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1.  Understanding fertilization through intracytoplasmic sperm injection (ICSI) 
Cell calcium  2013;55(1):24-37.
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.
PMCID: PMC4046257  PMID: 24290744
failed fertilization; oocyte activation; ICSI; PLCζ; assisted oocyte activation; sperm cytosolic factor; calcium influx
2.  Intracytoplasmic Sperm Injection (ICSI) in Extreme Cases of Male Infertility 
PLoS ONE  2014;9(12):e113671.
Severely compromised spermatogenesis typical of men with virtual azoospermia or non-obstructive azoospermia requires an extreme search for spermatozoa. Our goal was to evaluate the usefulness of a meticulous search carried out in ejaculated or surgically retrieved specimens in achieving pre- and post-implantation embryo development.
Patients and Methods
In a retrospective cohort study carried out in an academic institution, intracytoplasmic sperm injection (ICSI) outcomes were reviewed as a function of length of microscopic sperm search in ejaculated and surgically retrieved specimens. Couples whose male partner presented with either virtual or non-obstructive azoospermia were treated by ICSI and categorized according to the time spent in identifying and retrieving enough spermatozoa to inject all the oocyte cohort. Semen parameter, fertilization, pregnancies, deliveries, and child welfare in relation to increasing search time were analyzed and compared.
The maternal and paternal ages were comparable in both ejaculated and testicular sperm extraction (TESE) groups along with the oocytes retrieved. The fertilization rates for both ejaculated and TESE progressively decreased with increasing time (P<0.0001). Clinical pregnancies in the ejaculated cohort remained satifactory. In the TESE cohort, there was a decrease in pregnancy rate with increasing time, from 44% to 23%. In a limited number of cases, offspring health was evaluated in both semen sources and appeared reassuring.
An extensive and at time exhaustive sperm quest yields kinetically and morphologically impaired spermatozoa without apparent impact on embryo developmental competence. Retrieval of spermatozoa from the seminiferous tubules provided more consistent fertilization and pregnancy outcomes than those retrieved from the ejaculate. A trend indicated that pregnancy rate decreased as search time increased in the TESE group. The utilization of the scarce and unselected spermatozoa did not obviously impair embryo development or cause post-implantation errors.
PMCID: PMC4249967  PMID: 25437298
The Journal of urology  2013;191(1):175-178.
Introduction and Objectives
Men with azoospermia and severe testicular atrophy may be counseled to avoid sperm retrieval due to perceived limited success. We evaluated the outcomes of microdissection testicular sperm extraction (micro-TESE) in men with severe testicular atrophy (volume ≤ 2 mL).
We reviewed the records of 1127 men with nonobstructive azoospermia who underwent micro-TESE followed by intracytoplasmic sperm injection. The men were classified into three groups based on average testicular volume (mL), ≤2, >2–10, >10. Sperm retrieval, clinical pregnancy, and live birth rates were calculated. The clinical features evaluated included age, FSH level, history of cryptorchidism, Klinefelter syndrome, varicocele, and testicular histology from diagnostic biopsy.
Testicular sperm were successfully retrieved in 56% of the men. Sperm retrieval rates (SRR) in men with testicular volumes of ≤ 2, >2–10, and >10 mL was 55%, 56% and 55% respectively. Of those men who had sperm retrieved, clinical pregnancy and live birth rates were similar in the three groups (55.2%, 50.0%, and 47.0%; and 47.2%, 43.0% and 42.2% respectively). Of the 106 men with average testis volume ≤ 2 mL bilaterally, men who had sperm retrieved were younger (31.1 vs. 35.2 years), and were more likely to have a history of Klinefelter syndrome (82.2% vs 55.6%) compared to those in whom sperm was not found (p < 0.05). Men in this group had a higher prevalence of Klinefelter syndrome than men with testis >2 mL (72.6% vs 5.3%, p<0.0001). Men younger than 30 years with Klinefelter syndrome had a higher SRR (81.8%) compared to men older than 30 without Klinefelter syndrome (33%, p<0.01). There was no cut-point for age beyond which sperm could not be retrieved in men with small testes. On multivariable analysis, younger age was the only preoperative factor associated with successful sperm retrieval in men with small testes (<2mL).
Testicular volume does not affect sperm retrieval rates at our center with micro-TESE. For men with the smallest volume testes, those younger men with Klinefelter syndrome had the highest sperm retrieval rates. Severe testicular atrophy should not be a contraindication for micro-TESE.
PMCID: PMC4077600  PMID: 23911635
Azoospermia; testis; biopsy; microorchidism; volume
5.  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 
To 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.
Intact 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.
Although 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.
Achieving 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.
PMCID: PMC1479373  PMID: 16681851
6.  Non-obstructive azoospermia and maturation arrest with complex translocation 46,XY t(9;13;14)(p22;q21.2;p13) is consistent with the Luciani-Guo hypothesis of latent aberrant autosomal regions and infertility 
Cell & Chromosome  2005;4:2.
To describe clinical and histological features observed in the setting of an unusual complex translocation involving three autosomes (9, 13, and 14) identified in an otherwise healthy male referred for infertility consultation.
Materials and methods
The patient was age 30 and no family history was available (adopted). Total azoospermia was confirmed on multiple semen analyses. Peripheral karyotype showed a 46,XY t(9;13;14)(p22:q21.2;p13) genotype; no Y-chromosome microdeletions were identified. Cystic fibrosis screening was negative. Bilateral testis biopsy revealed uniform maturation arrest and peritubular fibrosis.
Formal genetic counseling was obtained and the extant literature reviewed with the couple. Given the low probability of obtaining sperm on testicular biopsy, as well as the high risk of any retrieved sperm having an unbalanced genetic rearrangement, the couple elected to proceed with fertility treatment using anonymous donor sperm for insemination.
Although genes mapped to the Y-chromosome have been established as critical to normal testicular development and spermatogenesis, certain autosomal genes are now also recognized as important in these processes. Here we present clinical evidence to support the Luciani-Guo hypothesis (first advanced in 1984 and refined in 2002), which predicts severe spermatogenic impairment with aberrations involving chromosomes 9, 13, and/or 14, independent of Y-chromosome status. Additional study including fluorescent in situ hybridization and molecular analysis of specific chromosomal regions is needed to characterize more fully the contribution(s) of these autosomes to male testicular development and spermatogenesis.
PMCID: PMC1253518  PMID: 16162283
7.  Identification and isolation of embryonic stem cells in reproductive endocrinology: theoretical protocols for conservation of human embryos derived from in vitro fertilization 
Embryonic stem cells (ESC) are pluripotent cells obtained from the inner cell mass (ICM) of blastocysts derived from in vitro culture associated with reproductive endocrinology therapy. Human ESCs are regarded as highly significant since they retain the capacity to differentiate into any of approximately 200 unique cell types. Human ESC research is controversial because to acquire such cells, the ICM of human blastocysts must be manipulated in a way that renders embryos nonviable and unsuitable for transfer in utero. Techniques to yield competent ESCs with conservation of source blastocysts would satisfy many objections against ESC research, but at present such approaches remain largely untested.
Results and discussion
We contrast experimental culture of single blastomeres obtained by 1) non-destructive biopsy of embryos destined for transfer, and 2) isolation of karyotypically normal blastomeres from disaggregated ("dead") embryos considered unsuitable for transfer, and evaluate these approaches with regard to production of ESCs. Pluripotency was confirmed by morphological criteria and by quantification of divergent homeodomain proteins specific to undifferentiated cell development. Following ESC isolation and identification, assessment was conducted according to a novel ESC grading system, also proposed here.
The role of reproductive endocrinology in ESC research remains paramount. In this report, we hypothesize new and expand on existing strategies having the potential to enhance human ESC isolation, identification and in vitro maintenance.
PMCID: PMC1185568  PMID: 16026616
8.  Article processing charges, funding, and open access publishing at Journal of Experimental & Clinical Assisted Reproduction 
Journal of Experimental & Clinical Assisted Reproduction is an Open Access, online, electronic journal published by BioMed Central with full contents available to the scientific and medical community free of charge to all readers. Authors maintain the copyright to their own work, a policy facilitating dissemination of data to the widest possible audience without requiring permission from the publisher. This Open Access publishing model is subsidized by authors (or their institutions/funding agencies) in the form of a single £330 article processing charge (APC), due at the time of manuscript acceptance for publication. Payment of the APC is not a condition for formal peer review and does not apply to articles rejected after review. Additionally, this fee is waived for authors whose institutions are BioMed Central members or where genuine financial hardship exists. Considering ordinary publication fees related to page charges and reprints, the APC at Journal of Experimental & Clinical Assisted Reproduction is comparable to costs associated with publishing in some traditional print journals, and is less expensive than many. Implementation of the APC within this Open Access framework is envisioned as a modern research-friendly policy that supports networking among investigators, brings new research into reach rapidly, and empowers authors with greater control over their own scholarly publications.
PMCID: PMC546227  PMID: 15649322
9.  Journal of Experimental & Clinical Assisted Reproduction: shaping the future of research and practice in reproductive endocrinology/infertility 
Journal of Experimental & Clinical Assisted Reproduction is an open access, online, peer-review journal publishing papers on all aspects of research into reproductive endocrinology, infertility, bioethics and the advanced reproductive technologies. The journal reports on important developments impacting the field of human reproductive medicine and surgery. The field exists as a sub-specialty of obstetrics & gynecology, focusing on the diagnosis and treatment of complex human reproductive problems. The continued growth of this relatively new field depends on quality research by proven scientists as well as junior investigators who, together, make contributions to this area of medical and surgical practice. The publishing revolution made possible by internet technology presages a bright future for continued interdisciplinary collaboration among researchers. Against this background, Journal of Experimental & Clinical Assisted Reproduction exists for the scientific community to facilitate this scholarly dialogue.
PMCID: PMC524035  PMID: 15507153
publishing; reproductive medicine; internet; research; trends
10.  Preimplantation Genetic Diagnosis for Elective Sex Selection, the IVF Market Economy, and the Child—Another Long Day's Journey into Night? 
The promise of medical innovation has long evoked social commentary, particularly when personal reproductive autonomy may be involved. Development of the oral contraceptive, effective and safe surgical sterilization, and later IVF and ICSI are among the revolutionary developments where the initial reactions were dubious but were accorded mainstream status with sufficient clinical experience. In each instance, debate about the moral and social implications of these treatments accompanied their introduction into the medical marketplace. This pattern appears to be repeating itself in connection with the use of preimplantation genetic diagnosis (PGD) for elective sex selection of human embryos. As with prior challenges in reproductive medicine, the development of meaningful “guidelines” for this latest controversy has proven to be a contentious task. Indeed, the progression of ethics committee reports from the Society for Reproductive Medicine seems to echo the ambivalence within society at large regarding this issue. In this report, we chronicle sex selection claims based on sperm sorting, and describe how flow cytometry and especially PGD have facilitated this selection at the gamete and embryo stage, respectively. In doing so, we also explore market forces and practitioner considerations associated with the application of PGD for this; related ethical issues with particular emphasis on the progeny derived from such treatment are also.
PMCID: PMC3455545  PMID: 12408539
Ethics; IVF; PGD; sex selection
11.  Intrauterine pregnancy following low-dose gonadotropin ovulation induction and direct intraperitoneal insemination for severe cervical stenosis 
We present a case of primary infertility related to extreme cervical stenosis, a subset of cervical factor infertility which accounts for approximately 5% of all clinical infertility referrals.
Case presentation
A 37 year-old nulligravida was successfully treated with ovulation induction via recombinant follicle stimulating hormone (FSH) and direct intraperitoneal insemination (IPI). Anticipating controlled ovarian hyperstimulation with in vitro fertilization/embryo transfer (IVF), the patient underwent hysteroscopy and cervical recanalization, but safe intrauterine access was not possible due to severe proximal cervical stricture. Hysterosalpingogram established bilateral tubal patency and confirmed an irregular cervical contour. Since the cervical canal could not be traversed, neither standard intrauterine insemination nor transcervical embryo transfer could be offered. Prepared spermatozoa were therefore placed intraperitoneally at both tubal fimbria under real-time transvaginal sonographic guidance using a 17 gage single-lumen IVF needle. Supplementary progesterone was administered as 200 mg/d lozenge (troche) plus 200 mg/d rectal suppository, maintained from the day following IPI to the 8th gestational week. A singleton intrauterine pregnancy was achieved after the second ovulation induction attempt.
In this report, we outline the relevance of cervical factor infertility to reproductive medicine practice. Additionally, our andrology evaluation, ovulation induction approach, spermatozoa preparation, and insemination technique in such cases are described.
PMCID: PMC139980  PMID: 12450413
cervical factor infertility; intraperitoneal insemination; ovulation induction
12.  A Seminar on Human Cloning: Reprogramming Somatic Cell Differentiation and the Hayflick Limit: Contrasting Two Modern Molecular Bioengineering Aims and Their Impact on the Future of Mankind 
The molecular biology of human cloning and aging research depend on the closely related laboratory techniques supported by a thorough understanding of cell-signaling processes. Unfortunately, the link between these two research fields has received only marginal attention in the lay press. Cloning is possible when somatic cell differentiation is successfully reprogrammed, and clinical control of cellular senescence depends on a proper reconfiguration of the predetermined number of divisions permitted during the cell life-cycle (the so-called “Hayflick Limit”). In this paper, we discuss these two concepts and compare the impact likely to be associated with bioengineering studies that facilitate both human cloning and longevity therapy.
PMCID: PMC3455520  PMID: 11599467
cloning; ethics; research; relativism

Results 1-12 (12)