Development of ICSI.

The first conception outside of the human body that led to the birth of Louise Brown was a tremendous accomplishment, which opened the door to the utilization of assisted reproductive techniques globally. This brought the understanding that accomplishing life in a dish required several steps, the most obvious being the timing and characteristics of fertilization. It soon became obvious in the 1980s that the most disappointing phenomenon was unexpected and complete fertilization failure. Among the approaches that were attempted to treat male factor infertility, ICSI surfaced as the technique that brought the ratio of the gametes to 1:1 and was also able to grant consistent fertilization and a higher pregnancy rate. ICSI has now been implemented for a quarter of a century, proving itself as the ultimate technique utilizing ejaculated spermatozoa independent of the semen parameters and is the sole insemination method to be used with surgically retrieved spermatozoa. There are currently various indications for ICSI that are widely adopted, rendering it the most popular insemination method worldwide. The reliability of ICSI ensures its employment in upcoming techniques involving in vitro spermatogenesis and neogametogenesis.

A treatment algorithm for couples with unexplained infertility based on sperm chromatin assessment.

OBJECTIVE: To design a reproductive treatment algorithm based on the sperm DNA fragmentation (SDF) for couples with unexplained infertility following a poor intrauterine insemination (IUI) outcome. DESIGN: Couples that failed IUI with no apparent reproductive issue in both partners were allocated to diverse reproductive treatments on the basis of SDF. SETTING: Reproductive medical center in an academic setting. PATIENT(S): Over 4 years, couples with an unexpected poor IUI outcome and no apparent female or male partner reproductive issues were recruited. INTERVENTION(S): IUI, IVF, and ICSI were performed in the standard fashion following sperm SDF assays. MAIN OUTCOMES MEASURE(S): Fertilization rate, implantation rate, pregnancy characteristics, and delivery rates. RESULT(S): A total of 354 couples with unexplained infertility and normal semen parameters underwent 1133 IUI cycles. Clinical pregnancy rate (CPR) with IUI at our center in an age-matched cohort is 23.9% while the study cohort had 1.8%. Following SDF assessment, couples with failed IUI attempts but normal SDF (SCSA 9.8 ± 4.6%; TUNEL 11.8 ± 6.2%) underwent IVF with a CPR of 12.7%; those with abnormal SDF underwent ICSI with ejaculated spermatozoa, resulting in a CPR of 18.7%. This group included couples with normal SDF that had failed IVF. Couples with abnormal SDF that failed ICSI with ejaculated spermatozoa achieved a CPR of 31.0% with surgically retrieved spermatozoa. CONCLUSION(S): Couples with unexplained infertility that present with unexpectedly poor IUI outcomes can be funneled into a treatment algorithm guided by the integrity of the sperm genome for higher chances of pregnancy using an alternate method of insemination.

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.

Treatment options for impaired spermatogenesis: germ cell transplantation and stem-cell based therapy.

Advances in infertility treatment had the most extraordinary breakthrough with the birth of the first in vitro fertilization baby in 1978. Fourteen years later, intracytoplasmic sperm injection has been introduced for the treatment of male factor infertility. Intra cytoplasmic sperm injection in combination with testicular sperm extraction has allowed men with azoospermia to father children. In fact, as long as a fully developed spermatozoon is identified, it can be utilized or can even be duplicated to inseminate several oocytes while providing information on its genomic content. There are, however, men who are suffering from spermatogenic arrest, where no post-meiotic germ cells are retrieved, and therefore, unable to generate their own offspring. More recently, the successful isolation and cultivation of spermatogonial stem cells has allowed the exploration of their biological characteristics and their application in therapeutic approaches following transplantation or in vitro maturation. Finally, men diagnosed with germ cell aplasia can only be treated by donor or de novo generated gametes. In the past several years, we have attempted to manufacture gametes by inducing haploidization of somatic cells and more recently, generating sperm-like cells through embryonic stem cell differentiation.

Surgical excision of Essure® devices with ESHRE Class IIb uterine malformation: sequential hysteroscopic-laparoscopic approach to the septate uterus.

OBJECTIVE: While contraindications to Essure® placement have been provided by the manufacturer, there is no consensus on how best to remove these contraceptive devices. Here, we describe a non-hysterectomy removal of Essure® for a patient with a septate uterus (ESHRE Class IIb uterine malformation). CLINICAL CASE: A 35yr old G4 P2 presented for removal of Essure® implants after three years of gradually increasing pelvic pain, weight gain, headache, dizziness, lower extremity paresthesia, and fatigue which followed hysteroscopic sterilization (HS). Prior to HS, the patient was in good general health. She did not smoke and had never had a miscarriage. HS was performed under general anesthesia in October 2012. HSG obtained three months later, confirmed bilateral tubal occlusion but revealed an abnormal uterine cavity. A repeat HSG in 2015 showed minimal device migration, no contrast dye spill and a deeply bifid uterine cavity. At our center laparoscopic cornual dissection and bilateral partial tubal resection achieved removal of both devices intact and the patient was discharged three hours after surgery. Her postoperative recovery was uneventful. CONCLUSION: The presence of a Müllerian anomaly is a relative contraindication to the Essure® procedure. This is the first reported description of successful removal of Essure® coils in the setting of an ESHRE Class IIb uterine anomaly, and underscores the importance of careful patient selection, accurate pre-operative imaging and a conservative technique which renders hysterectomy unnecessary.

ICSI and its outcome.

Since its introduction in 1992, intracytoplasmic sperm injection (ICSI) has become a popular assisted fertilization technique proved very efficient in treating male factor infertility. Many healthy children have been born worldwide from this procedure, and their physical and mental development appears to be within the normal limits. However, because of the peculiarity of the technique and the poor characteristics of the spermatozoa used, concern about the safety of ICSI still exist. In this article, we analyze the in vivo development of embryos conceived after ICSI as well as the obstetric outcome, occurrence of chromosomal abnormalities, and rate of congenital malformations in neonates born as a result of this treatment. A total of 2435 couples were studied in whom the male partners were presumed to be the cause of repeated failed attempts at in vitro fertilization (IVF) or had semen parameters that were unacceptable for conventional IVF treatment. Pregnancies resulting from 3573 ICSI cycles were analyzed; pregnancy outcome data were obtained from the records of obstetrician-gynecologists and/or pediatricians. The overall clinical pregnancy (fetal heartbeat) rate was 44.8% with a resultant delivery rate of 39.2% per ICSI cycle (n = 1388). In 37 of the 77 miscarriages for which cytogenetic data were available, an autosomal trisomy was found in each and 29 additional pregnancies were terminated because of a chromosomal abnormality revealed by prenatal diagnosis. There was an equal distribution of vaginal deliveries and cesarean sections (n = 682 and n = 658, respectively). Of the 2059 neonates resulting from ICSI treatment, 38 (1.8%) presented with congenital abnormalities (22 major and 16 minor). When the frequency of miscarriages and congenital malformations was analyzed in terms of semen origin, the outcome was no different between ICSI and IVF. The course of pregnancies and occurrence of congenital malformations following treatment by ICSI are within the ranges obtained following conventional IVF.

Micropuncture retrieval of epididymal sperm with in vitro fertilization: importance of in vitro micromanipulation techniques.

OBJECTIVES: To evaluate the importance of in vitro micromanipulation techniques, specifically intracytoplasmic sperm injection (ICSI), for the fertility treatment of men with congenital absence of the vas deferens (CAV) or other unreconstructable male reproductive tract obstruction. METHODS: Results using ICSI during in vitro fertilization (IVF) were compared to previously published results of IVF alone and IVF with other micromanipulation techniques at the same infertility center. Main outcome parameters evaluated were: fertilization rate per oocyte, clinical pregnancy rate, and ongoing pregnancies and deliveries. RESULTS: IVF with ICSI yielded a fertilization rate per oocyte of 140 of 312 (45%) and a clinical pregnancy rate of 14 of 27 (52%) per cycle of sperm and egg retrieval. Ongoing pregnancies or deliveries have occurred for 13 of 27 (48%) cycles with ICSI. These results were better than our previously published results of IVF alone or in conjunction with the micromanipulation techniques of subzonal insertion (SuZI) or partial zona dissection (PZD) that yielded a 119 of 631 (19%; P < 0.0001) fertilization rate, clinical pregnancy rate of 14 of 51 (27%; P < 0.001) and ongoing pregnancy or delivery for 12 of 51 cycles (24%; P < 0.001). CONCLUSIONS: Epididymal sperm retrieval should be performed only when micromanipulation is available in conjunction with IVF to maximize chances of fertilization and subsequent pregnancies. The use of ICSI for epididymal sperm appears to maximize chances of pregnancy for couples with surgically unreconstructable obstructive male infertility.

Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia.

OBJECTIVES: To provide fertility for men with nonobstructive azoospermia. METHODS: A retrospective review of treatment results at a university infertility center was undertaken. Sixteen couples entered an attempted in vitro fertilization (IVF)-intracytoplasmic sperm injection (ICSI) cycle for treatment of nonobstructive azoospermia. Each man was azoospermic, and the male factor diagnosis of nonobstructive azoospermia was made on testis biopsy for 14 men and on clinical grounds for 2 men. Sperm were retrieved by testicular biopsy on the day of oocyte retrieval. Results of testicular examinations, serum follicle-stimulating hormone levels, and testicular histology as well as evaluation of the success rates of sperm retrieval, fertilizations, and pregnancies were made. RESULTS: Sperm were extracted from testis biopsies in 10 of 16 (62%) testicular sperm extraction (TESE) attempts. For cycles in which sperm were retrieved, normal fertilizations were achieved for 51 of 98 (52%) mature oocytes injected with testicular sperm in 10 couples. Biochemical pregnancies were achieved for 6 of 16 (38%) couples, with clinical pregnancies during 5 of 16 (31%) attempts at sperm retrieval, and ongoing pregnancy and subsequent live delivery for 4 of 16 (25%) attempts. CONCLUSIONS; Pretreatment clinical parameters are unable to predict which men with nonobstructive azoospermia will have spermatozoa retrieved by TESE. When sperm are found, clinical pregnancies can occur for half (5/10) of these couples using TESE with ICSI, with ongoing pregnancy and delivery for 4 of 10 (40%). Many men with nonobstructive azoospermia will have retrievable sperm with testis biopsy that are suitable for ICSI; however, 6 of 16 (38%) couples will not have sperm retrieved with TESE and may undergo an unnecessary IVF procedure.

Intracytoplasmic sperm injection: a powerful tool to overcome fertilization failure.

OBJECTIVE: To describe the novel micromanipulation technique known as intracytoplasmic sperm injection (ICSI), which has been applied successfully to treat male factor infertility, even in patients with severely impaired sperm characteristics. This paper reviews the historical aspects that led to the development of ICSI in the animal model as well as the current experience in the human. DESIGN: Before using assisted fertilization techniques to enhance fertilization of human gametes, it is imperative that practitioners gain extensive experience in the animal model. In addition, criteria for accepting individuals for treatment with ICSI are discussed along with other applications of the procedure in infertile couples who do not benefit from standard IVF. RESULTS: Because ICSI resulted in limited success in animal models, it seemed unlikely that it would be successful in humans. Yet, ICSI now appears to be the most successful and significant innovation developed for dealing with male factor infertility since the emergence of IVF itself. To date, a relatively large group of healthy children have been born from this technology and there appears to be no increased incidence of congenital malformations. CONCLUSIONS: The consistently high success rate resulting from the application of ICSI to treat couples with male factor infertility is comparable to the results obtained using standard IVF techniques performed in nonmale factor couples. This finding indicates that spermatozoa obtained from subfertile men selected for intracytoplasmic injection are usually genotypically normal.

Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility.

OBJECTIVE: To demonstrate the effectiveness of intracytoplasmic sperm injection to treat male factor infertility and to report on the achievement of fertilization and pregnancy compared with standard in vitro insemination. DESIGN: Controlled clinical study. SETTING: Couples suffering from male factor infertility treated in an academic research environment. PATIENTS: Two hundred twenty-seven couples in whom the male partners were presumed to be the cause of repeated failed attempts at IVF or whose semen parameters were unacceptable for conventional IVF. INTERVENTIONS: Oocytes for this study were harvested after superovulation with GnRH agonist and gonadotropins. After removing the cumulus cells, a single spermatozoon was injected directly into the cytoplasm of metaphase II oocytes, and the outcome was analyzed in terms of semen parameters and origin of semen sample. MAIN OUTCOME MEASURES: Fertilization and pregnancy rates (PRs) in relation to sperm parameters and origin of semen sample. RESULTS: Two hundred twenty-seven couples were treated by intracytoplasmic sperm injection resulting in an ongoing pregnancy rate of 37.0% per retrieval (84/227). There were 47 singleton pregnancies (5 of which were vanishing twin pregnancies), 30 twin gestations, 6 triplet pregnancies, and 1 quadruplet pregnancy. The concentration of the total motile spermatozoa present in the ejaculate as well as the origin of the samples influenced the fertilization rate but not the pregnancy outcome. CONCLUSIONS: Intracytoplasmic sperm injection can be used successfully to treat couples who have failed IVF or who have too few spermatozoa for conventional methods of in vitro insemination. Sperm parameters do not clearly affect the outcome of this technique.

Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome.

OBJECTIVE: To evaluate the incidence of sperm aneuploidy in men screened for infertility and identify any eventual relation with assisted reproductive outcome. DESIGN: Controlled prospective study. SETTING: University hospital-based IVF program. PATIENT(S): Infertile couples who were screened for sperm aneuploidy and evaluated for IVF treatment. INTERVENTION(S): Fluorescence in situ hybridization was used to identify chromosomes 18, 21, X, and Y. The assisted reproductive techniques of IVF and intracytoplasmic sperm injection were used for infertility treatment. MAIN OUTCOME MEASURE(S): The incidence of sperm aneuploidy, semen parameters, fertilization rate, pregnancy characteristics, and rate of neonatal malformations were determined. RESULT(S): Oligozoospermic and teratozoospermic men had a significantly higher incidence of chromosomal abnormalities than men with normal semen parameters (2.7% vs. 1.8%). The increased frequency of sperm aneuploidy did not appear to affect pregnancy losses or the occurrence of neonatal malformations. CONCLUSION(S): Suboptimal semen samples had a higher incidence of aneuploidy. In this study, the increased frequency of chromosomal abnormalities did not have a direct effect on the fertilization rate, pregnancy characteristics, or neonatal outcome.

Use of electively cryopreserved microsurgically aspirated epididymal sperm with IVF and intracytoplasmic sperm injection for obstructive azoospermia.

OBJECTIVE: To investigate the efficacy of using intentionally cryopreserved epididymal sperm in selected cases of obstructive azoospermia. DESIGN: A retrospective, nonrandomized study. SETTING: Academic research environment. PATIENTS: One hundred forty-one couples undergoing first-time IVF/ICSI using either fresh or cryopreserved epididymal sperm. INTERVENTIONS: The epididymides were microsurgically aspirated. MAIN OUTCOME MEASURES: Clinical pregnancy rates. RESULTS: Motile sperm were obtained from all men. For the fresh group, the mean total sperm aspirated was 99 x 10(6) with 5.5 vials frozen per patient after ICSI and 82 x 10(6) with 4.7 vials frozen per patient in the cryopreserved group. No statistically significant difference in oocyte fertilization rate or number of embryos transferred was noted between groups. Of 108 patients using freshly aspirated sperm, 72 (66.7%) achieved clinical pregnancy. Of 33 patients in the group using cryopreserved sperm, 20 (60.6%) achieved clinical pregnancy (P=0.47). CONCLUSIONS: In selected ideal cases of unreconstructable azoospermia, elective open microsurgical epididymal sperm aspiration with cryopreservation yields pregnancy rates similar to that employing fresh sperm. The advantages of this method are: (1) Use of cryopreserved sperm obviates the logistics problems associated with the use of fresh sperm. (2) Abundant high-quality sperm can be cryopreserved in a single procedure for all future attempts at IVF/ICSI. Rarely, viable sperm will not be present after thawing, and fresh retrieval will be necessary.

Cystic fibrosis gene mutations do not affect sperm function during in vitro fertilization with micromanipulation for men with bilateral congenital absence of vas deferens.

OBJECTIVE: To assess the effects of cystic fibrosis transmembrane-conductance regulator (CFTR) gene mutations on sperm function and fertility in men with bilateral congenital absence of the vas deferens. DESIGN: Prospective. SETTING: Division of urologic microsurgery and associated hospital-based IVF unit. MAIN OUTCOME MEASURES: Fertilization and pregnancy rates. PATIENTS: Men referred to our fertility unit for treatment of bilateral congenital absence of the vas deferens, using sperm surgically retrieved from the epididymis with IVF and micromanipulation. RESULTS: Of 45 men with bilateral congenital absence of the vas, 54% (19/35) tested were found to be carriers of CFTR gene mutations, with one compound heterozygote. Epididymal sperm from men affected with CFTR mutations fertilized 19% (29/152) of oocytes, whereas men without mutations fertilized 22% (44/204) of oocytes. Pregnancy rates (PRs) were 36% (5/14) for cycles involving men with CFTR mutations and 33% (5/15) for other patients with congenital absence of the vas deferens but without detectable CFTR mutations. CONCLUSIONS: The presence of detectable CFTR mutations does not affect fertilization rates or PRs for men with bilateral congenital absence of the vas deferens when IVF and micromanipulation are applied.

Development and implementation of intracytoplasmic sperm injection (ICSI).

The purpose of this paper is to elucidate the experimental steps that led to the development of intracytoplasmic sperm injection (ICSI) and its application in the human. ICSI has become the most successful micromanipulation procedure for treating male infertility. A total of 355 in vitro fertilization (IVF) cycles utilizing ICSI are described; 180 couples were previously treated in 509 IVF cycles but achieved no fertilization and 175 couples could not be treated by IVF because of extremely poor semen parameters. Of the 3063 metaphase II (M II) oocytes retrieved, 2970 were injected with a survival rate of 93.6%, yielding 1917 bipronuclear zygotes (64.5%). In 148 patients, a foetal heart was evidenced by ultrasound; 11 of these patients miscarried between 7 and 13 weeks of gestation. The ongoing pregnancy rate was 38.6% (137/355) per retrieval and 40.5% (137/338) per embryo replacement. At the time of writing, there were 22 deliveries and one therapeutic abortion for a trisomy 21 chromosomal abnormality. In addition, 66 singleton, 37 twin, 10 triplet and 1 quadruplet pregnancies were ongoing. The concentration of motile spermatozoa in the ejaculate only slightly influenced the fertilization rate (P < 0.001) and the pregnancy outcome (P < 0.01). A preliminary injection procedure utilizing intracytoplasmic injection of isolated sperm heads was performed in 35 M II human oocytes with resultant fertilization and cleavage rates of 74% and 73% respectively. Skills in ICSI were acquired by injecting hamster and unfertilized human oocytes with human sperm. ICSI can be used to successfully treat couples who have failed IVF or who have too few spermatozoa for conventional in vitro insemination.(ABSTRACT TRUNCATED AT 250 WORDS)

Correction of hyperinsulinemia in oligoovulatory women with clomiphene-resistant polycystic ovary syndrome: a review of therapeutic rationale and reproductive outcomes.

Polycystic ovary syndrome (PCOS) describes a convergence of chronic multisystem endocrine derangements, including irregular menses, hirsutism, obesity, hyperlipidemia, androgenization, large and cystic-appearing ovaries, insulin resistance and subfertility. Few PCOS patients exhibit all of these features, and often only one sign or symptom is evident. The sequelae of PCOS reach beyond reproductive health, as women affected with PCOS have increased relative risks for myocardial infarction, hypertension, ischemic heart disease, thromboembolic disease and diabetes. Although the adverse health consequences associated with PCOS are substantial, unfortunately most women are not aware of these risks. Indeed, in infertility practice such concerns are secondary as most patients are referred for treatment specifically to achieve a pregnancy. Impairments in insulin metabolism appear central to the physiologic cascade of PCOS, yet clomiphene therapy fails to remedy this defect. Several investigators have described satisfactory reproductive outcomes for PCOS patients treated with oral insulin-lowering agents. In this report, we outline a diagnostic and therapeutic approach for women with PCOS refractory to clomiphene with attention to the underlying insulin imbalance associated with impaired fertility.

IVF: the new era.

In-vitro fertilization (IVF) has made great strides in the past 25 years including the development of micromanipulation techniques. Intracytoplasmic sperm injection (ICSI) for treatment of severe male factor infertility paved the way for the use of the micromanipulator. Micromanipulation techniques are now an integral part of an IVF program and are now also employed to biopsy embryos from patients harboring genetic abnormalities. Micromanipulation has also been used in the development of nuclear transfer techniques--a potential solution for creating "younger" oocytes for women of advanced maternal age and spermatozoa for azoospermic men. Other recent developments include the sequential media enabling the embryo to survive to blastocyst stage and also successful oocyte vitrification to preserve future fertility. This paper describes the latest technologies in in-vitro fertilization and relates them to the total problem of infertility, age-related and otherwise.

Indications for corticosteroids prior to epididymal sperm retrieval.

OBJECTIVE: To evaluate the effectiveness of low-dose intermittent prednisone treatment designed to reduce antisperm antibodies for men prior to epididymal sperm retrieval and in vitro fertilization. STUDY DESIGN: This was a retrospective review of 75 subfertile men with reproductive obstruction due to congenital absence of the vas deferens, or other reproductive tract obstruction, who underwent a total of 98 episodes of microsurgical epididymal sperm aspiration (MESA) with in vitro fertilization. RESULTS: For couples in whom the man was pretreated with prednisone, better fertilization rates (39% vs. 21%, P < .0001) and pregnancy rates (48% vs. 26%, p = 0.06) were obtained than in couples in whom the man was not pretreated. The benefit of prednisone treatment was most noticeable for men with preoperatively detectable antisperm antibodies. Prednisone-treated men with preoperative antisperm antibodies showed improved fertilization rates (40% vs. 9%, p = 0.005). Men without antibodies who were treated with prednisone showed a decrement in fertilization rate, from 73% to 24% (fertilizations per oocyte, P < .0005). Success of prednisone treatment could not be correlated with specific assisted-reproduction technique (IVF, PZD, SuZI, or ICSI). CONCLUSIONS: Preoperative evaluation of antisperm antibody status and treatment of antibody-positive men prior to epididymal sperm retrieval may play a role in improvement of the fertilization and pregnancy results achieved by centers that provide MESA with assisted reproduction. These results are most important when MESA is performed without the assistance of intracytoplasmic sperm injection. Empiric treatment with prednisone may be detrimental to the fertility of men who have no antisperm antibodies.

Intracytoplasmic sperm injection. Accomplishments and qualms.

AIM: Intracytoplasmic sperm injection (ICSI) is now the preferred technique for treatment of male factor infertility and many children have been born worldwide. However, concerns about the risk of transmitting genetic defects and development of ICSI children have been raised. We report clinical outcome of ICSI in Cornell University and results of screening for genetic defects in ICSI parents and their children. METHODS: Pregnancy and obstetrical outcomes as well as congenital malformations were analyzed. Chromosomal karyotyping and Yq deletion assessments were performed on blood samples. In addition, medical and developmental outcome were assessed in 3 and 5 year old ICSI children. RESULTS: We have performed 8 575 ICSI cycles with ejaculated spermatozoa that resulted in a 75.4% fertilization and a 42.8% clinical pregnancy rates while for surgically retrieved specimen resulted in 66%, 48.8% respectively. The incidence of Y deletion was within the expected range for an infertile population, with identical deletions transmitted to the male offspring. There were no differences in cognitive, motor and behavioral development observed between ICSI children and these conceived naturally. CONCLUSION: The large majority of infertile men were treatable by ICSI, which resulted in the transmission of a specific abnormality but did not enhance the incidence of de novo deletions. There is no indication that ICSI children develop more congenital defects or express a lower psychomotor development that children conceived naturally. Nonetheless, genetic screening and counseling of couples undergoing ICSI would seem to be appropriate.

A comparison of human spermatozoa immunolabeling features using xenogenic reagents for centrosomal proteins.

The centrosome is an organelle essential to proper chromosomal migration and normal cell growth. In the human, the centrosome is comprised of two centrioles and the pericentriolar cytosol; its control of embryo cleavage processes derives from its role as a locus for spindle organisation. At fertilisation, it is the human sperm centrosome that is responsible for ordering these processes, as the oocyte appears not to contain working centrosomal structures. Abnormalities in fertilisation or early embryo cleavage could be related to impaired sperm centrosome structure or function in some cases. While potential future treatments of infertility due to a defective centrosome could involve use of a donor centrosome to restore normal cell development, such an approach would depend on accurate localisation of this organelle for subsequent transplantation. To locate centrosomal components in the heads and tails of human spermatozoa, labeling was performed on intact spermatozoa using antibodies of known specificity to highly-conserved centrosomal elements. Following general mapping of immunofluorescent signals, unlabeled sperm were dissected to form head/tail sperm fragments which were then separately tested. Distribution of centrosomal proteins in head and tail fragments was assayed for each separation method. Three reagents were compared: 1) rabbit anti-mitotic spindle protein (anti-MSP) antibody, 2) rabbit polyclonal centriole-specific antibodies, and 3) mouse monoclonal anti-MPM-2 (a centrosome phospoprotein) antibody. Of these, anti-MPM-2 antibody appeared to be the most reliable, labeling centrosomal elements in 63% (n = 1,386) of treated spermatozoa. Sequential utilization of n-butylamine to effect head/tail separation followed by anti-MPM-2 antibody labeling was a satisfactory method of centrosome localisation. Microextraction of centrosomes and pericentriolar matrix identified by this method awaits further testing.