Donor oocyte cytoplasmic transfer did not enhance implantation of embryos of women with poor ovarian reserve.

PURPOSE: To determine whether donor oocyte cytoplasm transferred into the oocytes of women < or = 40 years or with diminished ovarian reserve would enhance embryo quality, implantation, or pregnancy rates. METHODS: Study subjects included women > or = 40 years (15) or with abnormal FSH levels (3). Healthy volunteers (18) produced oocytes for cryopreservation. Donor oocytes were thawed and cytoplasm from surviving oocytes was injected with a single sperm into the cytoplasm of recipient oocytes. Outcome measures included embryo quality scores, implantation, and pregnancy rates. RESULTS: Eighteen donors produced 213 oocytes for cryopreservation and 39/171 (22.8%) survived thawing. Eighteen recipients initiated 25 IVF cycles with embryo transfer in 20 cycles after cytoplasmic transfer (CT). Four cycles resulted in three biochemical losses and one aneuploid clinical loss. Embryo quality did not improve with CT compared to pre-CT IVF cycles in six recipients. CONCLUSIONS: CT with cryopreserved donor oocyte cytoplasm did not enhance success in women with advanced reproductive age or low ovarian reserve.

The number of embryos available for transfer predicts successful pregnancy outcome in women over 39 years with normal ovarian hormonal reserve testing.

PURPOSE: The purpose was to determine whether the number of embryos available for transfer following IVF in women over age 39 predicted a successful pregnancy outcome. METHODS: Retrospective analysis of 455 consecutive IVF cycles in women > or = 40 years of age. RESULTS: Few cycles were canceled (29/455, 6.4%) or produced no embryos (5/455, 1.1%). Women 40-43 years of age with normal ovarian reserve had a significantly greater delivery rate when > or = 4 embryos were available for transfer than when < 4 embryos were available (17.8% versus 2.4%, P = 0.002). Subsequent IVF cycles, from women with normal FSH whose first cycle produced < 4 embryos, produced delivery rates of 13.0% when > or = 4 embryos were available. Women with abnormal ovarian reserve or age > or = 44 years had very low delivery rates (1.2% and 1.4% respectively). CONCLUSIONS: The number of embryos available for transfer significantly predicts delivery from IVF-ET among reproductively older women. Many women age 40-43 with normal ovarian reserve can achieve pregnancy through IVF.

Pregnancy rates in sequential in vitro fertilization cycles by oocyte donors(1).

OBJECTIVE: To evaluate the clinical outcome of in vitro fertilization (IVF) treatment cycles from individual oocyte donors who underwent multiple sequential donations. METHODS: We reviewed clinical outcome data from sequential anonymous oocyte donation cycles using donors who underwent multiple IVF stimulations. Donors were grouped by the interval between cycles and the cycle number (rank). The primary outcome measure was delivery rate by individual donor per retrieval from the combined derivative fresh and frozen embryo transfers. RESULTS: Duration and amount of gonadotropin therapy and the fertilization rates did not correlate significantly with the interval between cycles or cycle rank. Cumulative delivered pregnancy rates for cycles 1-6 were 51.5%, 54.6%, 50.5%, 51.5%, 51.1%, and 57.6%, respectively. Delivered pregnancy rates did not vary by interval between cycles. CONCLUSION: Young healthy presumed or proven fertile women can reliably donate oocytes for at least six cycles with the expectation of consistently high pregnancy rates.

Mutational analysis of DAX1 in patients with hypogonadotropic hypogonadism or pubertal delay.

Although delayed puberty is relatively common and often familial, its molecular and pathophysiologic basis is poorly understood. In contrast, the molecular mechanisms underlying some forms of hypogonadotropic hypogonadism (HH) are clearer, following the description of mutations in the genes KAL, GNRHR, and PROP1. Mutations in another gene, DAX1 (AHC), cause X-linked adrenal hypoplasia congenita and HH. Affected boys usually present with primary adrenal failure in infancy or childhood and HH at the expected time of puberty. DAX1 mutations have also been reported to occur with a wider spectrum of clinical presentations. These cases include female carriers of DAX1 mutations with marked pubertal delay and a male with incomplete HH and mild adrenal insufficiency in adulthood. Given this emerging phenotypic spectrum of clinical presentation in men and women with DAX1 mutations, we hypothesized that DAX1 might be a candidate gene for mutation in patients with idiopathic sporadic or familial HH or constitutional delay of puberty. Direct sequencing of DAX1 was performed in 106 patients, including 85 (80 men and 5 women) with sporadic HH or constitutional delay of puberty and patients from 21 kindreds with familial forms of these disorders. No DAX1 mutations were found in these groups of patients, although silent single nucleotide polymorphisms were identified (T114C, G498A). This study suggests that mutations in DAX1 are unlikely to be a common cause of HH or pubertal delay in the absence of a concomitant history of adrenal insufficiency.

Mutation analysis of the EMX2 gene in Kallmann's syndrome.

OBJECTIVE: To investigate the possibility that a mutation in the human EMX2 gene may be involved in Kallmann's syndrome. DESIGN: In vitro experiment. SETTING: Academic Medical Center. PATIENTS: One hundred and twenty patients with Kallman's syndrome or idiopathic hypogonadotrophic hypogonadism (IHH). INTERVENTION: Peripheral blood leukocytes were used to obtain DNA. MAIN OUTCOMES MEASURES: Single-stranded conformational polymorphism (SSCP) analysis was used to identify possible mutations of the EMX2 gene. RESULTS: One hundred and twenty patients with Kallmann's syndrome or IHH, had no mutations noted in this gene. CONCLUSION: It is unlikely that EMX2 mutations are a clinically significant cause of IHH or Kallman's syndrome.

Percutaneous testicular sperm aspiration: a convenient and effective office procedure to retrieve sperm for in vitro fertilization with intracytoplasmic sperm injection.

PURPOSE: After intracytoplasmic sperm injection was established to facilitate in vitro fertilization in men with the most severe semen abnormalities, the use of testicular sperm to achieve conception became feasible. We investigated the use of a method of percutaneous needle aspiration previously used for diagnostic purposes to obtain testicular sperm for intracytoplasmic sperm injection. MATERIALS AND METHODS: A method of percutaneous aspiration of sperm was developed to facilitate intracytoplasmic sperm injection. A total of 69 testicular aspirations were performed for diagnostic purposes and 179 to obtain sperm on the day of egg retrieval for couples undergoing in vitro fertilization with intracytoplasmic sperm injection. The procedures were performed in an outpatient facility. Most patients received intravenous sedation and a few received only local anesthesia. RESULTS: Sperm adequate for intracytoplasmic sperm injection were obtained in all men with obstructive azoospermia, including those with significant testicular atrophy and those with anejaculation or necrospermia. Adequate numbers of sperm for intracytoplasmic sperm injection were retrieved less reliably in men with nonobstructive azoospermia. The number of sperm correlated positively with testicular size. Morbidity and discomfort were nonexistent. Sperm were obtained from 43 of 69 men undergoing diagnostic and 170 of 179 men undergoing therapeutic aspiration. Sperm motility ranged from 0 to 20% and viability from 55 to 85%. CONCLUSIONS: Percutaneous testicular sperm aspiration is a cost-effective method to retrieve sperm for intracytoplasmic sperm injection in select men with obstructive azoospermia, anejaculation and necrospermia, and some with nonobstructive azoospermia.

Preservation of reproductive function before therapy for cancer: new options involving sperm and ovary cryopreservation.

In summary, sperm cryopreservation for future ICSI and ovarian tissue cryopreservation for future autotransplantation are new opportunities to preserve reproductive options of great importance to patients with newly diagnosed cancer. Since patients must utilize these strategies before cancer therapy is initiated, and these patients will not have a future chance to benefit once therapy has damaged gonadal function, awareness of these technologies among oncologists, radiation therapists, and other colleagues who interface with the victims of cancer is a high priority.

Simple, rapid staining method for immediate intraoperative examination of testicular biopsies.

Before patients with obstructive azoospermia undergo surgical reconstruction, testicular biopsy is required to establish that spermatogenesis is normal. To perform the corrective surgical procedure at the same time as the testicular biopsy, a touch imprint method of processing the biopsy specimen has replaced frozen section processing, which distorts the germinal cells. We report a rapid, simple method of staining a touch imprint specimen in the operating room. It requires less than 5 minutes and can be performed by the andrology laboratory technologist, operating room staff, or the surgeon, thereby avoiding the need to transport the biopsy specimen to the pathology laboratory.

Comparisons of pregnancy loss patterns after intracytoplasmic sperm injection and other assisted reproductive technologies.

OBJECTIVE: To compare outcome of pregnancies after intracytoplasmic sperm injection (ICSI) with those of other assisted reproductive technologies. DESIGN: Pregnancy outcomes after ICSI were followed prospectively and compared with pregnancy outcomes after IVF with fresh and frozen ETs and donor oocyte cycles. SETTING: A private tertiary referral center for genetics and infertility in Fairfax, Virginia. PATIENTS: One hundred thirty-six couples achieving pregnancy after undergoing ICSI, 71 after IVF, 35 donor oocyte recipients, and 19 after transfer of frozen-thawed embryos. INTERVENTIONS: In vitro fertilization and/or ET for all couples. Dilatation and curettage to obtain products of conception for chromosome analysis in 28 women experiencing spontaneous abortion. MAIN OUTCOME MEASURES: Pregnancy outcomes were classified as preclinical loss, clinical loss, and ongoing pregnancy. RESULTS: The mean frequency of preclinical pregnancy loss was 26% after ICSI, 28% after IVF, 3% after ET using donor oocytes, and 11% after frozen ET. The rate of clinical loss after ICSI (21%) was compared with IVF (18%), donor oocyte cycles (11%), and frozen ETs (21%). CONCLUSIONS: Intracytoplasmic sperm injection is not associated with an increase in pregnancy losses, clinical or preclinical, compared with conventional IVF.

Ultrastructural and biochemical analysis of sperm flagella from an infertile man with a rod-dominant retinal degeneration.

This study examined the ultrastructural morphology and posttranslationally modified alpha-tubulin isoforms in the sperm flagella of a patient presenting with infertility and retinal degeneration. Clinical evaluation showed impaired motility and gross morphological abnormalities of the sperm and a rod-dominant retinal degeneration with midperipheral pigment clumping and scattered bone spicules. Other neurological indications included delayed neuroelectric transmission in the auditory brainstem and a temporal lobe seizure disorder. Ultrastructural analysis showed that 46% of sperm axonemes had missing and/or misplaced doublets compared with 10% to 12% in control subjects. ELISA analysis showed hypoacetylation of alpha-tubulin (30% of control) but normal levels of alpha-tubulin tyrosination. Tubulin acetyl-transferase specific activity was also 30% of control activity. These characteristics may be indicative of microtubule instability leading to the pathological consequences described.

Recent advances in reproductive genetic technologies.

New possibilities for the diagnosis and treatment of reproductive and genetic disorders are becoming available as a result of a series of recent technical advances. Intracytoplasmic sperm injection (ICSI) allows treatment of numerous infertile men whose sperm cannot penetrate the egg to initiate fertilization. Molecular genetic testing provides clients of reproductive age with additional information that permits prevention of genetic diseases such as fragile X syndrome, the leading cause of inherited mental retardation. Preimplantation genetic testing (PGT) offers couples who carry genetic disorders the prospect of having children with a greatly decreased risk of initiating a pregnancy involving an affected individual. Flow-cytometric sperm separation offers a new, effective approach for prevention of X-linked genetic disorders. Two major causes of recurrent pregnancy loss (RPL) involve recurrent trisomies and immunological disorders. Of the latter, 70% of studied populations of patients can attain live births with simple treatment protocols. Maternal serum assays involving multiple markers reduce both false positives and false negatives in detection of trisomies. Despite these advances in research, many safe and effective methods of diagnosis and treatment remain under-utilized in the clinical arena.

Intracytoplasmic sperm injection facilitates fertilization even in the most severe forms of male infertility: pregnancy outcome correlates with maternal age and number of eggs available.

OBJECTIVE: To evaluate, in a prospective study, the fertilization and pregnancy rates after intracytoplasmic sperm injection (ICSI) in infertile couples with severe male infertility. DESIGN: Intracytoplasmic sperm injection was performed in 229 consecutive IVF cycles on 190 couples with rigorously defined severe male infertility or proven failure of fertilization in prior IVF cycles. Neither male nor female partners were chosen from a waiting list or on any other selective basis, including age, prior or anticipated ovarian response, or oocyte number or quality. There were no upper age limits, in no instance was donor sperm used for ICSI, and cycle cancellation rate was minimal. SETTING: Private genetics and fertility center in Fairfax, Virginia. MAIN OUTCOME MEASURES: Fertilization, transfer, and pregnancy rates were measured in ICSI-treated couples, and comparisons were made regarding both female age and strictly defined semen categories. RESULTS: Two hundred six cycles (90%) resulted in ETs, with initiation of 52 pregnancies (25% per transfer, 23% per cycle). Thirty-eight of 52 (18% per transfer) were clinical pregnancies with established gestational sacs or were ongoing or delivered. Pregnancies were achieved even in older women but were more readily established in younger women producing larger numbers of metaphase II oocytes. The severity of semen abnormalities had some small effect on fertilization rate, but only actual necrospermia was associated with markedly decreased frequency of embryo formation. Pregnancy per transfer was similar across groups. In some cases, pregnancy was initiated with fewer than 100 viable sperm in the ejaculate. CONCLUSIONS: Intracytoplasmic sperm injection is a very powerful new treatment for severe male infertility. Paradoxically, egg number and probably egg quality are now the main determinants of success in treating male infertility.

Pregnancy with microsurgical vas sperm aspiration from a patient with neurologic ejaculatory dysfunction.

Patients with neurologic ejaculatory dysfunction who wish to have a child commonly undergo electroejaculation, which, unfortunately, may fail to stimulate either antegrade or retrograde ejaculation of sperm. Even when sperm are obtained with electroejaculation, conception still may not be achieved. We describe the achievement of a pregnancy after the intrauterine insemination of sperm obtained from microsurgical aspiration of the vas deferens. The intrauterine insemination of electroejaculated sperm had failed to achieve a pregnancy on three previous occasions. The relative merits of vas sperm aspiration and electroejaculation as part of the assisted reproduction regimen for men who have neurologic ejaculatory dysfunction are discussed.

Characterization and frequency distribution of sperm acrosome reaction among normal and infertile men.

We studied the acrosome reaction (AR) in human spermatozoa from 58 normal fertile men and 232 infertile patients. The median AR in the normal population was 17%; AR was > 9% in 83% of the subjects studied and only 2% of fertile individuals had a failed AR (< 5%). We calculated the within person variability using multiple specimens from each subject; the 95% confidence interval for each subject was 4 AR units above and 4 AR units below his mean value. In contrast, the median AR for the infertile population was 3%. AR values were below the normal range (< 5%) in 60% of the patients and only 26% of the patients had AR values > 9%. Because patients entered the study sequentially, without regard to the aetiology of infertility, the patient population comprised a wide variety of subjects ranging from individuals with good to extremely poor semen quality. There was a progressively greater number of individuals with a failed AR as semen quality deteriorated (P < 0.01). However, among patients with good count, motility and morphology, 20% unexpectedly had a failed AR, and among patients with severely impaired semen quality, 29% had a positive AR. We also analysed the relationship of AR values with other semen measurements. The frequency of acrosomal reactions was significantly correlated (Spearman, P < 0.001) with morphology (r = 0.509), concentration (r = 0.524), progression (r = 0.416) and percentage motility (r = 0.356), but not with the percentage of sperm moving at a curvilinear velocity of < or = 40 microns/s.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome reaction inducibility predicts fertilization success at in-vitro fertilization.

We prospectively studied the ability of acrosome reaction (AR) inducibility to predict fertilization success in a group of 232 infertile patients presenting sequentially for in-vitro fertilization (IVF). The median percentage of eggs fertilized for the overall patient population was 25% (interquartile range 5-58%), with one to 29 oocytes available for insemination (median, five oocytes). The median percentage of eggs fertilized at IVF increased as the percentage of spermatozoa able to undergo AR became greater: spermatozoa with a failed AR (< or = 5%) fertilized only 12% of eggs, while spermatozoa with AR values > 9% fertilized 50% of eggs. The assay had a specificity of 0.75, a sensitivity of 0.55 and an odds ratio of 2.9; thus, AR-positive patients are 2.9 times more likely to achieve fertilization than patients with a failed AR. Receiver operator characteristic (ROC) curves were constructed for AR, sperm concentration and percentage of normal forms in semen. All three parameters proved to be potentially useful in predicting the occurrence of fertilization, although AR and morphology appeared to be better than sperm concentration by ROC analysis. Patients were divided into four clearly defined subgroups according to their traditional semen characteristics, including morphology. The median percentage of eggs fertilized decreased as traditional semen characteristics deteriorated, from a median of 46% for patients with excellent sperm concentration, motility and morphology, to a median of 29% for patients with suboptimal semen quality and a median of 0% for patients with severely impaired semen.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome reaction inducing activity in follicular fluid correlates with progesterone concentration but not with oocyte maturity or fertilizability.

Follicular fluid is a potent mediator of sperm acrosome reaction (AR) in vitro. The aim of this study was to investigate whether individual follicular fluids vary quantitatively in their ability to stimulate an AR, and whether such variability relates to fertilizability of the corresponding egg, its maturational level and/or progesterone content. Individual follicular fluids were obtained from 24 women undergoing in-vitro fertilization and assayed for their ability to induce an AR in normal human spermatozoa. After incubation in capacitation medium for 18 h, spermatozoa were challenged with the individual follicular fluids for 30 min. AR was detected by immunofluorescence, using fluorescein-labelled Pisum sativum lectin. We found that individual follicular fluids varied markedly in their ability to induce AR. Acrosome reaction correlated linearly with progesterone concentration (Spearman's r = 0.735, P = 0.01) at constant protein level, but no correlation was found between AR and protein concentration at constant progesterone level. Progesterone concentrations were not only higher (ANOVA, P = 0.002) in fluids from mature oocytes compared to those from less mature or post-mature eggs but also in fluids from fertilized compared to unfertilized eggs (ANOVA, P = 0.015, n = 13 patients with both fertilized and unfertilized eggs). In contrast, AR-inducing ability of individual follicular fluids did not differ for fertilized and unfertilized eggs. While AR-inducing ability appeared to increase with maturational stage of the egg, this trend was not statistically significant, probably due to small sample size. Our data suggest that progesterone rather than protein is the principal mediator of acrosome reaction induced by follicular fluid in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical composition and protein source in the capacitation medium significantly affect the ability of human spermatozoa to undergo follicular fluid induced acrosome reaction.

We studied the effect of media composition on sperm capacitation, using Biggers-Whitten-Whittingham (BWW) medium, Ham's-F10 and a modified Tyrode's medium (HSM) supplemented with bovine serum albumin (BSA) or fetal cord serum (FCS). We evaluated the effect of chemical environment and protein supplementation on the sperm motion parameters of curvilinear velocity and linearity, and on the ability of incubated spermatozoa to undergo follicular fluid induced acrosome reaction. Neither chemical composition nor protein supplementation of capacitation media greatly affected motion parameters after 2 h incubation. Furthermore, chemical composition had only a small effect on the ability of spermatozoa to undergo the acrosome reaction upon exposure to follicular fluid. A higher proportion of spermatozoa underwent acrosome reaction after incubation in HSM (8% control (C); 28% follicular fluid) than in BWW (8% C, 17% follicular fluid) or Ham's F-10 (6% C, 19% follicular fluid). By contrast, protein source proved critical in determining acrosome reaction inducibility. Spermatozoa incubated in BSA-supplemented media showed a 4-fold increase in acrosomal discharge when exposed to follicular fluid (6% C, 22% follicular fluid) compared to controls while spermatozoa incubated in FCS were unable to undergo acrosome reaction (6% C, 6% follicular fluid). Simultaneous addition of FCS to BSA capacitation medium blocked acrosome reaction inducibility and the late addition of BSA, after sperm incubation in FCS, did not facilitate acrosome reaction. We propose that an inhibitor of sperm capacitation is present in FCS and therefore, the selection of optimum incubation conditions for spermatozoa may be of critical importance when evaluating or treating infertile patients.