Identification of protein changes in human spermatozoa throughout the cryopreservation process.

Cryoinjury is a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, morphology, and viability. This study was designed to identify potential proteomic changes in human sperm cells throughout the cryopreservation process. Comparisons made within this study included the detection of the sperm proteomic changes induced by incubation of the sperm cells with a protein-free cryoprotectant (with and without CryoSperm), and the proteomic changes induced by freezing, thawing, and subsequent after-thawing incubation at two different temperatures (0 °C vs. 23 °C). Tandem Mass Tag (TMT) peptide labeling coupled with LC-MS/MS was used for protein quantification. LC-MS/MS resulted in the identification of 769 quantifiable proteins. The abundance of 105 proteins was altered upon CryoSperm incubation. Freezing and thawing also induced substantial protein changes. However, fewer changes were observed when semen was thawed and then maintained after-thawing at approximately 0 °C than when it was maintained after-thawing at 23 °C, with 60 and 99 differential proteins detected, respectively, as compared to unfrozen semen incubated in CryoSperm. Collectively, these differences indicate that substantial changes occur in the sperm proteome at every stage of the cryopreservation process which may ultimately impair the sperm fertilizing capability. This is the first study to compare protein levels in fresh and cryopreserved semen using the TMT technology coupled to LC-MS/MS.

Identification of endogenous metabolites in human sperm cells using proton nuclear magnetic resonance ((1) H-NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS).

The objective of this study was to contribute to the first comprehensive metabolomic characterization of the human sperm cell through the application of two untargeted platforms based on proton nuclear magnetic resonance ((1) H-NMR) spectroscopy and gas chromatography coupled to mass spectrometry (GC-MS). Using these two complementary strategies, we were able to identify a total of 69 metabolites, of which 42 were identified using NMR, 27 using GC-MS and 4 by both techniques. The identity of some of these metabolites was further confirmed by two-dimensional (1) H-(1) H homonuclear correlation spectroscopy (COSY) and (1) H-(13) C heteronuclear single-quantum correlation (HSQC) spectroscopy. Most of the metabolites identified are reported here for the first time in mature human spermatozoa. The relationship between the metabolites identified and the previously reported sperm proteome was also explored. Interestingly, overrepresented pathways included not only the metabolism of carbohydrates, but also of lipids and lipoproteins. Of note, a large number of the metabolites identified belonged to the amino acids, peptides and analogues super class. The identification of this initial set of metabolites represents an important first step to further study their function in male gamete physiology and to explore potential reasons for dysfunction in future studies. We also demonstrate that the application of NMR and MS provides complementary results, thus constituting a promising strategy towards the completion of the human sperm cell metabolome.

Polymorphisms, haplotypes and mutations in the protamine 1 and 2 genes.

Protamines are the most abundant nuclear proteins and alterations in their expression have been described in infertile patients. Also, protamine haplo-insufficient mice have been described as infertile. Therefore, the protamine 1 and 2 genes have been considered important candidates in different mutational studies. In this article, we review all published articles related to protamine gene mutations and report new data on mutations from patients and controls drawn from the Spanish and Swedish populations. Sequencing of the protamine 1 and 2 genes in a total of 209 infertile patients and 152 fertility-proven controls from the Spanish and Swedish populations identified two novel and rare non-pathogenic missense mutations (R17C and R38M) in the protamine 1 gene and several additional polymorphisms. Furthermore, we have identified and we report for the first time five novel rare haplotypes encompassing the protamine 1 and 2 genes. A review of all available protamine gene mutational studies indicates that none of the reported missense mutations can be considered of proven pathogenicity. However, it is interesting to note that rare protamine 1 promoter variants have been reported only in infertile patients, but not in fertile control groups. Pathogenic high penetrance protamine gene missense mutations, if any, must be extremely rare. However, the detected presence of rare variants and haplotypes in infertile patients deserves further investigation.

Phenotypic variation within European carriers of the Y-chromosomal gr/gr deletion is independent of Y-chromosomal background.

BACKGROUND: Previous studies have compared sperm phenotypes between men with partial deletions within the AZFc region of the Y chromosome and non-carriers, with variable results. In this study, a separate question was investigated, the basis of the variation in sperm phenotype within gr/gr deletion carriers, which ranges from normozoospermia to azoospermia. Differences in the genes removed by independent gr/gr deletions, the occurrence of subsequent duplications or the presence of linked modifying variants elsewhere on the chromosome have been suggested as possible causal factors. This study set out to test these possibilities in a large sample of gr/gr deletion carriers with known phenotypes spanning the complete range. RESULTS: In total, 169 men diagnosed with gr/gr deletions from six centres in Europe and one in Australia were studied. The DAZ and CDY1 copies retained, the presence or absence of duplications and the Y-chromosomal haplogroup were characterised. Although the study had good power to detect factors that accounted for >or=5.5% of the variation in sperm concentration, no such factor was found. A negative effect of gr/gr deletions followed by b2/b4 duplication was found within the normospermic group, which remains to be further explored in a larger study population. Finally, significant geographical differences in the frequency of different subtypes of gr/gr deletions were found, which may have relevance for the interpretation of case control studies dealing with admixed populations. CONCLUSIONS: The phenotypic variation of gr/gr carriers in men of European origin is largely independent of the Y-chromosomal background.

Chromosomal abnormalities and polymorphisms in infertile men.

The aim of this study was to determine the prevalence of alterations and normal variable chromosome features in males from infertile couples. Karyotyping was performed to 84 men attending the infertility clinic at the Hospital Clinic i Provincial of Barcelona (Spain). Sex chromosome abnormalities were detected in 19 patients (26.62%): 14 (16.67%) aneuploidies 47,XXY and 47,XYY, 3 (3.57%) Y-chromosome long arm deletions; 1 (1.19%) mosaic 45,x/46,XY and 1 (1.19%) Robertsonian translocation (45.X-15-Y+t(15p: Yq). Chromosomal polymorphisms were observed in 29 patients. Yqh+ was the most frequent variant in sex chromosomes and increased length in heterochromatin and satellites were present in autosomal chromosomes. The high prevalence of chromosomal abnormalities observed in infertile men justify the use of karyotyping to evaluate males enrolled in new assisted reproductive technologies programs.

Y-chromosomal DNA haplotypes in infertile European males carrying Y-microdeletions.

We have determined Y-chromosomal DNA haplotypes in 73 infertile European males carrying Y microdeletions and compared them with the haplotypes of 299 infertile males lacking microdeletions. Chromosomes were typed with a set of 11 binary Y markers, which identified 8 haplogroups in the sample. Haplogroup frequencies were compared between 3 microdeletion classes and the non-deleted infertile males. Deletions arise on many different haplotypic backgrounds. No statistically significant differences in frequency were seen, although the small number of AZFa deletions lay predominantly on one branch of the Y haplotype tree.

Serum inhibin B determination is predictive of successful testicular sperm extraction in men with non-obstructive azoospermia.

Recent work indicates that serum inhibin B is a useful marker of spermatogenesis and inhibin B production sufficient to maintain detectable serum concentrations in adults depends on spermatogenic activity. The purpose of the present study was to investigate the usefulness of serum inhibin B measurement to predict the success of testicular sperm extraction (TESE) in 17 men with nonobstructive azoospermia to be treated by intracytoplasmic sperm injection (ICSI) (group 1). Two additional groups were used as positive controls; group 2 comprised 22 infertile men having obstructive azoospermia, and group 3, which included 29 semen donors having normal seminal parameters. Follicle stimulating hormone (FSH) was significantly higher (P < 0.01) and inhibin B significantly lower (P < 0.001), in group 1 as compared with groups 2 and 3. Serum inhibin B concentrations were significantly higher (P < 0.001) among successful TESE men as compared with those having failed TESE. In contrast, no differences were detected between these two groups with respect to serum FSH or testicular size. In addition, serum inhibin B but not FSH discriminated between successful and failed TESE in group 1 subjects as compared with control groups. According to the receiver operating characteristics curve analysis, the best inhibin B value for discriminating between successful and failed TESE was >40 pg/ml (sensitivity 90%, specificity 100%). It is concluded that inhibin B measurement is a useful non-invasive predictor of spermatogenesis and thus, all azoospermic males should have serum inhibin B concentrations determined in addition to FSH measurement and karyotyping prior to undergoing TESE.

Factor masculino, factor seminal y factor espermático: ¿nuevos diagnósticos o nuevos enfoques en esterilidad? / Male factor, seminal parameters and spermatic factor: new diagnoses or a new approaches on infertility?

La patología masculina en los casos de esterilidad conyugal parece incrementarse en las últimas décadas. En el presente trabajo se revisan los nuevos enfoques en la valoración diagnóstica de esta patología reproductiva, consecuentes a las nuevas técnicas de reproducción asistida. Pero estos nuevos procedimientos no deben distorsionar al clínico en su intento de lograr un correcto diagnóstico etiológico, especialmente en lo que a factores genéticos se refiere, lo que facilitará el posible empleo previo de eficaces tratamientos médicos, y en los casos de que estos no sean factibles, orientará acerca de los posibles riesgos de alteraciones genéticas. Por otro se insiste en la necesidad de persistir en la investigación básica, lo que facilitará la creación de programas preventivos que frenen el aparente deterioro progresivo de la fertilidad del varón (AU)

Management of HIV serodiscordant couples. The clinician point of view.

BACKGROUND: Couples where the male partner is HIV-infected but the female partner is not often express a desire for children. Assisted reproduction techniques (ART) with sperm "wash" can reduce the risk of acquisition of infection for the woman. The information about the characteristics of such couples coming to these clinics for advice and support is scant. OBJECTIVE: To describe the characteristics of a cohort of HIV-discordant couples seeking reproductive assistance in a referral clinic in Barcelona, Spain. METHODS: Demographic, social and HIV related information were collected prospectively according to a standard protocol. Blood tests, a full evaluation for STDs and sperm analysis were performed. RESULTS: Between April 1994 and February 1999, 140 couples contacted the clinic for reproductive advice, 100 couples were fully assessed and were offered reproductive assistance. The vast majority were white, with a history of injecting drug use. Nearly half of the men had been diagnosed during their present relationship. STDs were uncommon and abnormalities among sperm analysis were frequent. CONCLUSION: An increasing number of HIIV infected individuals desire to have children. With the introduction of HAART and the safety of sperm wash in such cases, the need to have children is more frequently expressed. These couples are in general healthy and accept to be included in an ART program.

Prevalence of Y chromosome microdeletions in oligospermic and azoospermic candidates for intracytoplasmic sperm injection.

OBJECTIVE: To determine the prevalence and type of Y chromosome microdeletions in 136 consecutively seen intracytoplasmic sperm injection (ICSI) candidates and in 50 consecutively seen azoospermic men attending an infertility clinic. DESIGN: Controlled clinical study. SETTING: Genetics laboratory and infertility clinic at a University hospital. PATIENT(S): One hundred eighty-six men who were seen at an infertility clinic and who were referred to a genetics counseling service for genetic assessment before ICSI. INTERVENTION(S): Collection of semen and blood samples. MAIN OUTCOME MEASURE(S): Semen analysis; serum FSH, LH, and T levels; karyotype analysis; and presence or absence of several single tagged site markers along the Y chromosome (sY274, sY238, sY276, sY84, sY102, sY143, sY153, sY254, sY269, sY202, sY158, sY160). RESULT(S): Yq chromosome microdeletions were detected in 10 (5.4%) of 186 consecutively seen ICSI candidates. The number of microdeletions was much higher in azoospermic patients (16%; 8 of 50) than in oligospermic patients (1.5%; 2 of 136). Two of the azoospermic patients with a Yq microdeletion also had sex chromosome aneuploidy mosaicism. No microdeletions were detected in 100 consecutively seen fathers who were included as controls. CONCLUSION(S): The prevalence of Yq microdeletions in the azoospermic group was much higher than in the oligospermic group and was consistent with the prevalence of Yq microdeletions detected in other series of azoospermic men in different geographic areas. All Yq microdeletions found in our patients belong to the AZFc region, indicating that microdeletions of the AZFa and AZFb regions are infrequent among oligospermic ICSI candidates or azoospermic males in our population.

Detection of P2 precursors in the sperm cells of infertile patients who have reduced protamine P2 levels.

OBJECTIVE: To determine whether the reduction in the protamine P2 content (increased P1/P2 ratio) reported in some infertile patients could result from incomplete processing of protamine P2 precursors. DESIGN: Analysis of samples with a marked reduction in the protamine P2 content using polyacrylamide gel electrophoresis and subsequent detection of protamine P2 precursors through Western blot analysis. SETTING: University departments and laboratories. PATIENT(S): One hundred eighty-four men undergoing an evaluation for infertility. MAIN OUTCOME MEASURE(S): Comparative Western blot analysis of nuclear sperm proteins using specific antibodies to protamine P1 and protamine P2. RESULT(S): After selection of the samples with a marked reduction of the protamine P2 content and subsequent analysis by Western blot, a small proportion of putative P2 precursors was detected in most samples, whereas a significant increase was detected in two of them. CONCLUSION(S): In some infertile men, a reduction in the protamine P2 content relative to protamine P1 (increased P1/P2 ratio) is detected concomitant with an increase in the amount of putative P2 precursors. This could represent the first report of incomplete processing of a nuclear sperm protein in humans.

Human chorionic gonadotrophin luteal support overcomes luteal phase inadequacy after gonadotrophin-releasing hormone agonist-induced ovulation in gonadotrophin-stimulated cycles.

Gonadotrophin-releasing hormone agonist (GnRHa)-induced ovulation after gonadotrophin ovarian stimulation is used to prevent ovarian hyperstimulation syndrome and multiple pregnancy in polyfollicular cycles. However, one of the major problems to be resolved is corpus luteum function after follicular maturation and ovulation by mid-cycle GnRHa administration. The present report investigated the luteal phase in non-conceptual polyfollicular cycles in 26 patients (group 1) receiving a single dose of 0.5 mg leuprolide acetate to induce ovulation and in a control group of patients (n = 26) (group 2) who were given human chorionic gonadotrophin (HCG) (10,000 IU i.m.) for ovulation induction. All of them were normal ovulatory women undergoing gonadotrophin ovarian stimulation because of unexplained infertility or male factor. In both groups of patients two doses of 2500 IU HCG i.m. were given 6 and 10 days after the ovulatory dose of HCG or GnRHa to support the luteal phase. All cycles were ovulatory as shown by mid-luteal serum progesterone concentrations >10 ng/ml. Mean serum progesterone concentrations were 62% higher in group 2 than in group 1, but this difference was not statistically significant. The mean length of the luteal phase was similar in groups 1 and 2. It is concluded that HCG luteal support is a useful tool to overcome the luteal phase inadequacy that characterizes GnRHa-triggered cycles after gonadotrophin stimulation.

Testicular function in asymptomatic chronic alcoholics: relation to ethanol intake.

To evaluate the effect of ethanol on testicular function in chronic alcoholics without chronic liver disease, we studied 38 asymptomatic chronic alcoholics and 19 age-matched controls. Detailed clinical history, nutritional status, hormonal analysis, and seminal studies were conducted in each case and control. Alcoholic patients had an average of 39 +/- 2 years old (range: 26 to 60) and reported a daily ethanol consumption from 100 to 350 g (mean: 198 +/- 15) over a period of 18.0 +/- 1.2 years. Alcoholics exhibited a significant increase of the luteinizing hormone (p < 0.001) and a decrease of the Free Androgen Index, compared with controls (p < 0.05) that related significantly with the total lifetime dose of ethanol (p < 0.01, both). Seminal studies indicate that 39.4% of alcoholics had significantly reduced their spermatozoa count (p < 0.01), whereas significant morphological abnormalities were observed in 44.7% of the alcoholics (p < 0.01). Spermatozoa motility from alcoholics was also found to be altered in half of the patients (p < 0.01). A significant increase of serum luteinizing hormone, follicle-stimulating hormone, and sex hormone binding globulin levels, and a decrease of Free Androgen Index were observed in alcoholics with morphology and motility abnormalities (p < 0.05, all). In multivariate analysis, the only independent factor that determined the alterations in sperm (count, morphology abnormalities, and motility alterations) was the total lifetime of ethanol intake (p < 0.001, all). We conclude that alcoholics frequently develop a situation of primary hypogonadism related to a lifetime of ethanol consumption.

The role of luteinizing hormone in human follicle development and oocyte fertility: evidence from in-vitro fertilization in a woman with long-standing hypogonadotrophic hypogonadism and using recombinant human follicle stimulating hormone.

To evaluate the relative importance of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in follicular development and oocyte fertility in the human species, the use of recombinant human FSH, human menopausal gonadotrophin (HMG), and very highly purified urinary human FSH (FSH-HP) plus oestradiol valerate for ovarian stimulation and in-vitro fertilization (IVF) were compared in three cycles in a woman with isolated congenital gonadotrophin deficiency who had never been treated with ovarian stimulating agents. The total number of ampoules of gonadotrophins used was lower in the HMG treatment cycle. Ovarian response and IVF outcome in the three treatment cycles were as follows: (i) HMG cycle: normal follicular growth, normal pattern of oestradiol and inhibin through the menstrual cycle, high fertilization rate (93%); (ii) recombinant FSH cycle: normal follicular growth, low oestradiol and abnormal inhibin, finally poor rate of fertilization (28%); (iii) FSH-HP plus oestradiol valerate cycle: normal follicular growth, normal pattern of inhibin and poor fertilization rate (27%). Luteal plasma progesterone concentrations were much higher in the HMG treatment cycle. This case shows that FSH is the only factor required in order to induce follicular growth in the human, although LH or a product derived from its action may assist in order to achieve full follicular maturity and oocytes capable of fertilization. Though oestradiol might have a mediatory role in the process of follicular maturation, our results favour a direct primary role of LH in complete maturation of the follicle.

Late low-dose pure follicle stimulating hormone for ovarian stimulation in intra-uterine insemination cycles.

At present, there is general agreement that ovarian stimulation improves pregnancy rates after intra-uterine insemination (IUI). Also, ovulation induction with gonadotrophins is associated with higher success rates than clomiphene citrate in IUI cycles. However, the drawbacks to the use of gonadotrophin stimulation before IUI include the risks of ovarian hyperstimulation and multiple gestation, and the relative cost of a treatment cycle in a view of the medication costs and the need for increased monitoring by hormone assays and ultrasonographic measurements. In the present prospective randomized trial, the efficacy and safety of ovarian stimulation with clomiphene citrate (50 mg/day for 5 days) and IUI (clomiphene/IUI group) were compared with those of late low-dose pure follicle stimulating hormone (FSH, 75 IU/day from day cycle 7 until the leading follicle reached > 17 mm in diameter) and IUI (FSH/IUI group) in ovulatory women who were infertile because of unexplained infertility (n = 40) or male subfertility (n = 60). The mean length of treatment in the FSH group was 6.4 +/- 2.5 days. Multiple follicular development was seen in 25% of clomiphene-stimulated cycles but only in 8% of those treated with FSH. Pregnancy rate per cycle in clomiphene/IUI and FSH/IUI groups was 4% (4/98) and 13% (12/94) respectively (P = 0.02). All pregnancies obtained were singleton. There were two and one clinical abortions in the clomiphene/IUI (50%) and FSH/IUI (8%) groups respectively. No patient developed ovarian hyperstimulation syndrome. Use of our therapeutic scheme, which proved to be efficacious, safe and economic for ovarian stimulation in IUI cycles, is advocated before the institution of in-vitro fertilization (IVF) or gamete intra-Fallopian transfer (GIFT) therapy in infertile patients with patient Fallopian tubes. This late low-dose technique of administering pure FSH is suitable for use in offices without immediate access to oestradiol results.

Intra-uterine insemination, cervical pregnancy and successful treatment with methotrexate.

Cervical pregnancy is rare but one of the most dangerous of all pregnancy complications. The aetiology of cervical implantation is unclear and because of the fear of profuse haemorrhage abdominal hysterectomy has been historically considered the standard therapy. We report here an unusual case of an infertile woman with cervical ectopic pregnancy after ovulation induction with gonadotrophins and intrauterine insemination. The patient was successfully treated with parenteral methotrexate without significant morbidity. It is concluded that chemotherapy should be initially considered in most cervical pregnancies either as the sole form of treatment or to improve the chances of success in alternative conservative measures.

Triggering of ovulation by a gonadotropin releasing hormone agonist in gonadotropin-stimulated cycles for prevention of ovarian hyperstimulation syndrome and multiple pregnancy.

Ovarian hyperstimulation syndrome (OHSS) and multiple pregnancies are the two main complications of ovulation induction using gonadotropins. Withholding an ovulatory dose of human chorionic gonadotropin (hCG) remains the safest option for prevention of both complications. However, this policy frustrates both patient and physician, wastes time and money due to cancelled treatment, and results in cancellation of a high proportion of cycles that would not have progressed to clinical OHSS. As gonadotropin releasing hormone analogs (GnRH-a) may elicit surges of endogenous luteinizing hormone and follicle stimulating hormone, we investigated the usefullness of a single s.c. injection of leuprolide acetate (0.5 mg) to trigger ovulation, without inducing OHSS or multiple pregnancy, in 23 consecutive gonadotropin-stimulated cycles which would otherwise have been cancelled. All patients had at least 4 mature follicles (> or = 14 mm in diameter) and plasma estradiol levels > 1000 pg/ml on the day of GnRH-a injection. No luteal support was given. Seventeen of the 23 (74%) cycles were ovulatory and four singleton pregnancies resulted, giving a pregnancy rate of 17.4% per cycle. The remaining six patients (26%) clearly had defective or short luteal phases. No patient developed OHSS. It is concluded that GnRH-a may be an acceptable substitute for hCG to salvage treatment cycles in patients thought to be at risk for OHSS or multiple pregnancy. However, further studies are necessary for optimization of this approach in order to improve ovulatory and conceptional results.

Complete selective absence of protamine P2 in humans.

Male sterility due to abnormal sperm morphology or motion has been widely reported, although relatively little has been published on the sperm nuclear protein abnormalities. We report the first cases worldwide of infertile patients having a complete selective absence of protamine P2 in the sperm nucleus. This provides a selective phenotype that will aid understanding of the mechanisms of synthesis, processing, or function of the P2 protamines. In addition, it is of marked immediate relevance to medicine as it allows the diagnosis of this type of human male sterility and the opportunity to understand the basis of this defect.

Transvaginal intratubal insemination, ectopic pregnancy and treatment by single-dose parenteral methotrexate.

We report a case of a woman with ectopic pregnancy with fetal cardiac activity after ovulation induction and transvaginal intratubal insemination by tactile sensation. The patient was successfully treated by single-dose methotrexate (MTX) (77 mg or 50 mg/m2 given intramuscularly). Control hysterosalpingograms showed no tubal patency on the involved side. Potential advantages and hazards of transvaginal intratubal insemination and single-dose MTX for ectopic pregnancy are discussed.

Human in vitro fertilization in couples with unexplained infertility and a poor postcoital test.

The results of in vitro fertilization (IVF) with the husband's semen were compared in 3 groups of fully investigated infertile couples. In those with tubal infertility (n = 43) 194 out of 273 (71.1%) oocytes were fertilized, at least 1 oocyte being fertilized in 42 of the 43 couples (97%). In couples with unexplained infertility and a 'good' postcoital test (PCT) (n = 9), 39 out of 64 (61.5%) oocytes were fertilized in 8 of the 9 (88%) couples. In couples with unexplained infertility and a 'poor positive' PCT despite normal semen analysis, 16 out of 44 (36.3%) oocytes were fertilized in 5 of the 6 (83%) couples. These results show a good correlation between in vivo sperm penetration of cervical mucus and human IVF in couples with unexplained infertility.