Blood Leukocyte ROS Production Reflects Seminal Fluid Oxidative Stress and Spermatozoa Dysfunction in Idiopathic Infertile Men.

A large proportion of infertile men do not receive a clear diagnosis, being considered as idiopathic or unexplained cases due to infertility diagnosis based on standard semen parameters. Particularly in unexplained cases, the search for new indicators seems mandatory to provide specific information. In the etiopathogenesis of male infertility oxidative stress displays important roles by negatively affecting sperm quality and function. In this study, performed in a population of 34 idiopathic infertile men and in 52 age-matched controls, redox parameters were assessed in blood, leukocytes, spermatozoa, and seminal fluid and related to semen parameters. The main findings indicate that blood oxidative stress markers reflect seminal oxidative stress. Interestingly, blood leukocyte ROS production was significantly correlated to sperm ROS production and to semen parameters. Overall, these results suggest the potential employ of blood redox markers as a relevant and adjunctive tool for sperm quality evaluation aimed to preconception care.

Sperm retrieval by conventional testicular sperm extraction for assisted reproduction in patients with Zinner syndrome.

We present data from three Caucasian men with Zinner syndrome who attended our center for the treatment of primary couple's infertility. Each patient was scheduled for conventional testicular sperm extraction (cTESE) and cryopreservation. Sperm analysis confirmed absolute azoospermia. Patient 1 had right and left testis volumes of 24 mL and 23 mL, respectively; left seminal vesicle (SV) agenesis, severe right SV hypotrophy with right renal agenesis. Follicle-stimulating hormone (FSH) was 3.2 IU/L. Patient 2 exhibited right and left testis volumes of 18 mL and 16 mL, respectively; a left SV cyst of 32 × 28 mm, ipsilateral kidney absence, and right SV agenesis. FSH was 2.8 IU/L. Patient 3 showed a testicular volume of 10 mL bilaterally, a 65 × 46 mm left SV cyst, right SV enlargement, and left kidney agenesis. FSH was 32.0 IU/L. Sperm retrieval was successful in all patients. Nevertheless, cTESE should be performed on the day of oocyte retrieval.

Cardiometabolic and Thrombotic Risk Profile in Women Undergoing Oocyte Donation for Assisted Reproduction.

Background: The last two decades have seen a growing number of pregnancies in women who needed the donation of oocytes. With oocyte donation pregnancies, studies on obstetric outcomes among these women revealed an increased incidence of pre-eclampsia and pregnancy-induced hypertension. Furthermore, several studies have found a higher incidence of low birth weight, preterm birth, and delivery by cesarean section in oocyte donation rather than in women subjected to assisted reproduction techniques (ART) with autologous oocytes. Numerous studies have also shown a deep connection between cardiovascular and thrombotic risk factors and adverse pregnancy outcomes. In this setting, to strictly assess the preconceptional risk for women who undergo egg donation to achieve pregnancy, the aim of our study is to draw a detailed assessment of the vascular risk profile of patients with gamete donation ART indications through the evaluation of comorbidities and cardiometabolic and thrombophilic markers Materials and Methods: Patients undergoing ART with oocyte or sperm donation or double donation of gametes underwent a careful clinical assessment through a detailed personal and family anamnesis and they were evaluated for cardiometabolic and thrombophilic profile. Clinical and demographic characteristics, comorbidities, and biohumoral parameters were collected. The study was approved by the Regional Ethical Committee(Em 2018-017 CINECA 10189). Results: We evaluated 525 women. Around 73.1% were >40 years and 35% of them were older than 45 years. There was a high prevalence of dyslipidemias (58.1%), smoking habit (24.6%), a body mass index >25 in 28.6% of patients, a high abdominal circumference in 58.1% of cases, a prevalence of acquired thrombophilia in about 7% and hereditary of 19.2%. Around 39.2% of patients had total cholesterol >200 mg/dL, 19.5% had high-density lipoprotein <48 mg/dL and 43.6% had low-density lipoprotein >115 mg/dL, and 6.9% had triglyceride values >150 mg/dL. Conclusions: A careful assessment of the preconceptional status of patients undergoing ART programs with oocyte donation can be highly recommended.

Correction: gr/gr deletion predisposes to testicular germ cell tumour independently from altered spermatogenesis: results from the largest European study.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sperm morphology: What implications on the assisted reproductive outcomes?

OBJECTIVE: To evaluate the impact of sperm morphology (SM) on laboratory and pregnancy outcomes in conventional intracytoplasmic sperm injection (c-ICSI) cycles, using the egg donation model to minimize female confounding variables. MATERIALS AND METHODS: We retrospectively collected data of oocyte donation cycles from October 2016 to February 2020. Median seminal parameters, total (1-2-3PN) fertilization rate (FR), 2PN FR, cleavage rate (CR), implantation rate (IR), pregnancy rate (PR), miscarriage rate (MR), and live birth rate (LBR) were collected. The study population was divided into three groups: Group 1 with SM < 4%, Group 2 with SM between 4% and 6%, and Group 3 with SM > 6%. RESULTS: Of 741 fresh ICSI cycles and 4507 warmed oocytes were included. Male age was 46.0 (31.0-72.0) years, and recipients' age was 44.0 (29.0-54.0) years. Normal SM was 5.0% (1.0%-15.0%). Male age was negatively correlated with normal SM (P = .002; Rho -0.113). Oocyte survival rate was 83.3% (16.7%-100.0%). Total FR was 75.0% (11.1%-100.0%), 2PN FR was 66.7% (11.1%-100.0%) %, and CR was 100% (0.0%-100%). Comparing samples with SM > 6% and those with SM < 4%, 2PN FR was significantly higher in the first group (P = .04). No significant associations were found among groups in terms of CR. IR was 27.7%, resulting significantly higher when normal SM was > 6% (P < .01). Clinical PR was 36.0%, MR was 23.9%, and LBR was 25.9%. PR and LBR were significantly higher in samples with normal SM > 6%, compared to other groups (P = .02 and P < .01, respectively). CONCLUSIONS: Although c-ICSI technique allows the embryologist to select the best quality spermatozoa, male factor plays a key role in achieving successful assisted reproductive outcomes. Normal SM has been shown to have implications not only for laboratory outcomes, in terms of fertilization, but also for clinical findings, as regards implantation, pregnancy, and live birth.

Can cytomegalovirus infection affect male reproductive function? Results of a retrospective single-centre analysis.

Our objective was to investigate whether the chronic cytomegalovirus (CMV) infection can affect semen parameters in men with couple infertility and to assess the impact of male CMV infection on the reproductive outcomes of CMV-seronegative women suffering from tubal factor. Group 1 included CMV IgG-seropositive men, Group 2 CMV IgG-seronegative patients. Seminal parameters, two-pronuclear (2PN) fertilization rate (FR), 1-2-3PN FR, cleavage rate (CR), miscarriage rate (MR), pregnancy rate (PR) and live birth rate (LBR) were collected. Two hundred and twenty-two men were included: 115 (51.8%) in Group 1 and 107 (48.2%) in Group 2. There was reported a low trend towards higher sperm concentration/ml, total sperm count and viability in CMV IgG-seronegative males, compared to CMV IgG-seropositive (p > .05). Semen volume, pH, motility and normal sperm morphology were similar among groups. Considering the subgroup of men, partners of CMV IgG-seronegative females, 65 couples (29.2%) were selected. Median 2PN FR was 67%, total FR 83%, CR 100%, PR/cycle 26.2%, MR 10.8%, LBR/cycle 15.4%. No significant differences were found regarding the reproductive outcomes between CMV IgG-seropositive men and those seronegative. CMV did not seem to play a key role in male reproductive function, as well as in influencing sperm fertility potential in the assisted reproductive outcomes.

Blood plasma miR-20a-5p expression as a potential non-invasive diagnostic biomarker of male infertility: A pilot study.

BACKGROUND: Recently, alterations in miRNAs expression profile in semen have been linked to damaged spermatogenesis, suggesting miRNAs could be used as potential infertility biomarkers. In previous animal studies, miR-20a-5p was found to be down-expressed in low motile spermatozoa, implying its potential target of genes associated with cell apoptosis. OBJECTIVE: To investigate miR-20a-5p expression in blood plasma of patients suffering from non-obstructive azoospermia (NOA), compared to normozoospermic controls. MATERIALS AND METHODS: Between January 2018 and December 2019, from 52 infertile couples eligible for the study, 24 couples were finally enrolled in this monocentric observational prospective pilot study. Patients were included into two groups: Group 1 comprised men with NOA (n = 14) and Group 2 fertile men partners of women with female tubal factor infertility (n = 10). All NOA patients underwent testicular sperm extraction. The expression of circulating miR-20a-5p in plasma samples was assessed by RT-qPCR. A relative quantification strategy was adopted using the 2-ΔCq method to calculate the target miR-20a-5p expression with respect to miR-16-5p as endogenous control. RESULTS: Median blood plasma miR-20a-5p was significantly higher in patients affected by NOA (0.16 2-ΔCt , range: 0.05-0.79 2-ΔCt ) than in fertile controls (0.06 2-ΔCt , range: 0.04-0.10 2-ΔCt ), P < .001. MiR-20a-5p was positively correlated with follicle-stimulating hormone (FSH) (rrho  = -0.490, P = .015) and luteinizing hormone (LH) (rrho  = -0.462, P = .023), and negatively correlated with serum total testosterone (TT) (rrho  = -0.534, P = .007) and right and left testicular size (rrho  = -0.473, P = .020 and rrho  = -0.471, P = .020, respectively). Successful sperm retrieval (SR) rate was 50.0%. Median value of miR-20a-5p did not differ significantly among patients with successful SR and those with negative SR. Testicular histological examination showed: hypospermatogenesis in 6/14 (42.8%), maturation arrest in 4/14 (28.6%), sertoli cell-only syndrome in 4/14 (28.6%). No significant differences in miR-20a-5p were found between histopathological patterns (P > .05). CONCLUSIONS: MiR-20a-5p could represent a novel non-invasive diagnostic biomarker of male infertility.

gr/gr deletion predisposes to testicular germ cell tumour independently from altered spermatogenesis: results from the largest European study.

The association between impaired spermatogenesis and TGCT has stimulated research on shared genetic factors. Y chromosome-linked partial AZFc deletions predispose to oligozoospermia and were also studied in TGCT patients with controversial results. In the largest study reporting the association between gr/gr deletion and TGCT, sperm parameters were unknown. Hence, it remains to be established whether this genetic defect truly represents a common genetic link between TGCT and impaired sperm production. Our aim was to explore the role of the following Y chromosome-linked factors in the predisposition to TGCT: (i) gr/gr deletion in subjects with known sperm parameters; (ii) other partial AZFc deletions and, for the first time, the role of partial AZFc duplications; (iii) DAZ gene dosage variation. 497 TGCT patients and 2030 controls from two Mediterranean populations with full semen/andrological characterization were analyzed through a series of molecular genetic techniques. Our most interesting finding concerns the gr/gr deletion and DAZ gene dosage variation (i.e., DAZ copy number is different from the reference sequence), both conferring TGCT susceptibility. In particular, the highest risk was observed when normozoospermic TGCT and normozoospermic controls were compared (OR = 3.7; 95% CI = 1.5-9.1; p = 0.006 for gr/gr deletion and OR = 1.8; 95% CI = 1.1-3.0; p = 0.013 for DAZ gene dosage alteration). We report in the largest European study population the predisposing effect of gr/gr deletion to TGCT as an independent risk factor from impaired spermatogenesis. Our finding implies regular tumour screening/follow-up in male family members of TGCT patients with gr/gr deletion and in infertile gr/gr deletion carriers.

A Biochemical Approach to Detect Oxidative Stress in Infertile Women Undergoing Assisted Reproductive Technology Procedures.

Oxidative stress plays a major role in critical biological processes in human reproduction. However, a reliable and biologically accurate indicator of this condition does not yet exist. On these bases, the aim of this study was to assess and compare the blood and follicular fluid (FF) redox status of 45 infertile subjects (and 45 age-matched controls) undergoing in vitro fertilization (IVF), and explore possible relationships between the assessed redox parameters and IVF outcomes. Reactive Oxygen Species (ROS) production, assessed by flow cytometry analysis in blood leukocytes and granulosa cells, significantly increased (p < 0.05) in infertile patients. Also, oxidative stress markers-ThioBarbituric Acid-Reactive Substances (TBARS) as an index of lipid peroxidation, and Oxygen Radical Absorbance Capacity (ORAC) to account for total antioxidant capacity, both assayed by fluorometric procedures-in blood and FF were significantly (p < 0.001) modified in infertile patients compared to the control group. Moreover, a significant correlation between blood redox markers and FF redox markers was evident. An ORAC/TBARS ratio, defined as the redox index (RI), was obtained in the plasma and FF of the patients and controls. In the patients, the plasma RI was about 3.4-fold (p < 0.0001) lower than the control, and the FF RI was about six-fold (p < 0.0001) lower than the control. Interestingly, both the plasma RI and FF RI results were significantly correlated (p < 0.05) to the considered outcome parameters (metaphase II, fertilization rate, and ongoing pregnancies). Given the reported findings, a strict monitoring of redox parameters in assisted reproductive techniques and infertility management is recommended.

Validation of a method for noninvasive prenatal testing for fetal aneuploidies risk and considerations for its introduction in the Public Health System.

OBJECTIVE: The aim of this study was to validate noninvasive prenatal testing (NIPT) for fetal aneuploidies by whole-genome massively parallel sequencing (MPS). METHODS: MPS was performed on cell-free DNA (cfDNA) isolated from maternal plasma in two groups: a first set of 186 euploid samples and a second set of 195 samples enriched of aneuploid cases (n = 69); digital PCR for fetal fraction (FF) assessment was performed on 178/381 samples. Cases with <10 × 106 reads (n = 54) were excluded for downstream data analysis. Follow-up data (invasive testing results or neonatal information) were available for all samples. Performances in terms of specificity/sensitivity and Z-score distributions were evaluated. RESULTS: All positive samples for trisomy 21 (T21) (n = 43), trisomy 18 (T18) (n = 6) and trisomy 13 (T13) (n = 7) were correctly identified (sensitivity: 99.9%); 5 false positive results were reported: 3 for T21 (specificity = 98.9%) and 2 for T13 (specificity = 99.4%). Besides FF, total cfDNA concentration seems another important parameter for MPS, since it influences the number of reads. CONCLUSIONS: The overall test accuracy allowed us introducing NIPT for T21, T18 and T13 as a clinical service for pregnant women after 10 + 4 weeks of gestation. Sex chromosome aneuploidy assessment needs further validation due to the limited number of aneuploid cases in this study.

16p11.2 de novo microdeletion encompassing SRCAP gene in a patient with speech impairment, global developmental delay and behavioural problems.

We describe a patient with speech impairment, global developmental delay, behavioural problems and a 186 kb de novo microdeletion on 16p11.2. There are four OMIM Phenotypes entries partially overlapping with the deleted region and related to recurrent microdeletions/microduplications in 16p11.2. A detailed review of published data shows that microdeletions/microduplications' boundaries do not include genes that are deleted in the case here reported. The deletion encompasses 9 RefSeq genes and includes SRCAP (Snf2-related CREBBP activator protein, OMIM*611421), a disease causing gene. Recently, truncating mutations in the SRCAP gene have been shown to cause Floating-Harbor syndrome (FHS, OMIM#136140), a rare disorder characterized by peculiar facial features, short stature with delayed osseous maturation and speech impairment. The patient reported here shows few subtle phenotypic features resembling that of FHS, but she does not have sufficient signs and symptoms for the clinical diagnosis and a clinical classification based on facial gestalt is not possible. This is the first report of a 16p11.2 deletion completely removing one copy of SRCAP, suggesting that haploinsufficiency of this gene could be associated to speech impairment, global developmental delay, behavioural problems and few subtle phenotypic features resembling FHS. However, further evidence for the putative causative role of SRCAP isolated deletion is needed.

X chromosome-linked CNVs in male infertility: discovery of overall duplication load and recurrent, patient-specific gains with potential clinical relevance.

INTRODUCTION: Spermatogenesis is a highly complex process involving several thousand genes, only a minority of which have been studied in infertile men. In a previous study, we identified a number of Copy Number Variants (CNVs) by high-resolution array-Comparative Genomic Hybridization (a-CGH) analysis of the X chromosome, including 16 patient-specific X chromosome-linked gains. Of these, five gains (DUP1A, DUP5, DUP20, DUP26 and DUP40) were selected for further analysis to evaluate their clinical significance. MATERIALS AND METHODS: The copy number state of the five selected loci was analyzed by quantitative-PCR on a total of 276 idiopathic infertile patients and 327 controls in a conventional case-control setting (199 subjects belonged to the previous a-CGH study). For one interesting locus (intersecting DUP1A) additional 338 subjects were analyzed. RESULTS AND DISCUSSION: All gains were confirmed as patient-specific and the difference in duplication load between patients and controls is significant (p = 1.65 × 10(-4)). Two of the CNVs are private variants, whereas 3 are found recurrently in patients and none of the controls. These CNVs include, or are in close proximity to, genes with testis-specific expression. DUP1A, mapping to the PAR1, is found at the highest frequency (1.4%) that was significantly different from controls (0%) (p = 0.047 after Bonferroni correction). Two mechanisms are proposed by which DUP1A may cause spermatogenic failure: i) by affecting the correct regulation of a gene with potential role in spermatogenesis; ii) by disturbing recombination between PAR1 regions during meiosis. This study allowed the identification of novel spermatogenesis candidate genes linked to the 5 CNVs and the discovery of the first recurrent, X-linked gain with potential clinical relevance.

Scoring human sperm morphology using Testsimplets and Diff-Quik slides.

OBJECTIVE: To compare two staining methods to assess sperm morphology: Diff-Quik (DQ), which is the fastest of the recommended techniques, and Testsimplets (TS), a technique that uses prestained slides and is quite popular in in vitro fertilization (IVF) centers. DESIGN: Prospective study. SETTING: Patients at the Sterility Center of the Obstetrics and Gynecology Unit of the Hospital of S.S. Cosma and Damiano (Azienda USL 3 of Pistoia, Italy). PATIENT(S): 104 randomly enrolled male patients evaluated by the seminology laboratory. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Statistical comparison of sperm morphology results obtained after staining of semen samples both with DQ and TS. RESULT(S): Our data show that TS gives a statistically significantly lower number of normal forms than DQ (median: 6% [range: 0-29%] vs. 12% [range: 0-40%], respectively) as well as an overestimation of sperm head defects (median: 92.0% [range: 67%-100%] vs. 82.3% [range: 55%-100%], respectively). CONCLUSION(S): The two staining methods should not be considered equivalent. Specifically, the lower reference limit established by the World Health Organization is not appropriate when sperm morphology is assessed by TS. The routine application of TS in the evaluation of sperm morphology is therefore not recommended because it leads to an overestimation of patients with sperm morphology values below the lower reference limit (4%), thus potentially influencing clinical decisions.

High resolution X chromosome-specific array-CGH detects new CNVs in infertile males.

CONTEXT: The role of CNVs in male infertility is poorly defined, and only those linked to the Y chromosome have been the object of extensive research. Although it has been predicted that the X chromosome is also enriched in spermatogenesis genes, no clinically relevant gene mutations have been identified so far. OBJECTIVES: In order to advance our understanding of the role of X-linked genetic factors in male infertility, we applied high resolution X chromosome specific array-CGH in 199 men with different sperm count followed by the analysis of selected, patient-specific deletions in large groups of cases and normozoospermic controls. RESULTS: We identified 73 CNVs, among which 55 are novel, providing the largest collection of X-linked CNVs in relation to spermatogenesis. We found 12 patient-specific deletions with potential clinical implication. Cancer Testis Antigen gene family members were the most frequently affected genes, and represent new genetic targets in relationship with altered spermatogenesis. One of the most relevant findings of our study is the significantly higher global burden of deletions in patients compared to controls due to an excessive rate of deletions/person (0.57 versus 0.21, respectively; p = 8.785×10(-6)) and to a higher mean sequence loss/person (11.79 Kb and 8.13 Kb, respectively; p = 3.435×10(-4)). CONCLUSIONS: By the analysis of the X chromosome at the highest resolution available to date, in a large group of subjects with known sperm count we observed a deletion burden in relation to spermatogenic impairment and the lack of highly recurrent deletions on the X chromosome. We identified a number of potentially important patient-specific CNVs and candidate spermatogenesis genes, which represent novel targets for future investigations.

Novel insights into DNA methylation features in spermatozoa: stability and peculiarities.

Data about the entire sperm DNA methylome are limited to two sperm donors whereas studies dealing with a greater number of subjects focused only on a few genes or were based on low resolution arrays. This implies that information about what we can consider as a normal sperm DNA methylome and whether it is stable among different normozoospermic individuals is still missing. The definition of the DNA methylation profile of normozoospermic men, the entity of inter-individual variability and the epigenetic characterization of quality-fractioned sperm subpopulations in the same subject (intra-individual variability) are relevant for a better understanding of pathological conditions. We addressed these questions by using the high resolution Infinium 450K methylation array and compared normal sperm DNA methylomes against somatic and cancer cells. Our study, based on the largest number of subjects (n = 8) ever considered for such a large number of CpGs (n = 487,517), provided clear evidence for i) a highly conserved DNA methylation profile among normozoospermic subjects; ii) a stable sperm DNA methylation pattern in different quality-fractioned sperm populations of the same individual. The latter finding is particularly relevant if we consider that different quality fractioned sperm subpopulations show differences in their structural features, metabolic and genomic profiles. We demonstrate, for the first time, that DNA methylation in normozoospermic men remains highly uniform regardless the quality of sperm subpopulations. In addition, our analysis provided both confirmatory and novel data concerning the sperm DNA methylome, including its peculiar features in respect to somatic and cancer cells. Our description about a highly polarized sperm DNA methylation profile, the clearly distinct genomic and functional organization of hypo- versus hypermethylated loci as well as the association of histone-enriched hypomethylated loci with embryonic development, which we now extended also to hypomethylated piRNAs-linked genes, provides solid basis for future basic and clinical research.

TSPY and Male Fertility.

Spermatogenesis requires the concerted action of thousands of genes, all contributing to its efficiency to a different extent. The Y chromosome contains several testis-specific genes and among them the AZF region genes on the Yq and the TSPY1 array on the Yp are the most relevant candidates for spermatogenic function. TSPY1 was originally described as the putative gene for the gonadoblastoma locus on the Y (GBY) chromosome. Besides its oncogenic properties, expression analyses in the testis and in vitro and in vivo studies all converge on a physiological involvement of the TSPY1 protein in spermatogenesis as a pro-proliferative factor. The majority of TSPY1 copies are arranged in 20.4 kb of tandemly repeated units, with different copy numbers among individuals. Our recent study addressing the role of TSPY1 copy number variation in spermatogenesis reported that TSPY1 copy number influences spermatogenic efficiency and is positively correlated with sperm count. This finding provides further evidence for a role of TSPY1 in testicular germ cell proliferation and stimulates future research aimed at evaluating the relationship between the copy number and the protein expression level of the TSPY1 gene.

Small variations in crucial steps of TUNEL assay coupled to flow cytometry greatly affect measures of sperm DNA fragmentation.

Techniques for assessing sperm DNA damage are numerous and various. There are 2 main types of assay: direct and indirect. The former directly detects the amount of sperm DNA damage, whereas the latter reveals the effects of an exogenous insult on sperm chromatin. In addition, even considering the same type of technique, different strategies to reveal or quantify sperm DNA damage, or both, are used. Finally, these techniques, except for sperm chromatin structure assay (SCSA), lack standardized protocols to which all users can adhere to minimize interlaboratory variations. In this study, we investigated the effects of some of the many ways the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) assay is performed when measuring sperm DNA fragmentation by flow cytometry. In addition, by using an established procedure, we determined the precision of the technique by calculating intra-assay coefficients of variation (CVs). We found that concentration of the fixative, the time of storage of fixed samples, the fluorochrome used to label DNA breaks, and the method used to analyze flow cytometric data all greatly affect the measures of sperm DNA fragmentation. In particular, we found that treatment with paraformaldehyde produced additional damage in most samples, suggesting that TUNEL also can be considered an indirect assay when performed in semen samples treated with such a fixative reagent. We also showed that 2 different methods used to analyze data yielded results that, albeit correlating, were different and associated differently to semen quality. On the contrary, the TUNEL assay, as measured here, showed low intraassay CVs, resulting in a quite precise technique when performed in established conditions.

TSPY1 copy number variation influences spermatogenesis and shows differences among Y lineages.

CONTEXT: TSPY1 is a tandemly-repeated gene on the human Y chromosome forming an array of approximately 21-35 copies. The testicular expression pattern and the inferred function of the TSPY1 protein suggest possible involvement in spermatogenesis. However, data are scarce on TSPY1 copy number variation in different Y lineages and its role in spermatogenesis. OBJECTIVES: We sought to define: 1) the extent of TSPY1 copy number variation within and among Y chromosome haplogroups; and 2) the role of TSPY1 dosage in spermatogenic efficiency. MATERIALS AND METHODS: A total of 154 idiopathic infertile men and 130 normozoospermic controls from Central Italy were analyzed. We used a quantitative PCR assay to measure TSPY1 copy number and also defined Y haplogroups in all subjects. RESULTS: We provide evidence that TSPY1 copy number shows substantial variation among Y haplogroups and thus that population stratification does represent a potential bias in case-control association studies. We also found: 1) a significant positive correlation between TSPY1 copy number and sperm count (P < 0.001); 2) a significant difference in mean TSPY1 copy number between patients and controls (28.4 +/- 8.3 vs. 33.9 +/- 10.7; P < 0.001); and 3) a 1.5-fold increased risk of abnormal sperm parameters in men with less than 33 copies (P < 0.001). CONCLUSIONS: TSPY copy number variation significantly influences spermatogenic efficiency. Low TSPY1 copy number is a new risk factor for male infertility with potential clinical consequences.

Partial AZFc deletions and duplications: clinical correlates in the Italian population.

The role of partial AZFc deletions of the Y chromosome in spermatogenic impairment is currently debated. Recently, it was also reported that duplications of the same region are associated with oligozoospermia in Han-Chinese men. The aims of this study were (1) to evaluate the clinical significance of partial AZFc deletions in a large study population and (2) to define if partial AZFc duplications are a risk factor for spermatogenic failure also in a Caucasian population such as the Italian. We screened 556 infertile patients and 487 normozoospermic controls for partial AZFc deletions with a combined method based on STS+/- followed by CDY1-DAZ gene dosage and copy analysis. For the second aim, we performed CDY1-DAZ gene dosage in 229 infertile patients and 263 normozoospermic controls. The frequency of gr/gr deletions in patients was significantly different from the controls (3.2 vs. 0.4%, respectively; P < 0.001), with an OR = 7.9 (95% CI 1.8-33.8). b2/b3 deletions were rare in both groups (0.5% in patients, 0.2% in controls). Concerning gr/gr duplications, we observed no significant differences in their frequency between cases (2.6%) and controls (3.8%). This is the largest study population in the literature in which all potential methodological and selection biases were carefully avoided to detect the clinical significance of partial AZFc deletions and duplications. Our study provides strong evidence that gr/gr deletion is a risk factor for impaired spermatogenesis, whereas we did not detect a significant effect of b2/b3 deletions and partial AZFc duplications on spermatogenesis in this Caucasian ethnic group.