S. Cheung

Intracytoplasmic sperm injection: state of the art in humans

Among infertile couples, 25% involve both male and female factors, while male factor alone accounts for another 25% due to oligo-, astheno-, teratozoospermia, a combination of the three, or even a complete absence of sperm cells in the ejaculate and can lead to a poor prognosis even with the help of assisted reproductive technology (ART). Intracytoplasmic sperm injection (ICSI) has been with us now for a quarter of a century and in spite of the controversy generated since its inception, it remains in the forefront of the techniques utilized in ART. The development of ICSI in 1992 has drastically decreased the impact of male factor, resulting in millions of pregnancies worldwide for couples who, without ICSI, would have had little chance of having their own biological child. This review focuses on the state of the art of ICSI regarding utility of bioassays that evaluate male factor infertility beyond the standard semen analysis and describes the current application and advances in regard to ICSI, particularly the genetic and epigenetic characteristics of spermatozoa and their impact on reproductive outcome.

Identifying Maternal Constraints on Fetal Growth and Subsequent Perinatal Outcomes Using a Multiple Embryo Implantation Model.

Introduction Although the majority of singleton births after in vitro fertilization (IVF) are uncomplicated, studies have suggested that IVF pregnancies may be independently associated with low birth weight (LBW), preterm birth (PTB), and perinatal mortality. These outcomes complicate multiple gestations as expected, but have also been reported in singletons. A multiple embryo implantation model allows for assessment of the early in utero environment, and therefore, assessment of any maternal constraints on developing fetuses. We question whether adverse perinatal outcomes associated with assisted reproductive techniques (ART) occur as a result of maternal physiologic adaptations. Patients and Methods This is a retrospective, single center study of ART cycles, specifically intracytoplasmic sperm injection (ICSI) cycles during a 16-year period. For each positive pregnancy test 9–11 days after embryo transfer, an ultrasonogram was performed at 7 weeks of gestation to record the number of implanted fetal poles with cardiac activity. Controlled ovarian stimulation (COS), hCG trigger, oocyte retrieval and sperm injection were performed as per our standard protocols. First trimester implantation sites that resulted in live births were defined as “true” to distinguish them from those that spontaneously reduced called “virtual.” Birth outcomes analyzed included birth weight and gestational age at delivery. Results A total of 17,415 cycles were analyzed. The average maternal age was 36.9 (±5.0) years. An overall fertilization rate of 73.4% generated approximately 48,708 good quality cleavage-stage embryos. In most patients (92.8%), an average of 3 embryos were transferred. The clinical pregnancy rate was 39.2% (n = 6,281). The overall occurrence of multiple gestations was 38.2% (n = 2,608) consisting of 2,038 twin, 511 triplet, and 59 quadruplet pregnancies. Of these multiple gestations, 18.6% of twin, 54.2% of triplet and 76.3% of quadruplet gestations spontaneously reduced. Failure of the implanted embryo to progress was not related to maternal age. Singleton newborns resulting from multiple implantation sites had lower birth weights (P<0.01) and shorter gestational ages (P<0.01) than those from a single implanted embryo. The number of embryos transferred did not affect the gestational length of singleton newborns. Although the birth weights of singletons from multiple implantation sites (virtual singletons) were lower than true singletons, the birth weight of virtual singletons were comparable to the birth weights of true twin, triplet, and quadruplet live births. Multiple logistic regression revealed that virtual singletons were an independent risk factor for PTB (odds ratio: 4.55, 95% CI 2.23–9.29) and LBW (odds ratio: 3.61, 95% CI 1.78–7.32), even after controlling for the number of oocytes, stimulation protocol type, sperm source, total gonadotropins administered, age, embryo quality, and day of embryo transfer. Conclusions Our study highlights that embryonic implantation sites during early gestation set the growth profile of each embryo, dictating later growth patterns. Specifically, spontaneous reduction of an embryo after multiple embryo implantations can confer greater perinatal risk in the form of LBW and PTB to the surviving fetus. Our findings suggest that maternal constraints or physiologic adaptations maybe one of the mechanisms mediating adverse perinatal outcomes when multiple embryo implantation occurs.

Lessons learned in andrology: from intracytoplasmic sperm injection and beyond.

Dr. Gianpiero D. Palermo is the pioneer of intracytoplasmic sperm injection (ICSI), a ground-breaking technique that has transformed the treatment of male factor infertility and fertilization failure. Ever since its inception in 1992, ICSI has assisted thousands of men to achieve biologic paternity. Beginning 1993, Dr. Palermo has been the Director of Assisted Fertilization and Andrology at the Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine, New York, one of the world’s largest ICSI programs. In his capacity as the Blavatnik Distinguished Professor of Reproductive Medicine, he leads a talented team of researchers comprising of andrologists and clinical fellows who investigate the molecular aspects of fertilization, genetic and epigenetic aspects of male infertility, follow-up of ICSI babies and the development and differentiation of embryonic stem cells. He has published over 76 book chapters, 327 abstracts, and 132 research papers, which have been featured in journals such as the New England Journal of Medicine, Lancet, JAMA, Human Reproduction, Fertility and Sterility, and PLOS One. He is the recipient of multiple awards, including the Jacob Heskel Gabbay Award in Biotechnology and Medicine awarded by the Rosenstiel Basic Medical Sciences Research Center and Brandeis University, the Barbara Eck Menning Founders Award, and the Francavilla Fontana Illustrious Citizen Award.

Shedding Light on the Nature of Seminal Round Cells

Introduction 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. Patients and Methods 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. Results 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. Conclusions 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.

Sperm DNA fragmentation as treatment guidance for infertile couples

ported only in 2009. Isolation of these cells was replicated by a small number of other groups in mouse, rat and extended to a human model. The objective of this study is to demonstrate the presence of OSC in non-human primates (NHPs), which can generate mature oocytes following transplantation into the ovary. DESIGN: Eggs from ovarian stem cell transplantation were characterized and explored their fertilization potential. MATERIALS AND METHODS: The ovarian cortex was digested with collagenase IV and DNase followed by FACS with the DDX4 antibody. The cells were expanded in culture, transfected with a GFP lentivirus, and transplanted into the remaining ovary of the rhesus monkey. Following transplantation, gonadotropins were used for ovarian hyperstimulation to isolate oocytes. Oocytes transplant derivations were assessed by fluorescence, PCR and nested PCR. True oocyte phenotype with appearance of intact zona pellucida and polar body was observed along with expression of oocyte specific genes. Intracytoplasmic sperm injection was performed with collectedMII oocytes and fertilization and embryo development potential assessed. RESULTS: Here we demonstrate, for the first time, the presence of adult stem cells in the primate ovary which form mature oocytes with fertilization capacity following orthotopic transplantation. Two out of 68 oocytes obtained by follicular aspiration were confirmed OSCs origin, whereas 17 out of 83 from microdissection. A mature oocyte originating from OSCs developed into 64-cells stage embryo. CONCLUSIONS: Stem cells from NHP adult ovaries can be transplanted and give rise to new oocytes that fertilize and develop into an embryo, suggesting that these stem cells could be a novel approach to treating infertility.

The Ideal Spermatozoon for ART

The spermatozoon is a very unique cell in that it is comprised of separate yet interrelated components, each of which plays a crucial role during conception. The utilization of an individual cell to treat severe male factor infertility by ICSI is on the rise, and the need for the assessment of surgically retrieved specimens has shifted the focus on the assessment of the sole gamete. Selection of the ideal sperm cell is carried out by assessing the acrosome, the chromosomal status of the male gamete, which is crucial, and another often overlooked organelle, the centrosome, which functions as the scaffold to ordain chromosomal segregation in the new conceptus. The fully developed spermatozoon is also characterized by defects in chromatin integrity, and its source and relevance are still object of intense investigation. Thus, the quest for the ideal spermatozoon has set the stage for future research to develop means and procedures to identify a male gamete with the best fitness to generate a healthy offspring.

Single Gamete Insemination Aiming at the Ideal Conceptus

About 15 % of couples are unable to achieve a pregnancy due to a male factor diagnosis, and ICSI is seemingly the ideal treatment. ICSI remains the most popular choice of insemination even for simple, routine IVF cases, and a standard semen analysis alone is no longer inadequate in the modern infertility evaluation. Accordingly, the arrival of more sophisticated assays to measure the full impact and competency of the spermatozoon is welcome. To allow the embryonic genome to be read and expressed, the spermatozoon must release its oocyte-activating factor. Various assays are now being utilized to more fully ascertain the male gamete and establish its genetic integrity and its ability to sustain proper embryo development. While these attempts to select the ideal spermatozoon are laudable, they are still being tested and debated. Of note, these testing methods still do not address the clinical context where sperm numbers are extremely limited. As novel sperm selection methods continue to emerge, information regarding the long-term intergenerational effects of utilizing suboptimal sperm in IVF continues to accumulate. The increased emphasis on the gamete has stimulated the quest for new tools to more accurately diagnose and select individual spermatozoa prior to direct injection. This enables better counseling and treatment of couples while at the same time assuages worries associated with potentially passing a nonideal paternal genome onto the next generation. Reproductive specialists can thus aim to provide an ideal, singleton gestation for all patients seeking advanced reproductive assistance.