A novel laboratory procedure to validate American Urological Association guideline on vasectomy success and to diagnose obstructive azoospermia.

The objective was to develop a laboratory procedure to validate American Urological Association (AUA) Guideline on vasectomy success when nonmotile spermatozoa are found in the post-vasectomy ejaculate. The neutral α-glucosidase (NAG) an epididymal protein assay modified to determine the activity at 30 and 90 min of incubation from 24 pre- and 47 post-vasectomy ejaculates. The difference between the two points in the relative activity was calculated and if the difference was nonsignificant will confirm vasectomy success. The mean differences in the relative NAG activity were significantly different in pre- and post-vasectomy ejaculates, respectively. The mean differences in the relative NAG activity were similar in post-vasectomy ejaculates with and without nonmotile spermatozoa. No difference in relative NAG activity in post-vasectomy ejaculates between two time points of incubation may be a reliable method to confirm occlusion of the vas deferens. It also validates the recommendation by AUA Guideline on vasectomy success in the presence of few nonmotile spermatozoa.

The hypo-osmotic swelling test: Is it a sperm vitality or a viability assay?

The first two editions of the World Health Organization laboratory manual described the determination of live spermatozoa by a dye exclusion method as a sperm "viability" test, whereas subsequent editions classified it as a "vitality" test, without providing an explanation for the reclassification. Additionally, the hypo-osmotic swelling (HOS) test, which assesses the functional integrity of the human sperm membrane, was placed in the same category as the dye exclusion test. Although the two terms might seem synonymous, the term "vitality" merely means "alive," whereas "viability" assesses qualities or physiological functions of a living entity. After comparing the morphological, physiological, and clinical findings obtained from dye exclusion testing vs. the HOS test, we conclude that the HOS test should be classified as a viability test, not merely as a vitality test.

Individual variation of the percentage of Y-chromosome bearing sperm content in human ejaculates.

The study aimed to determine the variation of Y-chromosome-bearing sperm content among individual ejaculates. A real-time polymerase chain reaction (qPCR) with unique primers was developed and used to calculate the percentage of Y-chromosome-bearing sperm in individual ejaculates from 50 randomly selected men. There was a significant difference in the overall mean ± SD between the proportion of Y-chromosome-bearing sperm and X-chromosome-bearing sperm (45.36 ± 7.88 vs. 54.42 ± 7.88). Of the 50 ejaculates, 17 had more than, and 14 had less than the 99% confidence interval of the mean of the Y-chromosome-bearing sperm (45.58 ± 2.87). These results suggest that the inconsistency in sperm-based sex-selection outcomes appears to be a function of differences in the ejaculates and highlights the need for further study in environmental and genetic factors contributing to X or Y bearing spermatozoan instability.Abbreviations: qPCR: real-time polymerase chain reaction; ROS: reactive oxygen species; DTT: dithiothreitol; SRY: sex-determining region Y.

Selecting the most competent sperm for assisted reproductive technologies.

This paper discusses the variety of effective sperm selection techniques that have been developed for use in assisted reproductive technologies. Available methods for isolating the competent sperm in an ejaculate are outlined, as well as techniques for selecting single sperm for use in intracytoplasmic sperm injection procedures. Case-specific methods for selecting the most competent sperm are discussed, with reference to the potential causes of male factor infertility and guidance for the embryologist based on the issues present for each couple seeking treatment.

Male fertility preservation and cancer treatment.

The diagnosis of cancer in men or women leads to prompt evaluation of the extent of the cancer, its treatment, and subsequent prognosis. However, relatively little emphasis is placed on fertility following the completion of therapy. As the effectiveness of cancer treatment has improved, men can enjoy a longer life that is free of cancer. However, chemotherapeutic regimens alone or in combination with radiation therapy frequently result in azoospermia or infertility. This paper reviews available methods to maintain male fertility in patients undergoing chemotherapy or radiation therapy. Certain chemotherapeutic agents that are less likely to cause azoospermia may be incorporated into potentially curative therapies. Hormonal suppression applied early (prior to) and during chemotherapy may protect future male fertility. Alternatively, cryopreservation of sperm enables men to reproduce in the future with the assistance of in vitro fertilization with intracytoplasmic sperm injection. Therefore, oncologists need to discuss male fertility preservation before initiating cancer treatment in reproductive-aged men. The emphasis for future cancer treatment and its research regarding male fertility preservation needs further attention.

The hypo-osmotic swelling test for evaluation of sperm membrane integrity.

A functional membrane is requisite for the fertilizing ability of spermatozoa, as it plays an integral role in sperm capacitation, acrosome reaction, and binding of the spermatozoon to the egg surface. The hypo-osmotic swelling (HOS) test evaluates the functional integrity of the sperm's plasma membrane and also serves as a useful indicator of fertility potential of sperm. The HOS test predicts membrane integrity by determining the ability of the sperm membrane to maintain equilibrium between the sperm cell and its environment. Influx of the fluid due to hypo-osmotic stress causes the sperm tail to coil and balloon or "swell." A higher percentage of swollen sperm indicates the presence of sperm having a functional and intact plasma membrane. Here, we present the detailed protocol for performing the HOS test and explain the results for interpretation.

Human chorionic gonadotropin from day 2 spent embryo culture media and its relationship to embryo development.

OBJECTIVE: To detect hCG in spent embryo culture media at day 2 after intracytoplasmic sperm injection and to assess the relationship of hCG to embryo development. DESIGN: Experimental study. SETTING: Fertility center and clinical diagnostic laboratory. SAMPLE(S): A total of 102 spent culture media from day 2 human embryos and corresponding unexposed media for blank control. INTERVENTION(S): The culture media samples were tested for hCG by ELISA. MAIN OUTCOME MEASURE(S): Quantity of hCG produced by embryos and correlation with the embryos' developmental status. RESULT(S): hCG was found in 93 of 102 culture media tested by enzyme-linked immunosorbent assay. The correlation analysis revealed that the concentration of hCG was independent of embryo developmental status. CONCLUSION(S): The ability to detect hCG from day 2 spent culture media may be used as a marker for embryo competence.

Prevalence of antiphospholipid antibodies among women experiencing unexplained infertility and recurrent implantation failure.

The prevalences of antiphospholipid antibodies (APAs) among 1,325 women with a history of unexplained infertility and 676 women experiencing recurrent implantation failure were compared with 789 women experiencing recurrent pregnancy loss and 205 fertile control women. Eight percent and 9% of women with a history of unexplained infertility and recurrent implantation failure had more than one positive APA compared with 1.5% of fertile negative control women and 11% of positive control women experiencing recurrent pregnancy loss.

Is apolipoprotien E codon 112 polymorphisms associated with recurrent pregnancy loss?

PROBLEM: To compare the prevalence of 112T>C point mutations among women experiencing RPL with fertile control women. METHOD OF STUDY: Buccal swabs were obtained from 232 individuals: 136 with a history of >or=2 abortions, 37 with at least 2 live births and 59 with a history of deep vein thrombosis (DVT). DNA was extracted and PCR amplification of Apo E codons was performed. RESULTS: The allelic frequency of a cytosine at position 112 was 11.4% (31/272) among patients experiencing RPL, compared with a frequency of 5.4% (4/74) among the fertile controls (P = 0.19) and 19.5% (23/118) among individuals with a history of DVT. However, significantly more E3/E4 and E4/E4 genotypes were seen among individuals experiencing RPL and DVT than fertile controls (P < 0.05). CONCLUSION: Apo E4 codon 112C point mutation is, by itself, not associated with an elevated risk of recurrent pregnancy loss, but rather codon 112C in association with codon 158C is a risk factor for RPL.

The relevance of neutral alpha-glucosidase activity in andrology.

To assess the usefulness of routine determination of neutral alpha-glucosidase (NAG) in andrology, 216 ejaculates were analyzed for NAG activity and semen quality. A correlation between NAG activity and semen volume and sperm concentration was determined; however, no correlation was observed between NAG activity and sperm motility or sperm morphology. The number of azoospermic ejaculates that had NAG activity below acceptable levels was significantly higher than the number of non-azoospermic ejaculates with similarly low NAG levels. Routine determination of NAG activity is not practical; however, when an epididymal pathology leading to a physiological or anatomical functional alteration is suspected, the determination of NAG activity is a valuable tool in the diagnosis, and would also aid in the prognosis.

Interleukin 1 receptor antagonist gene polymorphisms are not risk factors for recurrent pregnancy loss: evaluation of couples.

PROBLEM: The interleukin-1 system has been implicated in pregnancy outcome. Fetal carriage of interleukin-1 receptor antagonist (IL-1Ra) specific alleles has been associated with adverse pregnancy outcomes including spontaneous abortion and pre-term labor. This study was undertaken to compare the frequency of IL-1RN 2 alleles among both male and female partners of couples experiencing recurrent pregnancy loss with that of fertile control couples. METHOD OF STUDY: Buccal swabs were obtained from 42 couples experiencing recurrent pregnancy loss and from 20 fertile control couples. DNA was extracted from the buccal swabs and analyzed for the presence of IL-1RN variable number tandem repeat. RESULTS: No significant differences were found when the frequency of IL-1RN 2 polymorphisms were compared between fertile control couples and couples experiencing recurrent pregnancy loss. Similar results were also obtained when comparing women or men respectively from each group. CONCLUSION: IL-1RN 2 allele is not a risk factor for recurrent pregnancy loss.

Cryopreservation of human sperm in a lecithin-supplemented freezing medium.

The use of egg yolk and serum albumin as additive diluents for human sperm cryopreservation is routine. But because both diluents are of animal origin, they potentially may introduce microbial agents to the sample. To reduce the risk of contamination, the cryoprotective property of phospholipids extracted from lecithin was evaluated and found to be effective when supplemented with dimethyl sulfoxide and glycerol.

Comparison of thrombophilic gene mutations among patients experiencing recurrent miscarriage and deep vein thrombosis.

PROBLEM: Inherited thrombophilia has been shown to be a risk factor for cardiovascular disease including deep venous thrombosis as well as reproductive disorders including recurrent pregnancy loss. We have previously reported three out of the 10 thrombophilic mutations studied, plasminogen activator inhibitor-1 (PAI-1) 4G/5G, factor XIII V34L, and homozygous MTHFR C667T, correlated significantly with recurrent pregnancy loss compared with controls. This study was undertaken to compare the frequencies of nine inherited thrombophilias among women with a history of recurrent pregnancy loss with individuals experiencing deep venous thrombosis and fertile controls. METHOD OF STUDY: Six hundred thirty-four participants including 550 women with a history of recurrent pregnancy loss, 43 individuals with deep vein thrombosis and 41 fertile women without a history of recurrent miscarriage. All participants had buccal swabs taken for DNA analyses of nine gene polymorphisms including factor V G1691A, factor V H1299R (R2), factor II Prothrombin G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, human platelet antigen 1 a/b (L33P), MTHFR C677T, MTHFR A1298C. Frequencies of thrombophilic gene polymorphisms were compared among the three populations studied. RESULTS: Individuals with a history of DVT had a significantly higher frequency of all of the polymorphisms studied compared with women experiencing a history of recurrent pregnancy loss and the fertile controls. The frequencies of mutations for V34L and PAI-1 4G/5G were significantly increased among women experiencing recurrent pregnancy loss compared with controls. The most prevalent polymorphisms were factor XIII V34L and PAI-1 4G/4G for both individuals with a history of deep vein thrombosis and recurrent pregnancy loss compared with controls. CONCLUSION: Screening for risk factors for inherited thrombophilia with only polymorphisms for factor V von Leiden, factor II prothrombin and MTHFR may be missing the more prevalent identifiers of jeopardy.

The impact of male factor on recurrent pregnancy loss.

PURPOSE OF REVIEW: The present paper reviews the current literature on the impact of male factor on recurrent pregnancy loss. RECENT FINDINGS: Most clinicians focus their evaluation of recurrent pregnancy loss on the female, without much, if any, consideration of the other half of the couple - the male. Yet, the male contributes one-half of the genes for the embryo. Recent literature demonstrates that the male contributes to recurrent pregnancy loss due to genetic factors, semen factors or due to other factors such as age. SUMMARY: Recurrent pregnancy loss results as a factor of a couple. This paper emphasizes the contribution of the male to implantation failure, miscarriage, and congenital anomalies suggested by recent literature. The current data are preliminary. With further investigation, evaluation of the male may be considered a routine part of the evaluation in the near future.

Which thrombophilic gene mutations are risk factors for recurrent pregnancy loss?

PROBLEM: Thrombophilia has been associated with poor obstetrical outcomes. To determine the association of specific inherited thrombophilias and recurrent pregnancy loss, 10 thrombophilic genes were investigated. METHOD OF STUDY: A total of 550 women with a history of recurrent pregnancy loss had buccal swabs taken for DNA analyses of the following gene mutations: factor V G1691A, factor V H1299R (R2), factor V Y1702C, factor II prothrombin G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, HPA1 a/b(L33P), methylenetetrahydrofolate reductase (MTHFR) C677T, MTHFR A1298C. The frequencies of these mutations were compared with controls published in the literature. RESULTS: When examined individually, PAI-1 4G/5G (P = 0.009), factor XIII V34L (P < 0.0001), and homozygous MTHFR C667T (P < 0.0001) correlated significantly with recurrent pregnancy loss compared with controls. The frequency of the factor V Y1702C mutation was extremely low in patients and controls; thus, this gene was removed from further calculations. The remaining six mutated genes, when analyzed cumulatively, also corresponded with recurrent pregnancy loss (P < 0.0001). CONCLUSION: A panel of thrombogenic gene mutations consisting of factor V G1691A, factor V H1299R (R2), factor II prothrombin G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, HPA1 a/b(L33P), MTHFR C677T, and MTHFR A1298C can identify individuals at risk for recurrent pregnancy loss.

Y-chromosome microdeletions and recurrent pregnancy loss.

OBJECTIVE: To determine the prevalence of Y-chromosome microdeletions in recurrent pregnancy loss (RPL) couples as compared with couples with male factor infertility and fertile couples. DESIGN: Controlled clinical study. SETTING: Andrology laboratory and RPL clinic. PATIENT(S): Seventeen men from RPL couples, 18 men from couples with a live birth and no history of miscarriages, and 10 men from couples with male factor infertility. INTERVENTION(S): Buccal smears for Y-chromosome microdeletion testing. MAIN OUTCOME MEASURE(S): The DNA was tested for microdeletions in the proximal AZFc region by polymerase chain reaction (PCR). RESULT(S): Fourteen of the 17 men (82%) tested had microdeletions in one or more of the four segments studied. Two of the 10 male factor infertility patients (20%) had microdeletions in 2 different segments. None of the 18 fertile men had any microdeletions in the 4 segments of the proximal AZFc region studied. CONCLUSION(S): The prevalence of the Y-chromosome microdeletions in the proximal AZFc region was much higher in men from RPL couples than from fertile or infertile couples. Although these patients are from a tertiary referral center that may skew the population and findings, one may consider Y-chromosome microdeletion testing particularly of the AZFc region in the evaluation of RPL couples when all other tests fail to reveal the etiology.