Development Of Intracytoplasmic Sperm Injection Research Articles

Artificial oocyte activation with Ca2+ ionophore improves reproductive outcomes in patients with fertilization failure and poor embryo development in previous ICSI cycles.

Does artificial oocyte activation (AOA) by a calcium ionophore (ionomycin) improve the previous fertilization failure or poor embryo development of intracytoplasmic sperm injection (ICSI) account for male factor infertility or other infertility causes? This retrospective study involved 114 patients receiving ICSI-AOA in Shanghai First Maternity and Infant Hospital with previous ICSI fertilization failure or poor embryo development. The previous ICSI cycles of the same patients without AOA served as the control group. The fertilization rates, cleavage rates, transferable embryo rates and blastocyst formation rates of the two groups were compared. Additionally, the clinical pregnancy, implantation rate and live birth rates were also compared to assess the efficiency and safety of AOA. Furthermore, two subgroup analyses were performed in this study based on the cause of infertility and the reason for AOA. The fertilization rate, embryonic development potential and clinical outcome were compared among groups. Among 114 ICSI-AOA cycles, the fertilization rate, top-quality embryo rate, implantation rate, clinical pregnancy per patient and live birth rate per patient were improved significantly compared with previous ICSI cycles (p<0.05 to P< 0.001), and the miscarriage rate in the AOA group was significantly lower than that of the control group (p<0.001). In the AOA subgroups based on the cause of infertility, the fertilization rates of each subgroup were significantly improved compared with previous control cycles except for the mixed factor infertility subgroup (p<0.05 to p<0.001). In the AOA subgroups based on the reason for AOA, the fertilization rates of each subgroup were significantly increased compared with those in their previous ICSI cycle without AOA (p<0.001); however, there was no significant difference in the top-quality embryo rate. No significant improvement was found in the implantation rates and the clinical pregnancy rate in each subgroup except for the poor embryo development subgroup. In the 114 AOA cycles, 35 healthy infants (21 singletons and 7 twins) were delivered without major congenital birth defects or malformations. This study showed that AOA with the calcium ionophore ionomycin can improve the reproductive outcomes of patients with previous fertilization failure and poor embryo development after ICSI.

Fertility preservation in women: where we are now and the path we need to take

Background: In its short history, reproductive medicine has achieved many fertility-related milestones, including: the arrival of in vitro fertilization in the late 1970s, the development of intracytoplasmic sperm injection in the early 1990s, the first ovarian transplant a decade ago, and the first livebirth after uterine transplantation in 2014. This paper provides a brief overview of the indications and methods, as well as future perspectives, available for fertility preservation.Current Concepts: More women are undergoing fertility preservation for medical or social reasons. Fertility preservation aids cancer patients in retaining the ability to procreate. Despite the high survival rate of malignancies in young patients, chemotherapy and whole abdominal irradiation have cytotoxic effects on reproductive organs. Cancer treatments can significantly reduce a patient’s reproductive capacity and thus result in irreversible infertility. Early ovarian failure is also a common by-product of additional cancer treatment, bone marrow transplantation, or autologous transplantation. The current options for fertility preservation in cancer patients include cryopreservation (of embryos, oocytes, and ovarian tissue) and gonadotropic-releasing hormone agonist treatment (before and during chemotherapy). Fertility preservation is a means for female cancer patients to preserve their fertility and delay childbearing. Fertility preservation can also aid women who wish to delay childbearing for personal reasons. Since the indications for fertility preservation are different for each patient, individualized treatment strategies should be employed depending on the patient’s situation.Discussion and Conclusion: Health professionals must inform women who are undergoing cancer treatments or purposefully delaying childbearing about the risks of decreased fertility. Appropriate fertility preservation options must be provided for these female patients.

Severe male factor in in vitro fertilization: definition, prevalence, and treatment. An update.

Infertility affects 10%–15% of couples worldwide. Of all infertility cases, 20%–70% are due to male factors. In the past, men with severe male factor (SMF) were considered sterile. Nevertheless, the development of intracytoplasmic sperm injection (ICSI) drastically modified this scenario. The advances in assisted reproductive technology (ART), specifically regarding surgical sperm retrieval procedures, allowed the efficacious treatment of these conditions. Yet, before undergoing ICSI, male factor infertility requires careful evaluation of clinical and lifestyle behavior together with medical treatment. Epidemiologically speaking, women whose male partner is azoospermic tend to be younger and with a better ovarian reserve. These couples, in fact, are proposed ART earlier in their life, and for this reason, their ovarian response after stimulation is generally good. Furthermore, in younger couples, azoospermia can be partially compensated by the efficient ovarian response, resulting in an acceptable fertility rate following in vitro fertilization (IVF) techniques. Conversely, when azoospermia is associated with a reduced ovarian reserve and/or advanced maternal age, the treatment becomes more challenging, with a consequent reduction in IVF outcomes. Nonetheless, azoospermia seems to impair neither the euploidy rate at the blastocyst stage nor the implantation of euploid blastocysts. Based on the current knowledge, the assessment of male infertility factors should involve: (1) evaluation – to diagnose and quantify seminologic alterations; (2) potentiality – to determine the real possibilities to improve sperm parameters and/or retrieve spermatozoa; (3) time – to consider the available “treatment window”, based on maternal age and ovarian reserve. This review represents an update of the definition, prevalence, causes, and treatment of SMF in a modern ART clinic.

Diagnosis and Treatment of Male Infertility-Related Fertilization Failure.

Infertility affects approximately 15% of reproductive-aged couples worldwide, of which up to 30% of the cases are caused by male factors alone. The origin of male infertility is mostly attributed to sperm abnormalities, of which many are caused by genetic defects. The development of intracytoplasmic sperm injection (ICSI) has helped to circumvent most male infertility conditions. However, there is still a challenging group of infertile males whose sperm, although having normal sperm parameters, are unable to activate the oocyte, even after ICSI treatment. While ICSI generally allows fertilization rates of 70 to 80%, total fertilization failure (FF) still occurs in 1 to 3% of ICSI cycles. Phospholipase C zeta (PLCζ) has been demonstrated to be a critical sperm oocyte activating factor (SOAF) and the absence, reduced, or altered forms of PLCζ have been shown to cause male infertility-related FF. The purpose of this review is to (i) summarize the current knowledge on PLCζ as the critical sperm factor for successful fertilization, as well as to discuss the existence of alternative sperm-induced oocyte activation mechanisms, (ii) describe the diagnostic tests available to determine the cause of FF, and (iii) summarize the beneficial effect of assisted oocyte activation (AOA) to overcome FF.

In Vitro Culture of Embryos from Horses.

Establishment of optimal methods for equine embryo culture has been slow when compared to some domestic species. In part, this delay was caused by the failure of standard in vitro fertilization techniques in horses. However, the development of intracytoplasmic sperm injection (ICSI) for the assisted fertilization of equine oocytes has resulted in a renewed interest in establishing optimal methods for embryo culture. Currently, ICSI-produced equine embryos are cultured using media designed for other species or other cell cultures and, typically, with the addition of serum. Although systems specifically for horse embryo culture still have not been established, ICSI-produced embryos are developmentally competent and capable of producing live offspring.

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.

Are children born from singleton pregnancies conceived by ICSI at increased risk for congenital malformations when compared to children conceived naturally? A systematic review and meta-analysis.

Since 1992, the development of intra-cytoplasmic sperm injection (ICSI) has allowed infertile couples and couples affected by severe male factor infertility in particular, many of which with a history of failed traditional IVF, to become parents. This has generated considerable controversy over the safety of the procedure for the offspring. This systematic review seeks to determine whether evidence indicates that the use of ICSI increases the risk of congenital malformation in children born from singleton pregnancies versus naturally conceived children. Twenty-one of the 104 publications listed in the literature search were included in the analysis. Observational studies reported mostly an increased risk for congenital malformation; the risk of congenital malformations is 7.1% in ICSI and 4.0% in the general population (OR 1.99 (95% CI [1.87 - 2.11]). However, attributing higher risk solely to ICSI might seem far-fetched, as in vitro and simulation procedures, patient diseases, and ICSI indication may also be associated with higher risk of malformation.

Effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection in mouse mature oocytes

Better pregnancy outcomes can be obtained by human mature oocyte vitrification, but many problems remain to be resolved in human mature oocyte vitrification. Since mature oocyte development possesses its own maturity cycle, there should be the optimal timing for mature oocyte vitrification. The purpose of this study was to observe the effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection (ICSI) in vitrified mouse mature oocytes and explore its possible mechanism. Mouse oocytes were randomly divided into three groups according to different frozen timing including Groups A, B, and C in which oocytes were vitrified within 2 h after ovum pick-up, and 3-4 and 5-6 h after ovum pick-up, respectively. Spindle-related parameters were measured, ICSI was performed. The spindle occurrence rate of vitrified-thawed oocytes was 98.4% in Group A, 82.3% in Group B, and 75.8% in Group C, without statistical differences between pre-vitrification and post-thawing and among the three groups (P > 0.05). The angles between the polar body and spindle were larger after thawing than before vitrification (P < 0.01). The spindle retardance values were lower after thawing than before vitrification in Groups B and C (P < 0.05), but higher in Group A (P < 0.05). The spindle retardance values before vitrification were higher in Group B than in Groups A and C (P < 0.05), but the spindle retardance value, oocyte survival and two-cell rate after thawing were higher in Group A than in Groups B and C (P < 0.05). There were no statistical differences in ICSI fertility rate between the three groups (P > 0.05). The damage on the spindle is the slightest and embryo quality is the highest in the mouse oocytes vitrified within 2 h after ovum pick-up. The spindle retardance value is more valuable than the spindle occurrence rate in the evaluation of vitrified-thawed oocyte quality, and is positively correlated with embryo quality.

Is ICSI Risky?

As assisted reproductive technology (ART) methods become the mainstream of infertility treatment, it has become even more critical to reassess its safety. Following the results of a study published by the Robinson Institute in the New England Journal of Medicine, the risk of ART, especially intracytoplasmic sperm injection (ICSI), has never been so closely scrutinized. This paper traces the origins and development of ICSI, assesses the risks documented in the literature, and finally interprets the implications of the study for couples contemplating therapy. We support the need for continued vigilance towards ICSI and the importance in investigating male-factor infertility as a prequel to its use.

277 FERTILIZATION CAPABILITY OF BOAR SPERMATOZOA AFTER ALTERNATIVE SEMEN PRESERVATION METHODS AND INTRACYTOPLASMIC INJECTION INTO PORCINE OOCYTES

Long-term preservation of mammalian spermatozoa is regarded as being essential for the conservation of endangered species and for assisted reproduction. Although the method generally applied for semen preservation is storage at –196°C, liquid nitrogen and special equipment might not be available in every situation. Additionally, logistical difficulties make cryopreservation difficult under extreme conditions, such as in the wilderness. Fortunately, with the development of intracytoplasmic sperm injection (ICSI), preserved spermatozoa do not need to maintain their motility. The aim of the study was to investigate alternative preservation methods for spermatozoa without freezing in liquid nitrogen and to examine the effects of the preserved sperm on fertilization after injection into in vitro-matured pig oocytes. The pig was chosen as the model for investigations, and the alternative preservation methods included heat-drying, flame-drying, or freezing (–20°C, household refrigerator) of spermatozoa. After semen collection and swim up, sperm concentrations were adjusted to 0.5 to 1 × 105 cells mL–1. For heat-drying, aliquots of sperm suspension (50 µL) were dehumidified at 50, 56, or 90°C each for 45 min, or at 120°C for 20 min. Flame-drying was performed by flaming 5 µL of semen suspension or sperm-rich fraction for up to 2 s on glass slides. Dried semen samples were stored at 4°C. Further, sperm samples were frozen (100 µL/tube) without cryoprotectant and kept at –20°C. The specimens were stored up to 5 days. Before ICSI, heat-dried and flame-dried samples were rehydrated with 50 and 10 µL of ultrapure water (Millipore®, Millipore Corp., Billerica, MA, USA), respectively. Frozen sperm were thawed for 10 min at room temperature. Afterward, spermatozoa were injected into in vitro-matured pig oocytes at metaphase II, and sperm-injected oocytes were activated artificially by 10% ethanol. Subsequently, in vitro culture in mTCM-199® [20 µg mL–1 of insulin, 0.08 mg mL–1 of l-glutamine, 50 µg mL–1 of gentamicin, and 20% FCS (vol/vol)] with 10 µg mL–1 of cycloheximide was performed for 24 h. Afterward, oocytes were fixed, stained (aceto-orcein staining), and investigated for signs of fertilization. Positive fertilization criteria included extrusion of the second polar body, formation of two pronuclei, and the presence of a visible sperm tail. The fertilization ability of heat-dried samples achieved 9.0% (6/67), 8.8% (6/68), 2.7% (2/75), and 0% (0/60) at 50, 56, and 90°C for 45 min and 120°C for 20 min, respectively. Flame-drying reached a fertilization rate of 14.0% (6/43) v. 4.4% (3/69) for the sperm-rich fraction v. swim-up spermatozoa, respectively. With frozen semen (–20°C), 9.4% (13/138) fertilized oocytes were obtained. These results imply that ICSI with spermatozoa, which were alternatively preserved at various temperatures and circumstances in combination with artificial oocyte activation, may fertilize the oocytes. The best results were accomplished with the flame-dried sperm-rich fraction.

Contemporary aspects of male gamete (dys)function: aiming towards optimizing the treatment of fertilization disorders

At its inception, in vitro fertilization (IVF) was a relatively straightforward procedure encompassing the in vitro insemination of surgically retrieved oocytes and the trans-cervical uterine placement of the ensuing embryos with the goal of overcoming tubal infertility. Since that time, IVF has become the ultimate treatment modality for all infertility indications when standard treatments have failed. While IVF has revolutionized our clinical armamentarium, it has also served as a remarkable scientific tool allowing direct visualization of male and female gamete interaction and assessment of human embryo development, thus enabling reproductive specialists to dissect and better understand the early human reproductive processes. In its earliest stages, IVF was directed at the treatment of female infertility focusing on the role of the oocyte, later to be eclipsed by assisted fertilization techniques that revolutionized the treatment of male factor infertility. The advent of assisted fertilization techniques, from the most simplistic approaches to direct sperm injection, has made it possible for suboptimal male gametes to penetrate the oocyte vestments. The latter technique has shed light on the ultimate function of the spermatozoon and the fascinating process of syngamy. Moreover, the ability to temporally monitor the cleaving embryo in vitro has enabled us to ascertain the individual steps involved in the development of the conceptus from syngamy to blastocyst as well as to shed light on hitherto unapproachable infertility etiologies. After over 30 years of IVF and 20 years of intracytoplasmic sperm injection (ICSI), it seems appropriate to focus on our present understanding and the state of scientific knowledge of the male gamete. The present issue of Views and Reviews will describe the history and development of ICSI — the ultimate technique to treat male infertility — and review the current indications for assisted fertilization in terms of therapeutic outcome, safety, and future perspectives (1Palermo G.D. Neri Q.V. Monahan D. Kocent J. Rosenwaks Z. Development and current applications of assisted fertilization.Fertil Steril. 2012; 97: 248-259Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Other contributions will describe the currently available assays to screen for male factor infertility and sperm function. Although the semen analysis has been utilized for several decades as the cornerstone for assessing male fertility (dys)function, interpretation and clinical correlations still remain a challenge (2De Jonge C. Semen Analysis: Looking for an upgrade in class.Fertil Steril. 2012; 97: 260-266Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The recent understanding of the sperm epigenetic status in regards to protamination, specific localization/modifications of retained histones, and DNA methylation may delineate the role of the male gamete on embryogenesis and eventually in the establishment of totipotency of the embryo. This may help to unravel hitherto undiagnosable causes of impaired fertility and possibly provide insights into transmission of genetic risks (3Carrell D.T. Epigenetics of the male gamete.Fertil Steril. 2012; 97: 267-274Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). In fact, it has been documented that the nucleus of mature spermatozoa contains a complex population of RNAs that are transcriptionally inert. These small molecules which appear to orchestrate extraordinary spermiogenic modifications, may also aid in discriminating and assessing spermatozoal function that may serve as investigational and diagnostic tools for male infertility (4Hamatani T. Human spermatozoal RNAs.Fertil Steril. 2012; 97: 275-281Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). It has become evident that the role of the spermatozoon as a simple mobile device to deliver the male genome should be reconsidered and reinterpreted as a cell characterized by its unusual shape, extraordinary motility, resilience to environmental aggressors, and capable of its own reshaping during spermatogenesis. Upon reaching the oocyte, the spermatozoon triggers egg activation, orchestrates in a preprogrammed pattern peri- and post-fertilization events in the oocyte, thus, inevitably controlling early embryo development. Ideally, the utilization of novel diagnostic techniques will not only improve our abilities to assess sperm (dys)function, they will also allow us to discriminate the spermatozoon’s contribution to early embryo development.

Estrogenic compounds – endocrine disruptors

biphenyls, dichlorodiphenyl-trichloroethane [DDT], dioxin, and some pesticides) are estrogen-like and anti-androgenic chemicals in the environment. They mimic natural hormones, inhibit the action of hormones, or alter the normal regulatory function of the endocrine system and have potential hazardous effects on male reproductive axis causing infertility. Although testicular and prostate cancers, abnormal sexual development, undescended testis, chronic inflammation, Sertoli-cell-only pattern, hypospadias, altered pituitary and thyroid gland functions are also observed, the available data are insufficient to deduce worldwide conclusions. [1] The development of intra-cytoplasmic sperm injection (ICSI) is beyond doubt the most important recent breakthrough in the treatment of male infertility, but it does not necessarily treat the cause and may inadvertently pass on adverse genetic consequences. Many well-controlled clinical studies and basic scientific discoveries in the physiology, biochemistry, and molecular and cellular biology of the male reproductive system have helped in the identification of greater numbers of men with male factor problems. [1] Newer tools for the detection of Y

Surgical recovery of sperm in non-obstructive azoospermia

The development of intracytoplasmic sperm injection (ICSI) opened a new era in the field of assisted reproduction and revolutionized the assisted reproductive technology protocols for couples with male factor infertility. Fertilisation and pregnancies can be achieved with spermatozoa recovered not only from the ejaculate but also from the seminiferous tubules. The most common methods for retrieving testicular sperm in non-obstructive azoospermia (NOA) are testicular sperm aspiration (TESA: needle/fine needle aspiration) and open testicular biopsy (testicular sperm extraction: TESE). The optimal technique for sperm extraction should be minimally invasive and avoid destruction of testicular function, without compromising the chance to retrieve adequate numbers of spermatozoa to perform ICSI. Microdissection TESE (micro-TESE), performed with an operative microscope, is widely considered to be the best method for sperm retrieval in NOA, as larger and opaque tubules, presumably with active spermatogenesis, can be directly identified, resulting in higher spermatozoa retrieval rates with minimal tissue loss and low postoperative complications. Micro-TESE, in combination with ICSI, is applicable in all cases of NOA, including Klinefelter syndrome (KS). The outcomes of surgical sperm retrieval, primarily in NOA patients with elevated serum follicle-stimulating hormone (FSH) (NOA including KS patients), are reviewed along with the phenotypic features. The predictive factors for surgical sperm retrieval and outcomes of treatment were analysed. Finally, the short- and long-term complications in micro-TESE in both 46XY males with NOA and KS patients are considered.

Contemporary Management of Male Infertility

Knowledge of normal male reproductive function and familiarity with the diagnostic evaluation and treatment of male subfertility is beneficial for most physicians. Male subfertility is often correctable, may be genetically transmissible, and may be associated with occult health-threatening conditions. Herein we present an overview of male reproductive medicine, which has been revolutionized in the past two decades by dramatic scientific and therapeutic advances. The development of intracytoplasmic sperm injection and its successful application to sperm retrieved from the epididymis or testis have made biological paternity possible in men previously considered sterile. Microsurgical techniques for vasal-epididymal reconstruction and sperm retrieval have been refined. Novel tests of semen quality have been developed. Medical therapies to improve sperm production, such as estrogen receptor modulation and aromatase inhibition, have been used increasingly in clinical practice. Finally, associations between male subfertility and a spectrum of health-threatening conditions have been recognized.

¿Es la inyección intracitoplásmica de espermatozoides morfológicamente seleccionados una técnica con futuro?

Since its beginnings, assisted reproduction techniques (ART) have evolved considerably. The development of ICSI (intracytoplasmic sperm injection) made possible the treatment of many cases of male sterility. The morphologic observation of oocytes and embryos and their classification and selection resulted in an increase in the pregnancy rates.In the present a new technique has been developed for the observation of sperm under the microscope with high magnification, allowing a better selection of the sperm cells used for microinjection. The introduction of this technique called IMSI (intracytoplasmic morphologically selected sperm injection) has resulted in higher pregnancy rates and reduced abortion rates.

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.

271 THE EFFECT OF TEMPERATURE ON HUMAN TESTICULAR TISSUE TO OPTIMIZE THE IN VITRO MATURATION OF PRE-FREEZE MOTILITY

The development of intracytoplasmic sperm injection has made the use of testicular sperm a viable option for infertile men with obstructive and nonobstructive azoospermia. Over the past decade, testicular biopsies have been handled and processed using a variety of different methods. Whole biopsy pieces can be effectively cryopreserved in a 10% glycerol diluent (Schiewe et al. 1997 67, S115 abst); however, the ability to find viable, motile sperm post-thaw is improved when prefreeze motility exists. The purpose of this study was to comparatively document in vitro sperm motility enhancement over time at different temperatures, and to prove that an intermediate temperature (28 to 30°C) would optimize sperm longevity for up to 1 week. In this study, 10 men with obstructive azoospermia underwent a surgical, open testicular biopsy procedure. Each biopsy was placed in HEPES buffered-human tubal fluid (mHTF) medium supplemented with 5% human serum albumin (HSA; Irvine Sci., Santa Ana, CA, USA) and transported to the laboratory at room temperature. Each testis biopsy (TBx) was dissected into 8 equal pieces (approximately 2 × 2 × 1 mm). Five intact pieces of TBx were cryopreserved in separate cryovials for future use. The remaining TBx tissue was subdivided into 1 of 3 temperature treatment groups (24, 30, or 37°C) for extended IVM. The 30°C incubation condition was achieved by placing the dish(es) in a Styrofoam box placed on a 37°C warming plate. Each TBx was placed into a separate 100 × 35 mm Falcon dish in 150-μL droplets of mHTF under oil and shredded by needle dissection to disperse the contents of the seminiferous tubules. Reminant tissue was placed into another droplet for additional dissection, as needed. Sperm were analyzed for motility (graded as Type I = twitching, II = undulating, III = slow progression, and IV = rapid progression) at 0, 24, 96, and 144 h without replacing the IVM medium. The percentage increase in motility, compared with 0 h, was statistically contrasted across temperature treatments by chi-squared analysis. Testicular sperm motility ranged from 5 to 25% at 0 h and increased in all groups at 24 h. There was no difference in total motility at 24 h, but progressive motility was higher (P &lt; 0.05) at 37°C compared with 24°C. Significant differences (P &lt; 0.05) were observed in total percentage of motility/percentage of progressive motility at 96 h with treatment differences (*) being 30°C (66%*/44%*) &gt; 24°C (42%*/14%) &gt; 37°C (18%*/10%). This statistical trend continued at 144 h with 30°C (42%*/23%*) &gt; 24°C (24%*/8%) &gt; 37°C (9%*/5%). During this study, a viable pregnancy was achieved using a 30°C sample 8 days post biopsy, exceeding 2 previous healthy triplet pregnancies, which were successful using 4-day-old TBx specimens in 1996 and 2004. This study confirms that an intermediate IVM temperature of 30°C is optimal to enhance TBx cryopreservation, which in our laboratory is generally performed at 24 to 72 h of IVM. Our routine pregnancy success in applying ICSI with IVM, thawed TBx sperm discounts the concern some individuals express regarding DNA fragmentation of sperm over time.

Endocrine disruptors and estrogenic effects on male reproductive axis

Endocrine disruptors (e.g., polychlorinated biphenyls [PCBs], dichlorodiphenyl-trichloroethane [DDT], dioxin, and some pesticides) are estrogen-like and anti-androgenic chemicals in the environment. They mimic natural hormones, inhibit the action of hormones, or alter the normal regulatory function of the endocrine system and have potential hazardous effects on male reproductive axis causing infertility. Although testicular and prostate cancers, abnormal sexual development, undescended testis, chronic inflammation, Sertoli-cell-only pattern, hypospadias, altered pituitary and thyroid gland functions are also observed, the available data are insufficient to deduce worldwide conclusions. The development of intra-cytoplasmic sperm injection (ICSI) is beyond doubt the most important recent breakthrough in the treatment of male infertility, but it does not necessarily treat the cause and may inadvertently pass on adverse genetic consequences. Many well-controlled clinical studies and basic scientific discoveries in the physiology, biochemistry, and molecular and cellular biology of the male reproductive system have helped in the identification of greater numbers of men with male factor problems. Newer tools for the detection of Y-chromosome deletions have further strengthened the hypothesis that the decline in male reproductive health and fertility may be related to the presence of certain toxic chemicals in the environment. Thus the etiology, diagnosis, and treatment of male factor infertility remain a real challenge. Clinicians should always attempt to identify the etiology of a possible testicular toxicity, assess the degree of risk to the patient being evaluated for infertility, and initiate a plan to control and prevent exposure to others once an association between occupation/toxicant and infertility has been established.

The effects of age on the incidence of aneuploidy rates in spermatozoa of oligoasthenozoospermic patients and its relationship with ICSI outcome

The development of intracytoplasmic sperm injection (ICSI) for treatment of infertility as a result of severe male factor has improved the chances of achieving pregnancy in many infertile couples. However, concerns have been raised regarding the safety of this technique, because natural sperm selection is bypassed. In the present study, 25 oligoasthenozoospermic patients who were divided into two groups according to age: group A, 20-34 (n = 10) and group B, 35-50 (n = 15), were included. Pooling the data of the three semen parameters that were tested (volume, concentration and progressive motility) no statistically significant difference between the two age groups was found. A total of 50 883 decondensed spermatozoa was analysed using the dual and triple colour fluorescence in situ hybridization to estimate the rates of aneuploidy for chromosomes 13, 18, 21, X and Y in the two age groups. There was a significantly higher incidence of disomy for chromosome 21 compared to the other autosomes (chromosomes 13 and 18) in both age groups. The disomy rate of XY was significantly higher in the younger subject group (0.1%) compared to the older group (0.05%, p < 0.05). Statistically significant differences in the mean number of clinical pregnancies and abortions were not observed between the two age groups. The aneuploidy rates for all the analysed chromosomes did not differ significantly, both between and within the two age groups, and as a result there seems to be no effect of male age on chromosome numbers in the spermatozoa and on the ICSI outcome.

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos.

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 degrees C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p < 0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p < 0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p > 0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p < 0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p > 0.05) among methods 1, 3 and 4, but method 2 produced a higher (p < 0.05) blastocyst rate than method 3.