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• W1973249625 abstract "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. 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. Infertility affects approximately 12%–15% of couples of reproductive age (1CDCAssisted reproductive technology success rates. National summary and fertility clinic reports. National Center for Chronic Disease and Prevention and Health Promotion. Center for Disease Control and Prevention, Atlanta, Georgia2008Google Scholar). Although today we recognize that infertility is caused equally by male and female factors, where the male partner’s role has been neglected or underestimated since antiquity. Moreover, for thousands of years, societal and cultural attitudes seemed to impose responsibility for infertility on the female rather than the male partner. Indeed, in the mid-20th century, a lack of understanding and appreciation for the complexity of the male reproductive system led to the simplistic approach of treating male infertility with donor insemination (2Traina G. Minerva Ginecol. 1977; 29: 449-450PubMed Google Scholar, 3Walton A. The technique of artificial insemination. Imperial Bureau of Animal Genetics. Oliver and Boyd, Edinburg1933Google Scholar, 4Weller J. Zentralbl Gynakol. 1977; 99: 264-270PubMed Google Scholar). This approach was further advanced by the early success with cryopreservation of spermatozoa (5Foote R.H. Bratton R.W. The fertility of bovine semen cooled with and without the addition of citrate sulfanilamide yolk extender.J Dairy Sci. 1949; 32: 856-861Abstract Full Text PDF Google Scholar, 6Sherman J.K. Improved methods of preservation of human spermatozoa by freezing and freeze-drying.Fertil Steril. 1963; 14: 49-64Abstract Full Text PDF PubMed Google Scholar). The versatility of the male gamete was mostly due to the fact that male reproductive cells are often available in excess and therefore even the loss of about half of their population would still suffice to successfully inseminate a woman in spite of the rudimentary cryostorage procedures available (6Sherman J.K. Improved methods of preservation of human spermatozoa by freezing and freeze-drying.Fertil Steril. 1963; 14: 49-64Abstract Full Text PDF PubMed Google Scholar, 7Wales R.G. Whittingham D.G. Development of eight-cell mouse embryos in substrate-free medium.J Reprod Fertil. 1973; 32: 316-317Crossref PubMed Google Scholar). Much of our understanding of the mechanisms of mammalian reproduction and advancements in this area emanated from animal husbandry and the outstanding work performed by veterinarians (7Wales R.G. Whittingham D.G. Development of eight-cell mouse embryos in substrate-free medium.J Reprod Fertil. 1973; 32: 316-317Crossref PubMed Google Scholar) and early reproductive biologists (8Edwards R.G. Immunological control of fertility in female mice.Nature. 1964; 203: 50-53Crossref PubMed Scopus (34) Google Scholar, 9McLaren A. Biggers J.D. Successful development and birth of mice cultivated in vitro as early as early embryos.Nature. 1958; 182: 877-878Crossref PubMed Scopus (36) Google Scholar). However, it was not until the late 1970s when IVF became a reality that female infertility treat-ments ascended to the fore. It was soon realized that although IVF successfully treated tubal infertility, there were great limitations in achieving predictable fertilization rates after in vitro inse-mination in males with compromised semen parameters and in couples with other infertility etiologies (10Cohen J. Edwards R.G. Fehilly C.B. Fishel S.B. Hewitt J. Rowland G. et al.Treatment of male infertility by in vitro fertilization: factors affecting fertilization and pregnancy.Acta Eur Fertil. 1984; 15: 455-465PubMed Google Scholar). In fact, a 40% fertilization failure was observed in the most reputable centers (10Cohen J. Edwards R.G. Fehilly C.B. Fishel S.B. Hewitt J. Rowland G. et al.Treatment of male infertility by in vitro fertilization: factors affecting fertilization and pregnancy.Acta Eur Fertil. 1984; 15: 455-465PubMed Google Scholar). Micromanipulation burst onto the scene in the mid 1980s with zona drilling, first reported by Gordon and Talansky (11Gordon J.W. Talansky B.E. Assisted fertilization by zona drilling: a mouse model for correction of oligospermia.J Exp Zool. 1986; 239: 347-354Crossref PubMed Google Scholar), a procedure that involved the creation of a circumscribed opening in the zona by acid Tyrode’s solution (pH 2) applied through a fine glass micropipette. After insemination, more than one spermatozoon frequently entered the breached zona. As zona drilling was being tested, mechanical cutting of a hole in the zona emerged for nuclear manipulation of fertilized eggs (12Tsunoda Y. Yasui T. Nakamura K. Uchida T. Sugie T. Effect of cutting the zona pellucida on the pronuclear transplantation in the mouse.J Exp Zool. 1986; 240: 119-125Crossref PubMed Google Scholar). Similar procedures were zona cracking in which the zona was breached mechanically with two fine glass hooks controlled by a micromanipulator (13Odawara Y. Lopata A. A zona opening procedure for improving in vitro fertilization at low sperm concentrations: a mouse model.Fertil Steril. 1989; 51: 699-704Abstract Full Text PDF PubMed Google Scholar) and zona softening performed by a brief exposure to trypsin (14Gordon J.W. Use of micromanipulation for increasing the efficiency of mammalian fertilization in vitro.Ann N Y Acad Sci. 1988; 541: 601-613Crossref PubMed Google Scholar) or pronase. Partial zona dissection involved cutting the zona with glass pipettes just before exposure of the treated oocytes to spermatozoa (15Malter H.E. Cohen J. Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration.Fertil Steril. 1989; 51: 138-148Google Scholar). For all these techniques, spermatozoa had to be progressively motile and to have undergone or have the potential for an acrosome reaction. The techniques also carried a distinct risk of injury to the oocytes as it required producing an opening in the zona of optimal size. Localized laser photoablation of the zona has also been used to produce a gap of precise dimensions in the zona, and this has resulted in a few healthy offspring (16Antinori S. Versaci C. Fuhrberg P. Panci C. Caffa B. Gholami G.H. Seventeen live births after the use of an erbium-yytrium aluminum garnet laser in the treatment of male factor infertility.Hum Reprod. 1994; 9: 1891-1896PubMed Google Scholar, 17Feichtinger W. Strohmer H. Radner K.M. Erbium YAG laser for micromanipulation of oocytes and spermatozoa.Lancet. 1992; 340: 115-116Abstract PubMed Google Scholar). However, not only did all these early procedures bring modest fertilization rates, with partial zona dissection being the most useful, they also carried a significant incidence of polyspermy. Later, mechanical insertion of spermatozoa directly into the perivitelline space—subzonal sperm injection (18Laws-King A. Trounson A. Sathananthan H. Kola I. Fertilization of human oocytes by microinjection of a single spermatozoon under the zona pellucida.Fertil Steril. 1987; 48: 637-642Abstract Full Text PDF PubMed Google Scholar)—appeared as another way of overcoming inadequacies of sperm concentration and motility, and proved to be more effective than zona drilling or partial zona dissection, particularly after prior induction of the acrosome reaction (19Palermo G. Joris H. Devroey P. Van Steirteghem A.C. Induction of acrosome reaction in human spermatozoa used for subzonal insemination.Hum Reprod. 1992; 7: 248-254PubMed Google Scholar, 20Palermo G. Van Steirteghem A. Enhancement of acrosome reaction and subzonal insemination of a single spermatozoon in mouse eggs.Mol Reprod Dev. 1991; 30: 339-345Crossref PubMed Google Scholar, 21Liu L. Trimarchi J.R. Oldenbourg R. Keefe D.L. Increased birefringence in the meiotic spindle provides a new marker for the onset of activation in living oocytes.Biol Reprod. 2000; 63: 251-258Crossref PubMed Google Scholar). Subzonal sperm injection required that spermatozoa be normal, capacitated, and even hyperactivated or acrosome-reacted. The limitation of this technique was its inability to overcome acrosomal abnormalities or dysfunction of the sperm-oolemma fusion process, and, ultimately, by unacceptably low fertilization rates. The implementation of assisted fertilization had generated a remarkable effort in understanding the physiology of the individual spermatozoon and provided a method to bypass the limitations of a subfertile sibling sperm cell. Assisted fertilization brought a shift from the existing in vitro approach that aimed at generating a viable embryo outside the body to target a specific role—achieving fertilization. Assisted fertilization, particularly intracytoplasmic sperm injection (ICSI) has brought down the ratio between the parental gametes to 1:1, where in fact one individual spermatozoon was paired to an oocyte, paving the way to understanding the interaction between the two gametes. Because of ICSI, the scientific interest in studying the individual spermatozoon and oocyte was sparked. With ICSI, the oocyte was better visualized once void of the ancillary cells. Scanning of the ooplasm, observation of the meiotic spindle, for example by polarized microscopy (21Liu L. Trimarchi J.R. Oldenbourg R. Keefe D.L. Increased birefringence in the meiotic spindle provides a new marker for the onset of activation in living oocytes.Biol Reprod. 2000; 63: 251-258Crossref PubMed Google Scholar, 22Neri Q.V. Maggiulli R. Monahan D. Ermolovich E. Rosenwaks Z. Palermo G.D. Intracytoplasmic sperm injection. Surgical management of male infertility. Cambridge University Press, New York2012Google Scholar), as well as the clear identification and access to the polar body has allowed the proliferation of preimplantation genetic screening techniques (23Munne S. Gianaroli L. Tur-Kaspa I. Magli C. Sandalinas M. Grifo J. et al.Substandard application of preimplantation genetic screening may interfere with its clinical success.Fertil Steril. 2007; 88: 781-784Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The reliable evaluation of oocyte nuclear maturity has also allowed for the refinements of ovarian superovulation protocols (24Fauser B.C. Donderwinkel P. Schoot D.C. The step-down principle in gonadotrophin treatment and the role of GnRH analogues.Baillieres Clin Obstet Gynaecol. 1993; 7: 309-330Abstract Full Text PDF PubMed Google Scholar, 25Jones G.S. Garcia J.E. Rosenwaks Z. The role of pituitary gonadotropins in follicular stimulation and oocyte maturation in the human.J Clin Endocrinol Metab. 1984; 59: 178-180Crossref PubMed Google Scholar) by tailoring medications and dosages. In addition, sperm injection has the ability to precisely pinpoint the timing of fertilization of the oocyte and, therefore, has made it possible to appreciate the importance of synchronicity between nuclear maturation and ooplasmic readiness (26Maggiulli R. Neri Q.V. Monahan D. Hu J. Takeuchi T. Rosenwaks Z. et al.What to do when ICSI fails.Syst Biol Reprod Med. 2010; 56: 376-387Crossref PubMed Scopus (4) Google Scholar). Moreover, zona thickness variations and morphologic aspects of the oolemma (27Van Blerkom J. Bell H. Regulation of development in the fully grown mouse oocyte: chromosome-mediated temporal and spatial differentiation of the cytoplasm and plasma membrane.J Embryol Exp Morphol. 1986; 93: 213-238PubMed Google Scholar) inherent to patient characteristics or in response to superovulation medications are among the diverse gametic characteristics that have been beautifully studied in the course of performing assisted fertilization. Finally, the arbitrary beginning of the fertilization process has permitted the design of observational studies of ooplasmic dynamics confirmed by time lapse videoimaging (28Payne D. Flaherty S.P. Barry M.F. Matthews C.D. Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography.Hum Reprod. 1997; 12: 532-541Crossref PubMed Google Scholar). Recently, our attention has been directed toward the unicellular approach for studying the male gamete aiming at reading its chromosomal constitution (29Colombero L.T. Hariprashad J.J. Tsai M.C. Rosenwaks Z. Palermo G.D. Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome.Fertil Steril. 1999; 72: 90-96Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 30Hu J.C.Y. Monahan D. Neri Q.V. Rosenwaks Z. Palermo G.D. The role of sperm aneuploidy assay.Fertil Steril. 2011; 96: S24-S25Abstract Full Text Full Text PDF PubMed Google Scholar, 31Palermo G.D. Colombero L.T. Hariprashad J.J. Schlegel P.N. Rosenwaks Z. Chromosome analysis of epididymal and testicular sperm in azoospermic patients undergoing ICSI.Hum Reprod. 2002; 17: 570-575Crossref PubMed Google Scholar) or its chromatinic integrity (32Bungum M. Humaidan P. Spano M. Jepson K. Bungum L. Giwercman A. The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI.Hum Reprod. 2004; 19: 1401-1408Crossref PubMed Scopus (193) Google Scholar, 33Evenson D. Jost L. Sperm chromatin structure assay is useful for fertility assessment.Methods Cell Sci. 2000; 22: 169-189Crossref PubMed Scopus (163) Google Scholar, 34Fernandez-Gonzalez R. Moreira P.N. Perez-Crespo M. Sanchez-Martin M. Ramirez M.A. Pericuesta E. et al.Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring.Biol Reprod. 2008; 78: 761-772Crossref PubMed Scopus (95) Google Scholar). The method of suspending spermatozoa in viscous medium allows the observation of their 3D kinetic patterns (35Palermo G.D. Hu J.C.Y. Rienzi L. Maggiulli R. Takeuchi T. Yoshida A. et al.Thoughts on IMSI.in: Racowsky C. Schlegel P.N. Fauser B.C. Carrell D.T. Biennial review of infertility. Vol. 2. Springer, New York2011: 296Google Scholar) and to evaluate their morphologic characteristics at high magnification (36Bartoov B. Berkovitz A. Eltes F. Selection of spermatozoa with normal nuclei to improve the pregnancy rate with intracytoplasmic sperm injection.N Engl J Med. 2001; 345: 1067-1068Crossref PubMed Scopus (68) Google Scholar, 37Bartoov B. Berkovitz A. Eltes F. Kogosowski A. Menezo Y. Barak Y. Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome.J Androl. 2002; 23: 1-8PubMed Google Scholar). Although new insights are being established on surface markers of the spermatozoon (38Huszar G. Ozenci C.C. Cayli S. Zavaczki Z. Hansch E. Vigue L. Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status.Fertil Steril. 2003; 79: 1616-1624Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 39Jakab A. Sakkas D. Delpiano E. Cayli S. Kovanci E. Ward D. et al.Intracytoplasmic sperm injection: a novel selection method for sperm with normal frequency of chromosomal aneuploidies.Fertil Steril. 2005; 84: 1665-1673Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 40Yagci A. Murk W. Stronk J. Huszar G. Spermatozoa bound to solid state hyaluronic acid show chromatin structure with high DNA chain integrity: an acridine orange fluorescence study.J Androl. 2011; 31: 566-572Crossref Scopus (17) Google Scholar), a clearer understanding of the conformational chromatin structure characterized by the two forms of DNA present (protamine and histone bound) and the recent recognition of small-non-coding RNA (41Hamatani T. Spermatozoal RNA profiling towards a clinical evaluation of sperm quality.Reprod Biomed Online. 2011; 22: 103-105Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, 42Krawetz S.A. Kruger A. Lalancette C. Tagett R. Anton E. Draghici S. et al.A survey of small RNAs in human sperm.Hum Reprod. 2011; 26: 3401-3412Crossref PubMed Scopus (27) Google Scholar, 43Miller D. Ostermeier G.C. Krawetz S.A. The controversy, potential and roles of spermatozoal RNA.Trends Mol Med. 2005; 11: 156-163Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) will guide the treatment of infertility through the next generation. In this work we provide some insights on the circumstances that brought about the development of ICSI as well the recent accomplishments with ejaculated and surgically retrieved spermatozoa. We will review the current techniques available to gauge male genomic and epigenomic competence. This will include a survey regarding recent data on the safety of these treatments and future plans to tackle the most unexplored and intriguing aspects of male infertility. I still clearly remember the moment when I had my one-on-one encounter with the micromanipulator. The machine was under a plastic cover that instilled a sense of mystery to the device more than exerting a protective role. During my sabbatical in Brussels, I was allowed to work with the machine only one afternoon per week. Yes, the allotted time was actually distributed in half-days even if for the remainder of the week the micromanipulator was almost always beneath a dustcover. When my first afternoon dedicated to work on the manipulator arrived, I experienced an overwhelming excitement that triggered my instinctive curiosity focused at comprehending the meaning of all parts and components held together by a clutter of levers and screws. Since my early adolescence, I have been very fascinated by mechanical devices, and the prospect to carry out my work on such an instrument appeared to be unreal. The microinjector was an item all on its own; in fact it did not take long to figure out that this was the key instrument to deliver a steady pressure of viscous medium through a microneedle of 5 μm diameter in such a controlled manner to rein in the position of a spermatozoon within it. In about 2 months, working with Nikon and Narishige, I was able to replace the entire micromanipulation setup. Even the inverted microscope had the lenses and condenser replaced to work with plasticware, and the heating stage was redesigned to accommodate the injection dish. The choice of micro-tools represented another challenge—I needed to decide how to inject—whether on a slide with straight tools or in a dish with bent tools. Some investigators used the lid of a 60-mm dish, and when I looked at the Falcon catalogue, I identified a bacteriology dish, the 1006—now known as the ICSI dish (BD 351006). The dish impressed me because of the low profile allowing the injection in a dish—not in a lid—which also had the ability to be carried by its handles. It had an airtight cover allowing to be placed in an incubator while containing N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffered medium under oil. A major challenge was the fabrication of the injection pipette, starting from the grinding process, during which I added a water drip device to avoid glass debris from accumulating on and in the pipette tip, ending with a spike added at the outer bevel of the microneedle without closing its opening. The actual injection was interesting to learn, though somewhat intuitive, in unfertilized human and hamster eggs, turned out to be a nightmare in the mouse. The mouse injection was extremely difficult because of the length of the sperm flagellum in contrapose to the relatively smaller size and higher membrane fragility of the mouse egg. The injection in the mouse proved to be very tedious and labor intensive but finally paid off when one of the mouse pregnancies brought forth live pups (20Palermo G. Van Steirteghem A. Enhancement of acrosome reaction and subzonal insemination of a single spermatozoon in mouse eggs.Mol Reprod Dev. 1991; 30: 339-345Crossref PubMed Google Scholar)—which generated a tremendous source of pride. Treating the first patient was a stressful and an exciting experience. While performing subzonal injection and by creating a membrane invagination, this approach caused the breaching of the oolemma. In one injection, I had three sperm loaded and after penetrating the zona pellucida while pressing against the oolemma with the injection tool, I saw one sperm swim into the cytoplast. I believed that this egg would inevitably lyse, so I promptly labeled it with a question mark. The recurrence of the accidental oolemma breaching inspired me with the idea to purposely deposit the sperm deeply into the ooplasm but paying attention at creating a membrane depression sufficient enough to allow self-resealing after microtool withdrawal. I asked my close collaborator, “to leave a track in the history of medicine we should get a pregnancy with intracytoplasmic sperm injection?” He candidly replied, “We have one already!” It was the only egg in a cohort of 12 that actually fertilized, generating an embryo that resulted in the first ICSI baby. He was born in January 1992. He was the egg labeled with a question mark. Among the general population, the ability of a man to procreate appears to have progressively decreased during the past half century (44Stefankiewicz J. Kurzawa R. Drozdzik M. Ginekol Pol. 2006; 77: 163-169PubMed Google Scholar). An estimated 12%–15% of couples in their reproductive age are afflicted by a range of causes pertaining to their ability to reproduce, and about half of all infertility cases are directly attributed to the male partner. Approximately 6% of males between the ages of 15 and 44 are deemed infertile or have their fecundity severely compromised (45Wright V.C. Chang J. Jeng G. Macaluso M. Assisted reproductive technology surveillance—United States, 2003.MMWR Surveill Summ. 2006; 55: 1-22PubMed Google Scholar). Evaluation of endocrine data implies that the main causes of male infertility are hormonal disturbances and aberrations in the production of semen (44Stefankiewicz J. Kurzawa R. Drozdzik M. Ginekol Pol. 2006; 77: 163-169PubMed Google Scholar). Notwithstanding, the absence of spermatozoa in the ejaculate is the most dramatic form, termed azoospermia, whereas about 50% of these men are characterized by hypo-spermatogenesis, complete germ cell aplasia accounts for about 25%, and the remainder present with spermatogenic arrest (46Chan P.T. Schlegel P.N. Nonobstructive azoospermia.Curr Opin Urol. 2000; 10: 617-624Crossref PubMed Scopus (18) Google Scholar). Intracytoplasmic sperm injection has proven itself as the assisted fertilization technique that grants the best results in terms of fertilization and clinical pregnancy. It is equally successful independently of the semen specimens used whether fresh or after cryopreservation. Even immunologic cases where anti-sperm antibodies are present on the spermatozoa benefit from ICSI (47Mansour R.T. Aboulghar M.A. Serour G.I. Amin Y.M. Ramzi A.M. The effect of sperm parameters on the outcome of intracytoplasmic sperm injection.Fertil Steril. 1995; 64: 982-986Abstract Full Text PDF PubMed Google Scholar, 48Nagy Z.P. Liu J. Joris H. Verheyen G. Tournaye H. Camus M. et al.The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters.Hum Reprod. 1995; 10: 1123-1129PubMed Google Scholar, 49Palermo G. Joris H. Derde M.P. Camus M. Devroey P. Van Steirteghem A. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection.Fertil Steril. 1993; 59: 826-835Abstract Full Text PDF PubMed Google Scholar, 50Palermo G.D. Cohen J. Alikani M. Adler A. Rosenwaks Z. Development and implementation of intracytoplasmic sperm injection (ICSI).Reprod Fertil Dev. 1995; 7 (discussion 217–8): 211-217Crossref PubMed Google Scholar). Similarly, when spermatozoa are retrieved surgically from the epididymis or testis, the clinical outcome is not affected (51Palermo G.D. Cohen J. Alikani M. Adler A. Rosenwaks Z. Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility.Fertil Steril. 1995; 63: 1231-1240Abstract Full Text PDF PubMed Google Scholar, 52Tournaye H. Devroey P. Liu J. Nagy Z. Lissens W. Van Steirteghem A. Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens.Fertil Steril. 1994; 61: 1045-1051PubMed Google Scholar). Because of the dependable and versatile performance of ICSI, it has broadened its initial indication as a technique capable of overriding sperm dysfunction to a procedure that may partly address issues attributable to the female partner. Indeed, ICSI has allowed successful fertilization when only a few and/or abnormal oocytes are available (53Ludwig M. al-Hasani S. Kupker W. Bauer O. Diedrich K. A new indication for an intracytoplasmic sperm injection procedure outside the cases of severe male factor infertility.Eur J Obstet Gynecol Reprod Biol. 1997; 75: 207-210Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) and most importantly has rendered possible the successful insemination of cryopreserved oocytes (54Porcu E. Fabbri R. Seracchioli R. Ciotti P.M. Magrini O. Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes.Fertil Steril. 1997; 68: 724-726Abstract Full Text PDF PubMed Scopus (287) Google Scholar). This is explained by the notion that freezing can lead to premature exocytosis of cortical granules consequent to a zona pellucida molecular mechanism that inhibits natural sperm penetration (55Johnson L. Evaluation of the human testis and its age-related dysfunction.Prog Clin Biol Res. 1989; 302 (discussion 61–7): 35-60PubMed Google Scholar, 56Schalkoff M.E. Oskowitz S.P. Powers R.D. Ultrastructural observations of human and mouse oocytes treated with cryopreservatives.Biol Reprod. 1989; 40: 379-393Crossref PubMed Google Scholar, 57Van Blerkom J. Davis P.W. Merriam J. The developmental ability of human oocytes penetrated at the germinal vesicle stage after insemination in vitro.Hum Reprod. 1994; 9: 697-708PubMed Google Scholar, 58Vincent C. Pickering S.J. Johnson M.H. The hardening effect of dimethylsulphoxide on the mouse zona pellucida requires the presence of an oocyte and is associated with a reduction in the number of cortical granules present.J Reprod Fertil. 1990; 89: 253-259Crossref PubMed Google Scholar). Intracytoplasmic sperm injection is also the preferred conception method for treating cases scheduled for preimplantation genetic diagnosis (59Handyside A.H. Kontogianni E.H. Hardy K. Winston R.M. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification.Nature. 1990; 344: 768-770Crossref PubMed Scopus (621) Google Scholar). It avoids sperm DNA zona contamination and by enhancing the number of fertilized oocytes clearly increases the number of embryos available for genetic screening. Further, it has been instrumental in preimplantation genetic screening, particularly for polar body biopsy (60Verlinsky Y. Kuliev A. Preimplantation polar body diagnosis.Biochem Mol Med. 1996; 58: 13-17Crossref PubMed Scopus (17) Google Scholar, 61Wilton L. Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: a review.Prenat Diagn. 2002; 22: 512-518Crossref PubMed Scopus (80) Google Scholar). Finally, because a single spermatozoon is used, ICSI has allowed treatment of men who are virtually azoospermic (also defined as cryptozoospermic) (62Bendikson K.A. Neri Q.V. Takeuchi T. Toschi M. Schlegel P.N. Rosenwaks Z. et al.The outcome of intracytoplasmic sperm injection using occasional spermatozoa in the ejaculate of men with spermatogenic failure.J Urol. 2008; 180: 1060-1064Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). This scenario has fueled attempts to inject immature spermatozoa or germ cells (63Edwards R.G. Tarin J.J. Dean N. Hirsch A. Tan S.L. Are spermatid injections into human oocytes now mandatory?.Hum Reprod. 1994; 9: 2217-2219PubMed Google Scholar, 64Fishel S. Green S. Bishop M. Thornton S. Hunter A. Fleming S. et al.Pregnancy after intracytoplasmic injection of spermatid.Lancet. 1995; 345: 1641-1642Abstract Full Text PDF PubMed Google Scholar, 65Tesarik J. Mendoza C. Testart J. Viable embryos from injection of round spermatids into oocytes.N Engl J Med. 1995; 333: 525Crossref PubMed Scopus (5) Google Scholar, 66Tsai M.C. Takeuchi T. Bedford J.M. Reis M.M. Rosenwaks Z. Palermo G.D. Alternative sources of gametes: reality or science fiction?.Hum Reprod. 2000; 15: 988-998Crossref PubMed Google Scholar). However, once an ICSI conception is established, embryo implantation rates" @default.
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• W1973249625 title "Development and current applications of assisted fertilization" @default.
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