developmental genes, a high level of expression was identified in the GVT and “rescue”-derived fetal adnexa. Thus, one can induce in oocyte mitochondria a photosensitization-based type of damage, which consistently inhibits GV breakdown, meiotic spindle formation, chromosomal segregation, and polar body extrusion. Germinal vesicle transplanted and rescued oocytes were able to undergo maturation, fertilization, and embryonic cleavage and, ultimately, to develop to term. This approach may provide a model with which to study the age-related ooplasmic dysfunction seen in human oocytes.

Genetic and epigenetic characteristics of ICSI children

The outcome of pregnancy and the developmental wellbeing of children conceived from 12,866 consecutive intracytoplasmic sperm injection (ICSI) cycles was assessed. A total of 3277 couples delivered 5891 neonates. There was a higher than normal incidence of de-novo chromosomal abnormalities in a small sample of ICSI offspring. Controlling for maternal age showed that the incidence of low birth weight and gestational length were comparable with the naturally conceived counterpart. Rates of malformation in ICSI offspring ranged from 3.5 to 6.2%. At 3 years of age (n = 811), the proportion of children at risk for developmental delays was 10.4% in ICSI and 10.7% in IVF singletons. However, high order gestations were characterized by 19.4% of the children having compromised development. Epigenetic analysis of assisted reproductive technique conceptuses found minor imprinted gene expression imbalances. ICSI offspring presented with genetic defects that were inherited or arose de novo. Obstetric and neonatal outcomes of singleton pregnancies appeared to be dependent upon maternal age. ICSI and IVF appeared to exert a negative effect on the wellbeing of offspring mainly because of the association with multiple gestations. All assisted reproduction procedures should be monitored for the eventual effect of environmental aggressors on offspring epigenesis.

The Outcome of Intracytoplasmic Sperm Injection Using Occasional Spermatozoa in the Ejaculate of Men With Spermatogenic Failure

PURPOSE Men with spermatogenic failure so profound that they are considered as having nonobstructive azoospermia occasionally have spermatozoa in the ejaculate. We compared intracytoplasmic sperm injection outcomes following the injection of ejaculated or surgically retrieved spermatozoa from these men. MATERIALS AND METHODS A study was performed of intracytoplasmic sperm injection cycles with no spermatozoa on initial semen analysis and 100 or fewer following centrifugation (cryptozoospermia). Only 16 couples that underwent intracytoplasmic sperm injection cycles with ejaculated spermatozoa and cycles with testicular spermatozoa were included. RESULTS Initial analysis was done to compare outcomes between the 2 semen origins. There was no difference in the rate of normal or abnormal fertilization between the 2 groups. The rate of clinical pregnancies seemed to favor testicular spermatozoa (47.4% vs 20.8%), although results were not significant. When a comparison was performed between the first testicular cycle and the ejaculated cycle closest in time to the cycle with testicular spermatozoa, a higher rate of normal fertilization with testicular spermatozoa was observed (60.9% vs 48.5%, p <0.05). Also, in this comparison a clear trend toward a higher percent of clinical pregnancies and deliveries in the testicular group was observed (50.0% vs 14.3%). CONCLUSIONS Transit through the male genital tract did not enhance the ability of ejaculated spermatozoa to achieve fertilization with intracytoplasmic sperm injection compared to that of testicular spermatozoa in men with severely impaired production. In ejaculated samples a lower number of spermatozoa available resulted in an impaired chance of achieving pregnancy. Using testicular spermatozoa may be a reasonable alternative for couples in whom multiple attempts at intracytoplasmic sperm injection have failed using ejaculated sperm from men with cryptozoospermia.

Understanding fertilization through intracytoplasmic sperm injection (ICSI)

Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described.

Y chromosome assessment and its implications for the development of ICSI children

The aetiology of compromised spermatogenesis is often genetic in nature. There are only a few reports of father/son cohorts that have been evaluated to assess heritability of mutations associated with male factor infertility and the psychological well-being of the children. In the present study, multiple tissues were sampled from consenting male patients and their sons derived from intracytoplasmic sperm injection (ICSI) and underwent chromosomal and genetic analyses. Paediatric and psychological examinations were also conducted. In 87 men and 47 boys, 22 sequence tagged sites (STS) were analysed by multiplex PCR and deletion breakpoints were defined with additional loci. In one patient, the breakpoints map to the highly unstable palindrome-rich region within AZFb and proximal AZFc was investigated. A total of 86 blood, 26 semen, and 73 cheek cells samples were collected from adults, and 36 blood samples and 44 cheek cell specimens were obtained from the boys. Though all of the fathers had normal karyotypes, the incidence of chromosomal abnormalities in the somatic cells of male progeny was 8.3% (3/36). The incidence of germ line aneuploidy in these men was 0.5-2.8%. A CF mutation (Delta508) was detected in one of 87 men (1.2%) and microdeletions in Yq AZF were detected in 3.4% of 87 adults and in 2.1% of their sons (n = 47). In conclusion, screening for Y chromosome microdeletions provides crucial information in the counselling of couples seeking infertility treatment. Moreover, DNA extraction and Y deletion assessments of cheek cells provide a non-invasive approach. Inheritance of Yq deletions appears not to affect the psychological and physical development of children derived from ICSI.

Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model

BackgroundTo evaluate embryonic stem cell (ESC) harvesting methods with an emphasis on derivation of ESC lines without feeder cells or sera. Using a murine model, laser-assisted blastocyst dissection was performed and compared to conventional immunosurgery to assess a novel laser application for inner cell mass (ICM) isolation.MethodsIntact blastocysts or isolated ICMs generated in a standard mouse strain were plated in medium with or without serum to compare ESC harvesting efficiency. ESC derivation was also undertaken in a feeder cell-free culture system.ResultsAlthough ICM growth and dissociation was comparable irrespective of the media components, an enhanced ESC harvest was observed in our serum-free medium (p < 0.01). ESC harvest rate was not affected by ICM isolation technique but was attenuated in the feeder cell-free group.ConclusionAchieving successful techniques for human ESC research is fundamentally dependent on preliminary work using experimental animals. In this study, all experimentally developed ESC lines manifested similar features to ESCs obtained from intact blastocysts in standard culture. Cell/sera free murine ESC harvest and propagation are feasible procedures for an embryology laboratory and await refinements for translation to human medical research.

Perspectives on the assessment of human sperm chromatin integrity

Apoptosis plays a significant role in regulating germ cell development by removing damaged germ cells from seminiferous tubules, thereby safeguarding the genome of a given species. The unique chromatin-packing process of the spermatozoon has important implications for both the development of male infertility screening tests and understanding of sperm chromatin characteristics, which may affect assisted reproductive technology outcomes. Sperm deoxyribonucleic acid (DNA) integrity tests have been proposed as a means to assess male gamete competence. Although these assays are currently gaining popularity, and are more often used as a supplement to traditional semen analysis, the point at which DNA damage occurs during spermiogenesis, and to what degree, remains to be elucidated. Here, we examined current studies of DNA fragmentation, to understand its origin and import, as well as its impact on pre- and post-implantation development. As the DNA fragmentation index is strongly correlated with the motility characteristics of a semen specimen, controlling for this factor may be helpful. Utilization of more sensitive assays, possibly on the actual spermatozoa used for insemination, may generate healthier conceptuses.

Development and current applications of assisted fertilization.

Since the very early establishment of in vitro insemination, it became clear that one of the limiting steps is the achievement of fertilization. Among the different assisted fertilization methods, intracytoplasmic sperm injection emerged as the ultimate technique to allow fertilization with ejaculated, epididymal, and testicular spermatozoa. This work describes the early steps that brought forth the development of intracytoplasmic sperm injection and its role in assisted reproductive techniques. The current methods to select the preferential male gamete will be elucidated and the concerns related to the offspring of severe male factor couples will be discussed.

To ICSI or Not to ICSI

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.