Analysis of the functional and nuclear integrity of human spermatozoa.

The analysis of semen quality and sperm function has added a new dimension to the evaluation of male infertility. In the present investigation, some recent techniques were used to assess the nuclear, membrane, and acrosomal integrity of spermatozoa from semen of 15 subfertile males with low sperm count and motility. In the infertile semen, the live-to-dead ratio and percentage of spermatozoa showing swelling in hypoosmotic solution was lower than normal but correlated positively with the motility of spermatozoa (r = .85). Staining the spermatozoa with silver nitrate, using a modified technique developed in our laboratory, revealed a higher percentage of morphologically abnormal spermatozoa, in particular, with loss of acrosomal integrity in the subfertile males. Moreover, the percentage of green-fluorescing "fertile" spermatozoa, with intact double-stranded DNA (acridine orange fluorescence), was lower than normal, which correlated with the reduction in sperm nuclear DNA content. A low correlation was obtained between motility and the percentage abnormal spermatozoa (r = .44) and motility and percent green sperm (r = -.28), suggesting that both tests evaluate sperm functional properties independent of motility. The new parameters, assessed with recent techniques, indicate poor sperm fertilizability, which may therefore contribute to the low fertility of the cases studied.     

Initial 10-year experience of sperm cryopreservation services for cancer patients.

Offering sperm cryopreservation to preserve the fertility of male cancer patients is a relatively recent service in Asia. This study analyzed the types of cancer, timing of collection, sperm quality, and utilization for reproductive services by patients during a 10-year period at a medical center in Taiwan. A total of 75 oncology patients elected to freeze sperm for fertility preservation at our medical center during the initial 10 years of the availability of this service. The mean age of the patients was 25.7 years. Storage was discontinued in 13 (17%) patients and their survival duration was 13.1 +/- 11.1 months. The utilization rate of sperm cryopreservation was 2.8% (75/2642). The types of cancer varied, with leukemia (35%), lymphoma (25%), and testicular cancer (13%) comprising the largest groups. A significantly lower sperm count was found in patients with chronic myelogenous leukemia, suggesting the need for earlier sperm collection after initiation of cancer treatment. Only three (4%) patients utilized their specimens for reproductive purposes. There was no clinical pregnancy during the study period, although one biochemical pregnancy was achieved. The low rates of sperm cryostorage for fertility preservation in cancer patients in this study suggest that there is a need for greater emphasis of this option for male oncology patients whose fertility is likely to be affected by chemotherapeutic treatment.     

An examination of blood steroid and gonadotropin concentrations in relation to fertility status and testicular function in men

Plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), testosterone (T), and 17 beta-estradiol (E2) have been measured in men complaining of infertility in comparison with men of proven fertility. Subgrouping of patients was achieved on the basis of the presence or absence of sperm in the ejaculate and further by the concentration of sperm or by testicular score. The levels of plasma LH, FSH, PRL, and T were found to be significantly different in the fertile men, compared with both infertile men with sperm in their ejaculates and azoospermic men. There were no significant differences between the groups for E2. There appeared to be an inverse relationship between LH concentrations and sperm count in both fertile and infertile men. FSH levels did not vary significantly in the fertile men in relation to sperm count grouping but were significantly less than those found for the infertile men with sperm. Azoospermic patients with high testicular scores had FSH levels indistinguishable from those of the fertile men. The results are discussed in terms of testicular abnormalities and on the interrelationship between the hormones examined.     

轻度子宫内膜异位症伴不育患者腹腔液对精子穿卵及精子膜的影响

本研究对轻度子宫内膜异位症患者（３０例）及正常对照组（１５例）在腹腔镜直视下收集腹腔液,正常人精液按１：１比例稀释,以去透明带地鼠卵穿透试验（ＨＯＰ）、精子尾部低渗肿胀试验（ＨＯＳ）及精子头部ＤＮＡ荧光染色有效精子计数（ＥＳＣ）为指标,探讨轻度子宫内膜异位症（ＥＭ）患者的腹腔液对精子受精能力、精子膜及ＤＮＡ 双螺旋结构的影响。结果显示,ＥＭ组精子穿卵率和受精卵外精子附着数明显低于正常对照组（Ｐ＜０．０１）,ＥＭ组的 ＨＯＳ明显低于正常对照组（Ｐ＜０． ０５）,而 ＥＭ组的 ＥＳＣ虽有所下降,但与正常对照组相比无显著性差异。结果提示：轻度ＥＭ腹腔液对精子受精能力及精子膜结构的完整性有影响。     

Effects of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-controlled trial

OBJECTIVE: To study the effects of folic acid and zinc sulfate treatment on semen variables in fertile and subfertile men. DESIGN: Double-blind, placebo-controlled interventional study. SETTING: Two outpatient fertility clinics and nine midwifery practices in The Netherlands. PARTICIPANT(S): One hundred eight fertile and 103 subfertile men. INTERVENTION(S): Both groups were randomly assigned to receive one of four treatments for 26 weeks: folic acid and placebo, zinc sulfate and placebo, zinc sulfate and folic acid, and two placebos. Folic acid was given at a daily dose of 5 mg, and zinc sulfate was given at a daily dose of 66 mg. MAIN OUTCOME MEASURE(S): Before and after treatment, standardized semen and blood samples were obtained for determinations of sperm concentration, motility, and morphology according to World Health Organization guidelines; semen morphology according to strict criteria; and blood folate and zinc concentrations. Effects of the four interventions were evaluated separately in subfertile and fertile men. RESULT(S): Subfertile men demonstrated a significant 74% increase in total normal sperm count and a minor increase of 4% abnormal spermatozoa. A similar trend was observed in fertile men. Pre-intervention concentrations of folate and zinc in blood and seminal plasma did not significantly differ between fertile and subfertile men. CONCLUSION(S): Total normal sperm count increases after combined zinc sulfate and folic acid treatment in both subfertile and fertile men. Although the beneficial effect on fertility remains to be established, this finding opens avenues of future fertility research and treatment and may affect public health.     

Identification of a novel m.9588G > A missense mutation in the mitochondrial COIII gene in asthenozoospermic Tunisian infertile men

Purpose:

Infertility affects 10–15 % of the population, of which, approximately 40 % is due to male etiology consisting primarily of low sperm count (oligozoospermia) and/or abnormal sperm motility (asthenozoospermia). It has been demonstrated that mtDNA base substitutions can greatly influence semen quality.

Methods:

In the present study we performed a systematic sequence analysis of the mitochondrial cytochrome oxidase III (COIII) gene in 31 asthenozoospermic infertile men in comparaison to normozoospermic infertile men (n=33) and fertile men (n=150) from Tunisian population.

Results:

A novel m.9588G>A mutation was found in the mtDNA sperm’s in all asthenozoospermic patients and was absent in the normozoospermic and in fertile men. The m.9588G>A mutation substitutes a highly conserved Glutamate at position 128 to Lysine. In addition, PolyPhen-2 analysis predicted that this variant is “probably damaging”.     

Induction of infertility in adult male bonnet monkeys by immunization with phage-expressed peptides of the extracellular domain of FSH receptor

Active immunization of proven fertile adult male bonnet monkeys (Macaca radiata) with phage-expressed follicle-stimulating hormone receptor (FSHR)-specific peptides from the extracellular domain resulted in a progressive drop in sperm count with all animals becoming azoospermic by day 100. However, serum testosterone concentrations were unaltered during the entire course of study and animals exhibited normal mating behaviour. Breeding studies with proven fertile female monkeys revealed that all the immunized males were infertile. Following interruption of immunization on day 225, sperm counts returned to normal with restoration of fertility. These results indicate that infertility can be induced in adult male monkeys by interfering with the action of FSH using specific peptides of the extracellular domain of FSHR as antigens, without the risk of producing cross-reacting antibodies to the other glycoprotein hormones.     

The effect of cancer on sperm DNA fragmentation as measured by the sperm chromatin dispersion test

The percentage of spermatozoa with fragmented DNA from cancer patients before surgery, chemotherapy, or radiotherapy treatments was compared with infertile male patients in an assisted reproduction program and with sperm donors of proven fertility. The percentages of DNA fragmentation were 34.3% in cancer patients, 30.9% in infertile men whose partners did not become pregnant, 28.8% in men who partners became pregnant, and 10.8% in fertile sperm donors. The DNA fragmentation of sperm donors was statistically significantly lower compared the other groups. No statistically significant differences were found in the levels of DNA fragmentation when comparing cancer types, including those of testicular origin.     

Cigarette smoking and the risk of male factor subfertility: minor association between cotinine in seminal plasma and semen morphology

OBJECTIVE: To evaluate the impact of cigarette smoking on male factor subfertility and the semen parameters of sperm count, motility, and morphology by questionnaire and determination of the cotinine concentrations in blood and seminal plasma of fertile and subfertile males. DESIGN: Case-control study of 107 fertile and 103 subfertile males who provided a standardized blood and semen specimen and completed a self-administered questionnaire about their smoking habits. SETTING: Outpatient fertility clinic of the University Medical Centre St. Radboud, Nijmegen, The Netherlands. PATIENT(S): One hundred seven fertile and 103 subfertile males. INTERVENTION(S): Vena puncture and semen collection. MAIN OUTCOME MEASURE(S): Blood and seminal plasma cotinine levels in relation to semen parameters. RESULT(S): A higher frequency of cigarette smoking was observed in subfertile males than in fertile males, with an odds ratio of 1.7 (95% confidence interval, 0.9-3.2). The self-reported number of cigarettes smoked per day correlated with the cotinine concentrations in blood and seminal plasma for both groups. A small but statistically significant correlation was found between cotinine concentrations in seminal plasma and the percentage of abnormal sperm morphology, but not for other semen parameters (r(s) = 0.19). CONCLUSION(S): Although the mechanism of the toxicity of cotinine on sperm morphology is not clear, this study indicates only a minor effect of cigarette smoking on male factor subfertility, which is probably due to compounds in cigarette smoke other than nicotine (cotinine).     

The microsperm penetration assay: development of a sperm penetration assay suitable for oligospermic males

OBJECTIVE: To develop a specialized sperm penetration assay (SPA) for the evaluation of sperm from oligospermic patients. DESIGN: The development of the assay is in four parts: determine optimal sperm number; demonstrate quality control; establish statistical limits for fertile population; compare results to in vitro fertilization (IVF) outcome. SETTING AND PATIENTS: A group of 63 patients with oligospermia and/or poor motility and a group of 17 fertile donors were compared using the optimized SPA and the micro-SPA. Sperm from a third group of 35 patients were simultaneously incubated with human ova (IVF) and hamster ova (micro-SPA). MAIN OUTCOME MEASURES: Both types of SPA scores are expressed as a sperm capacitation index (penetrations per ovum). Outcome of IVF is expressed as a percent of ova fertilized. RESULTS: Using 25,000 sperm was found to be optimal. The normal fertile range was statistically determined to have a lower limit (-2 SD) of 2.0 penetrations per ovum. When scores from 63 male factor patients were compared using the optimized SPA, only 43% had sufficient swim-up sperm. However, the micro-SPA could accurately test 100% of the samples because it requires only one tenth the number of sperm. CONCLUSION: The micro-SPA provides a valuable diagnostic test for the evaluation of the male factor patient.     

Prognostic value of in vitro sperm penetration into hormonally standardized human cervical mucus

To analyze the prognostic value of the sperm cervical mucus penetration test (SCMPT), fresh semen samples of 99 male patients under infertility investigation were exposed to capillary tubes filled with freshly obtained cervical mucus (CM) of the patients' wives (WCM), fertile donors (DCM), and bovine CM (BCM). The quality of the human CM was standardized by oral administration of estrogens. The overall pregnancy rate after 6 months was 17.2% (17/99), and was significantly different in couples with poor and good SCMPT with WCM (1/44, 2.3% versus 16/55, 29%; P less than 0.001) in a prospective study. Human CM was superior to BCM as a penetration medium in providing more information about sperm function. The results suggest that in vitro sperm penetration testing with hormonally standardized CM of female partners adds an important dimension to sperm analysis with regard to fertility prognosis.     

生育与不育男性生育力指数的比较

目的:评估生育力指数(FI)在判断男性生育能力中的作用。方法:对不育组(n=124)和生育组(n=62)进行精液常规检查,并计算FI[FI=精子密度(106/ml)×精子活动力×精子正常形态率]。结果:生育组FI为13.23(24.16),高于不育组的5.69(10.62)(t=5.657,P=0.001)。生育组FI(P2.5,P97.5)的范围为2.06-56.85。结论:FI较单个精液参数更能客观反映男性生育能力,当FI<2.0时男性生育概率将下降。     

Germ cell transplantation

Transplantation of male germ line stem cells from a fertile donor to the testis of an infertile recipient restores donor-derived spermatogenesis in the recipient testis. The resulting sperm pass the donor genotype to the offspring of the recipient. Germ cell transplantation helped to elucidate the biology of male germ line stem cells and their niche in the testis, develop systems to isolate and culture spermatogonial stem cells, examine defects in spermatogenesis, correct male infertility, and introduce genetic changes into the male germ line. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals, including primates. Potential clinical applications include restoration of fertility in patients who underwent sterilizing treatments for cancer or targeted correction of genetic defects in testicular somatic cells. Recently, ectopic grafting of testis tissue from diverse donor species, including primates, into a mouse host has opened an additional possibility to study spermatogenesis and to produce fertile sperm from immature donors. Testis xenografts are ideally suitable to study toxicants or drugs with the potential to enhance or suppress male fertility without the necessity of performing experiments in the target species. Therefore, transplantation of germ cells and xenografting of testis tissue represent powerful approaches for the study, preservation, and manipulation of male fertility.     

Evaluation of subgroups of the human sperm hypoosmotic swelling test in normozoospermic male cases with recurrent fertilization failure: a prospective case-controlled study

Purpose

To compare subgroups of the human sperm hypoosmotic swelling test subgroups in both recurrent fertilization negative infertile cases with normal semen analysis and fertilization positive controls.

Methods

This was a prospective case-controlled study performed with normospermic 33 previously fertile male (secondary infertility) and 41 infertile men who had undergone two or three unsuccessful in vitro fertilization attempts. HOS test was investigated in 4 subgroups including HOS 1, HOS 2, HOS 3, and HOS 4 according to the degree of sperm tail swelling and compared between the two groups.

Results

Four subgroups were compared and statistical significance was demonstrated in HOS 1, HOS 3 and HOS 4 tests (p < 0.001) in fertile and infertile men. Highest HOS 1 and lowest HOS 4 grades were determined in Group A. However, no statistical significance was determined between two groups in HOS 2 test which was minimal swelling in sperm tails.

Conclusions

HOS 1, HOS 3 and HOS 4 subgroups of HOS test are reliable and useful methods providing important information regarding the sperm function. A high HOS test 1 grade plus a low HOS test 4 grade should suggest a fertility problem, despite a normal semen analysis. HOS test subgroups provide additional information in normospermic cases with unexplained infertility.     

Meeting report: World Congress on Practical Infertility Management

When male germ line stem cells are transplanted from the testis of a fertile donor animal to the testis of an infertile recipient they can establish donor-derived spermatogenesis in the recipient testis, and the resulting sperm can transmit the genotype of the donor to the offspring of the recipient. Germ cell transplantation provides a bioassay to study the biology of these stem cells, to develop systems for spermatogonial stem cell isolation and culture, to examine defects in spermatogenesis and to correct male infertility. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals, including primates. A potential clinical application is restoration of fertility in patients that underwent cytotoxic treatments for cancer. As an alternative to transplantation of isolated germ cells to a recipient testis, ectopic grafting of testis tissue from diverse mammalian donor species, including primates, into a mouse host represents a novel possibility to study spermatogenesis, to investigate the effects of toxins or drugs with the potential to enhance or suppress male fertility, and to produce fertile sperm from immature donors. Therefore, transplantation of germ cells or xenografting of testis tissue are uniquely valuable approaches for the study, preservation and manipulation of male fertility in mammalian species.     

Advances and applications of germ cell transplantation

When male germ line stem cells are transplanted from the testis of a fertile donor animal to the testis of an infertile recipient they can establish donor-derived spermatogenesis in the recipient testis, and the resulting sperm can transmit the genotype of the donor to the offspring of the recipient. Germ cell transplantation provides a bioassay to study the biology of these stem cells, to develop systems for spermatogonial stem cell isolation and culture, to examine defects in spermatogenesis and to correct male infertility. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals, including primates. A potential clinical application is restoration of fertility in patients that underwent cytotoxic treatments for cancer. As an alternative to transplantation of isolated germ cells to a recipient testis, ectopic grafting of testis tissue from diverse mammalian donor species, including primates, into a mouse host represents a novel possibility to study spermatogenesis, to investigate the effects of toxins or drugs with the potential to enhance or suppress male fertility, and to produce fertile sperm from immature donors. Therefore, transplantation of germ cells or xenografting of testis tissue are uniquely valuable approaches for the study, preservation and manipulation of male fertility in mammalian species.     

Glutathione and glutathione S-transferases A1-1 and P1-1 in seminal plasma may play a role in protecting against oxidative damage to spermatozoa

OBJECTIVE: To study the levels of glutathione, glutathione S-transferase A1-1, and glutathione S-transferase P1-1 in seminal fluid of fertile and subfertile men. DESIGN: Retrospective case-control study. SETTING: Departments of gastroenterology, obstetrics and gynecology, and epidemiology and biostatistics in a university medical center. PATIENT(S): Twenty-five subfertile men visiting the fertility clinic and 25 fertile men from midwife practices were recruited. INTERVENTION(S): Collection of semen of subfertile and fertile men. MAIN OUTCOME MEASURE(S): Plasma levels of glutathione and glutathione S-transferases A1-1 and P1-1 in relation to seminal characteristics. RESULT(S): Glutathione, glutathione S-transferase A1-1, as well as glutathione S-transferase P1-1 were found in considerable amounts in seminal fluid of subfertile and fertile men. No differences between groups were found for glutathione S-transferases A1-1 and P1-1. Also, no associations with sperm count, motility, or morphology could be detected. Fertile men had significantly higher glutathione levels as compared with the case of subfertile men. Associations of glutathione with sperm motility quality (r(s) = 0.321) and abnormal sperm morphology (r(s) = -0.496) were found. CONCLUSION(S): The presence of glutathione S-transferases A1-1 and P1-1 in seminal fluid suggests a role in the protection against (oxidative) damage of spermatozoa, whereas glutathione may play a role in male fertility.     

Antisperm antibodies in infertile men and their correlation with seminal parameters

Aim:  Antisperm antibodies (ASA) in males cause the autoimmune disease 'immune infertility'. The present study intended to detect the presence of ASA and their incidence in men with unexplained infertility, as well as to evaluate the correlation between the presence of ASA and semen parameter alterations.
Methods:  Blood and sperm assessment were collected to carry out a direct and indirect mixed antiglobulin reaction (MAR) test and semen analysis in infertile and fertile men from the University Hospital of the Faculty of Medicine, Sao Paulo State University, Sao Paulo.
Results:  In the MAR test, 18.18% of infertile men were positive for ASA. In fertile men, no positivity was found. A significant correlation between the presence of ASA with an increased white blood cell count plus a decreased hypoosmotic swelling test result was observed.
Conclusions:  The results indicate that ASA are involved in reduced fertility. It is not ASA detection per se that provides conclusive information about the occurrence of damage to fertility. The correlation between infertility and altered seminal parameters reinforce the ASA participation in this pathology. (Reprod Med Biol 2007; 6 : 33–38)     

Prognosis of fertility of men with sperm agglutinins in the serum

A retrospective study of 254 infertile men with serum sperm agglutination titers ranging from 4 to 1024 determined by the Kibrick agglutination test between 1954 and 1968 and reexamined in the 1970s is presented. 36 of the subjects had become fertile in the years following the testing, 30 of whom were normospermic. With titers of 32 or more, the probability of becoming fertile was considerably lower than with lower titers. It was confirmed that the longer infertility existed, the less probable was fertility to occur (p less than .01). The serum of 22 men who regained fertility was tested. 19 of the subjects showed no difference in titers between the first and last test. 2 of the 3 remaining fertile men had fathered 2 children each despite titers of 1024 in 1970. The 3rd subject's titer value decreased from 512 to 4 in 1970. It was concluded that sperm agglutinins are involved in the pathogenesis of male infertility.     

不明原因重复性自然流产妇女配偶精子DNA完整性

目的:探讨精子DNA完整性与重复性自然流产(RSA)的关系。方法:85例不明原因RSA妇女配偶(RSA组)和50例已生育的成年健康男性(对照组)的精液,应用精子染色质扩散实验(SCD)检测精子DNA完整性。将RSA组根据1年后怀孕结果分为3个亚组:怀孕组(30例)、流产组(26例)、未孕组(29例)。结果:RSA妇女配偶DNA损伤精子的百分率(14.6±6.9)%与对照组的(12.9±3.8)%相比无统计学差异(P0.05)。DNA损伤精子百分率大于20%视为精子DNA完整性异常,则有17.6%的RSA患者配偶的精子DNA完整性异常,6%的正常生育男性精子DNA完整性异常,但差异无统计学意义(P0.05)。怀孕组、流产组、未孕组配偶的DNA损伤精子百分率分别为(12.4±5.3)%,(14.6±6.5)%和(16.8±8.1)%,未孕组与对照组比较,差异有统计学意义(P0.05),怀孕组、流产组与对照组比较,差异无统计学意义(P0.05)。结论:精子DNA完整性异常与RSA继发不育有关,有必要筛查RSA患者配偶的精子DNA完整性。