Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays

BACKGROUND: Sperm DNA integrity (SDI) is an important factor in the prognosis of male fertility. Here we compare the toluidine blue (TB) image cytometry test, recently proposed by us for SDI assessment, with two other tests-the sperm chromatin structure assay (SCSA) and the terminal nick-end labelling (TUNEL) assay. METHODS: Sperm samples from 35 men were evaluated for standard sperm parameters and subjected to the TB test and SCSA. Eighteen of the 35 samples were also subjected to the TUNEL assay. RESULTS: The proportion of sperm cells with abnormal DNA integrity assayed by the TB test correlated strongly with the proportion of abnormal cells detected by the SCSA and TUNEL assay (rho=-0.84 and rho=0.80, P<0.001, respectively). Furthermore, the fractions of abnormal cells by the TB test corresponded closely to the sum of two SCSA parameters, the DNA fragmentation index (DFI) and the fraction of highly DNA-stainable cells (HDS) (medians 33.0 versus 32.0%, P=0.6). CONCLUSIONS: Abnormal cells in a TB test correspond to the sum of DFI and HDS fractions in the SCSA. TB-positive cells may represent sperm with fragmented DNA and/or abnormal chromatin structure. Because the TB test is an easy and inexpensive method, its potential use as a routine test for sperm DNA integrity, complementary to standard semen parameters, should be investigated further.     

120例精子发生障碍的遗传缺陷研究

目的探讨无精子症,严重少精子症和少、弱精子症患者的遗传缺陷与男性不育的关系。方法采用外周血染色体核型分析技术和Y染色体基因微缺失检测方法,对120例无精子症,严重少精子症和少弱精子的患者进行了遗传咨询。结果在被筛查患者中发现异常染色体核型13例,异常核型发生率为10.83%;而其Y染色体微缺失检测中存在AZFc/SPGY基因缺失31例,缺失率25.83%。结论染色体核型异常和Y染色体微缺失与精子生成障碍有直接逻辑关系。Y染色体AZFc/SPGY区域的微缺失是中国男性不育的重要原因,因此,中国男性不育症患者有必要进行Y染色体AZFc/SPGY微缺失的常规筛查。     

3-氯丙二醇及2,3-氧丙醇合并给药对大鼠附睾、肝、肾超微结构的影响

以每日5mg/kg的3-氯丙二醇及75mg/kg的2,3-氧丙醇同时给大鼠灌胃,连续给药2天后停药8天,共10天为一疗程。观察了3,6,9疗程结束时,附睾起始部主细胞的超微结构改变,及附睾管起始部的精子和肝脏、肾脏的超微结构。发现在3、6疗程末,附睾主细胞的高尔基复合体扁平囊变窄,大泡皱缩。在6疗程末,细胞表面的吞饮小泡和静纤毛减少。仅在3疗程见到粗面内质网扩张。9疗程的超微结构与对照比较无明显区别。结果提示,每一疗程药物引起的附睾主细胞超微结构的改变,在多数动物是可恢复的。但由于个体差异,部分动物于疗程之末,尚未完全恢复。在第6疗程末的肝脏,有部分肝细胞滑面内质网扩张,线粒体体积缩小,基质电子密度增高。这些变化可能是一种轻度的细胞损伤,这种损伤在3,9疗程均未发现,说明它与疗程长短不呈平行关系。肾尿细管上皮细胞及附睾起始部精子的超微结构,无可见改变。这一结果为阐明3-氯丙二醇及2,3-氧丙醇合并用药的抗生育作用,及对有关内脏的毒性作用,提供了形态学的依据。     

Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations

Although various genetic factors have been implicated in human male infertility, the causative genes for the different types of idiopathic male infertility have not been elucidated. Protamines, which are the major DNA-binding proteins in the sperm nucleus, package the DNA into the sperm head. Analysis of the human protamine-1 (PRM1) and -2 (PRM2) gene sequences in 226 sterile male patients and in 270 proven-fertile male volunteers revealed four single nucleotide polymorphisms (SNPs) in the PRM1 coding region, which did not cause any amino acid substitutions, and one SNP in the PRM2 gene, which produced translation termination. We also observed one SNP in the 3' non-coding region of the PRM1 gene, and two SNPs within the intron of the PRM2 gene. The prevalence of these SNPs was similar in both infertile patients and in proven-fertile volunteers, except that the c248t alteration in the PRM2 gene induced a nonsense codon under conditions of heterozygosity in one infertile patient. Although the PRM1 and PRM2 genes are highly conserved, the single SNP in the PRM2 gene that induces translation termination may result in male infertility due to haploinsufficiency of PRM2.     

Recombinant follicle stimulating hormone: development of the first biotechnology product for the treatment of infertility. Recombinant Human FSH Product Development Group

Genes encoding the common gonadotrophin subunit and folliclestimulating hormone (FSH)-specific ß subunit wereisolated from a DNA library derived from human fetal liver cells,and inserted into separate expression vectors containing a selectable/amplifiablegene. These vectors were inserted into the genome of the Chinesehamster ovary cell line, resulting in expression of large amountsof biologically active human (h)FSH. This cell line was culturedon microcarrier beads in a large-scale bioreactor. hFSH in thecell culture supernatant was purified to homogeneity by a multistepprocess. The mature ß subunit had seven fewer aminoacid residues than reported in the literature and three otherdifferences were found in the sequence. Similar oligosaccharidestructures were present on recombinant (r)-hFSH and a purifiedurinary (u)-hFSH preparation. In-vitro and in-vivo, the biologicalactivities of u- and r-hFSH were indistinguishable, r-hFSH wasformulated in ampoules containing 75 IU FSH activity ( 7.5 µgFSH), which accounts for >99% of the protein content of thepreparation. Studies in non-human primates and human volunteersshowed the pharmacokinetics of u- and r-hFSH to be similar.In healthy volunteers, r-hFSH stimulated follicular developmentand induced significant increases in serum oestradiol and inhibin.Clinical experience with r-hFSH has shown it is more effectiveat stimulating ovarian follicle growth than urinary gonadotrophins.It is also effective at initiating spermatogenesis when giventogether with human chorionic gonadotrophin.     

Computer image sperm selection as a novel approach to subzonal insemination in the human

Utilizing real-time computer image analysis, individual spermatozoawere selected using microaspiration. Selection criteria werebased on potential hyperactivation motility characteristics;the amplitude of lateral head displacement >7.5 µm,curvilinear velocity >70 µm/s and linearity of <30%.For this pilot study, 16 patients (eight in each group) wererecruited. Using subzonal insemination (SUZI), up to five (mean= 4.4 ± 0.3) spermatozoa selected using computer-imagesperm selection (CISS) were microinjected, or up to 15 (mean= 12.8 ± 1.3 SD) unselected spermatozoa. In the groupwhich utilized CISS, 28 out of 49 (57%) oocytes were fertilizedcompared with 13 out of 52 (25%) utilizing conventional SUZI(P < 0.04); polyspermy was 20% (n = 10) and 2% (n = 1) respectively.CISS with SUZI showed increased efficiency in achieving fertilizationand is a novel approach to studying individual sperm functionin a sperm egg bioassay where gamete ratios are close to unity.     

The diagnosis of male infertility--prospective time-specific study of conception rates related to seminal analysis and post-coital sperm--mucus penetration and survival in otherwise unexplained infertility

Infertile women without any inherent female infertility factorsand able to secrete normal cervical mucus were studied prospectivelyin relation to post-coital sperm—mucus penetration (PCT)and their partner's seminal analysis, excluding men with azoospermia.Time-specific cumulative conception rates calculated as forlife-table analysis were related to each measured seminal variableon routine analysis of 2–3 samples (volume, density, proportionwith progressive motility, and proportion with normal morphology);to various derivatives from combinations of these variables;to seminal findings after vital staining; and to the PCT results.The best seminal predictor of fertility was the motile normalsperm density (MNSD), the 18 month conception rates being 57.4%+ 4.6 (SE) and 30.2% + 5.9 (ratio 1.9, P < 0.001) above andbelow a derived threshold value of 4 x 10⁶/ml. The PCT led torates of 55.6% ± 4.3 and 14.9% ± 5.1 (ratio 3.73,P < 0.001) for positive and negative results, respectively.The PCT also gave rise to a significantly distinct intermediatepoor-psitive sub-group (conception rate 30.6% ± 9.0).Seminal analysis (the MNSD) did not affect the conception rateassociated with a positive PCT but helped to discriminate furtherwith a negative PCT (conception rates 22.5% ± 8.7 withan MNSD above 4 x 10⁶/ml versus 5.6% ± 4.8 below, P <0.05). The PCT was the single best predictor of fertility butseminal analysis (the MNSD) was of additional value after anegative PCT.     

Changes in anti-Müllerian hormone serum concentrations over time suggest delayed ovarian ageing in normogonadotrophic anovulatory infertility

BACKGROUND: Anti-Müllerian hormone (AMH), produced by growing pre-antral and early antral ovarian follicles, has been shown to be a useful marker for ovarian ageing. Serum AMH concentrations are elevated during reproductive life in anovulatory women, especially in those patients exhibiting polycystic ovaries (PCO). The current study was designed to investigate whether the decrease in AMH serum concentrations over time is different comparing women with normogonadotrophic anovulation [World Health Organization (WHO) group 2 (including polycystic ovary syndrome (PCOS)] and normo-ovulatory controls. METHODS AND RESULTS: AMH serum levels were assessed on two occasions in 98 patients suffering from WHO 2 anovulatory infertility as well as in 41 normo-ovulatory premenopausal women. Median time interval between both visits was 2.6 years (range 0.3-9.0) for WHO 2 patients compared with 1.6 years (range 1.0-7.3) in controls. Serum AMH concentrations were significantly (P < 0.0001) elevated on both occasions in WHO 2 patients (AMH1, median = 7.5 microg/l, range 0.1-35.8; and AMH2, median = 6.7 microg/l, range 0.0-30.6) compared with controls (AMH1, median = 2.1 microg/l, range 0.1-7.4; and AMH2, median = 1.3 microg/l, range 0.0-5.0). Regression analysis, corrected for age, indicated a significant relative decrease in serum AMH concentrations over time for both groups (P < 0.001). However, the decline in serum AMH in WHO 2 patients was significantly less compared with controls (P = 0.03). CONCLUSION: The present longitudinal study shows that serum AMH concentrations decrease over time both in women presenting with WHO 2 anovulatory infertility and in normo-ovulatory controls. The decrease in WHO 2 patients is less pronounced despite distinctly elevated concentrations. This observation may suggest retarded ovarian ageing and hence a sustained reproductive life span in these patients.     

Testicular sperm extraction in azoospermic men submitted to bilateral orchidopexy

BACKGROUND: This study was carried out to evaluate whether bilateral orchidopexy represents a poor or good prognostic factor in azoospermic men undergoing testicular sperm extraction (TESE). METHODS: One hundred and seven presumed non-obstructive azoospermia (NOA) patients, according to conventional clinical parameters (volume of testis, FSH, clinical history) were submitted to testicular biopsy with TESE. Thirty men (28%) had a history of bilateral orchidopexy for cryptorchidism. RESULTS: Normal spermatogenesis or mild hypospermatogenesis was diagnosed in 12/30 ex-cryptorchid patients and in 7/77 presumed NOA patients (P = 0.0004). Conversely, pure Sertoli cell-only syndrome or complete maturation arrest was found in 10/30 ex-cryptorchid patients and in 48/77 presumed NOA patients (P = 0.0094). In 53/107 patients (49.5%), TESE allowed a positive sperm retrieval. At least one spermatozoon was observed in 22/30 ( approximately 73%) ex-cryptorchid patients and in 31/77 ( approximately 40%) presumed NOA patients (P = 0.0026). A large number of spermatozoa (equivalent to an obstructive pathology) were retrieved in 13/30 ex-cryptorchid and in 10/77 presumed NOA patients (P = 0.001). A history of bilateral orchidopexy in presumed NOA patients correlates positively for the chance of retrieving testicular spermatozoa (odds ratio 3.8; 95% confidence interval 1.41-10.21; P = 0.008). CONCLUSIONS: Although bilateral cryptorchidism is usually considered a testicular secretive dysfunction, TESE permits retrieval of a large number of spermatozoa in almost 40% of cases. Our data suggest the existence of congenital or acquired obstructive anomalies of the seminal ducts in azoospermic orchidopexed men.     

Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa

In 25 patients (14 suffering from obstructive azoospermia, sixfrom non-obstructive azoospermia, three from astheno-azoospermiaand two from absence of ejaculation) spermatozoa were extractedfrom testicular biopsies. Intracytoplasmic sperm injection (ICSI)with fresh testicular spermatozoa was performed in 18 cases;spermatozoa in excess were cryopreserved in pills. No pregnancieswere achieved. In the remaining seven patients, testicular spermatozoawere retrieved and cryopreserved during a diagnostic testicularbiopsy. After thawing, sperm motility was assessed in 17 cases(68%), and 18 ICSI with cryopreserved testicular spermatozoawere performed. The mean two-pronuclear (2PN) fertilizationrate was 59%, the mean cleavage rate was 92%, and six clinicalpregnancies were achieved, all of them still ongoing (pregnancyrate 33%). A comparison of the results of ICSI carried out withfresh or cryopreserved testicular spermatozoa showed that themean 2PN fertilization rates per cycle (53 compared with 55%),mean cleavage rates per cycle (99 compared with 96%) and embryoquality were not significantly different In conclusion, cryopreservationof testicular spermatozoa is feasible, even in patients withnon-obstructive azoospermia, and the results of ICSI with frozen-thawedtesticular spermatozoa are similar to those obtained using freshtesticular spermatozoa. Cryopreservation of testicular spermatozoamay avoid repetition of testicular biopsies to retrieve spermatozoafor successive ICSI cycles in patients in whom the only sourceof motile spermatozoa is the testicle.