Non-invasive ultrasound localization microscopy (ULM) in azoospermia: connecting testicular microcirculation to spermatogenic functions.

Rationale: Azoospermia is a significant reproductive challenge. Differentiating between non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) is crucial as each type requires distinct management strategies. Testicular microcirculation plays a profound role in spermatogenic functions. However, current diagnostic methods are limited in their ability to effectively elucidate this crucial connection. Methods: We employed ultrasound localization microscopy (ULM) to visualize testicular microcirculation in NOA and OA patients and quantified the testicular hemodynamic parameters. Pearson correlation analysis was conducted to investigate the inner connection between parameters of testicular microcirculation and clinical spermatogenic functions. We conducted multiple logistic regression analysis to establish a new diagnostic model that integrates follicle-stimulating hormone (FSH) and mean vascular diameter to distinguish NOA from OA. Results: Our findings demonstrated significant differences in vascular parameters between NOA and OA, with NOA characterized by lower mean vascular diameter (p < 0.001), vessel density (p < 0.001), and fractal number (p < 0.001). Testicular volume showed a moderate positive correlation with mean vascular diameter (r = 0.419, p < 0.01) and vessel density (r = 0.415, p < 0.01); Mean vascular diameter exhibited negative correlations with both FSH (r = -0.214, p < 0.05) and age (r = -0.240, p < 0.05); FSH (r = -0.202, p < 0.05) and luteinizing hormone (LH) (r = -0.235, p < 0.05) were negatively correlated with mean blood flow velocity. The diagnostic model demonstrated an area under the curve (AUC) of 0.968. We also reported a method to map the vascular pressure distribution derived from the blood flow velocity generated by ULM. Conclusions: ULM provides a non-invasive and detailed assessment of testicular microvascular dynamics. The ULM-derived vascular parameters are able to connect testicular microcirculation to spermatogenic functions. The combination of FSH and mean vascular diameter enhances diagnostic precision and holds potential for distinguishing NOA from OA.

The therapeutic potential of adipose tissue-derived mesenchymal stromal cells in the treatment of busulfan-induced azoospermic mice.

PURPOSE: The generation of germ cells from mesenchymal stromal cells (MSCs) provides a valuable in vitro platform for infertility modeling. The establishment of these cells is a new approach for assisted reproductive technology (ART) to help infertile patients who lack functional gametes. METHODS: Human adipose-derived MSCs were isolated and then characterized for multipotency by flow cytometry, differentiation capacity, and cytogenetic assays. These cells were used in a male germ cell differentiation study. The expression of male germ cell markers was evaluated at day 21 of differentiation using an immunofluorescence assay, flow cytometry, and RT-qPCR. Undifferentiated MSCs were used for transplantation in busulfan-induced azoospermic mice. RESULTS: In this study, MSCs were successfully isolated from human adipose tissues which were positive for cell markers such as CD90, CD105, CD73, and CD29 but negative for CD34 and CD45. The results of flow cytometry, immunocytochemistry, and RT-qPCR analysis at day 21 of differentiation showed that the undifferentiated adipose-derived MSCs are able to differentiate into male germ cells. Additionally, transplantation of undifferentiated MSCs in busulfan-induced azoospermic mice caused spermatogenesis recovery in the majority of seminiferous tubules. CONCLUSION: In this study, we showed that differentiation of human adipose-derived MSCs into male germ cells is a useful tool for in vitro study of human germ cell development. Our results demonstrated that cell therapy with adipose-derived MSCs could help the repair of pathological changes in testicular seminiferous tubules. Therefore, it may have a clinical application for the treatment of azoospermia in infertile patients.

Meiotic Chromosome Synapsis and XY-Body Formation In Vitro.

To achieve spermatogenesis in vitro, one of the most challenging processes to mimic is meiosis. Meiotic problems, like incomplete synapsis of the homologous chromosomes, or impaired homologous recombination, can cause failure of crossover formation and subsequent chromosome nondisjunction, eventually leading to aneuploid sperm. These meiotic events are therefore strictly monitored by meiotic checkpoints that initiate apoptosis of aberrant spermatocytes and lead to spermatogenic arrest. However, we recently found that, in vitro derived meiotic cells proceeded to the first meiotic division (MI) stage, despite displaying incomplete chromosome synapsis, no discernible XY-body and lack of crossover formation. We therefore optimized our in vitro culture system of meiosis from male germline stem cells (mGSCs) in order to achieve full chromosome synapsis, XY-body formation and meiotic crossovers. In comparison to previous culture system, the in vitro-generated spermatocytes were transferred after meiotic initiation to a second culture dish. This dish already contained a freshly plated monolayer of proliferatively inactivated immortalized Sertoli cells supporting undifferentiated mGSCs. In this way we aimed to simulate the multiple layers of germ cell types that support spermatogenesis in vivo in the testis. We found that in this optimized culture system, although independent of the undifferentiated mGSCs, meiotic chromosome synapsis was complete and XY body appeared normal. However, meiotic recombination still occurred insufficiently and only few meiotic crossovers were formed, leading to MI-spermatocytes displaying univalent chromosomes (paired sister chromatids). Therefore, considering that meiotic checkpoints are not necessarily fully functional in vitro, meiotic crossover formation should be closely monitored when mimicking gametogenesis in vitro to prevent generation of aneuploid gametes.

Variant PNLDC1, Defective piRNA Processing, and Azoospermia.

BACKGROUND: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility. METHODS: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing. RESULTS: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1: the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations. CONCLUSIONS: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).

Iatrogenic effects of radical cancer surgery on male fertility.

Iatrogenic causes of male infertility can include medications, chemotherapy, radiation, and surgery. In this review, we discuss commonly performed urologic cancer surgeries and nonurologic surgeries that harbor a high risk of iatrogenic infertility. These include radical prostatectomy, radical cystectomy, retroperitoneal lymph node dissection, pelvic colon surgery, and anterior spine surgery. In addition, we review the anatomy and surgical strategies that help to reduce the risks of infertility. With an increase in life expectancy and improvements in fertility preservation, it is important to properly counsel patients about the risks of infertility and provide options for fertility preservation before surgery.

Poor intracytoplasmic sperm injection outcome in infertile males with azoospermia factor c microdeletions.

OBJECTIVE: To explore whether the presence of azoospermia factor c (AZFc) microdeletions adversely affects intracytoplasmic sperm injection (ICSI) outcome. DESIGN: Retrospective cohort. SETTING: University hospital. PATIENT(S): A total of 293 patients with azoospermia or severe oligozoospermia AZFc deletions underwent 345 ICSI cycles, and 363 idiopathic patients with normal Y chromosome underwent 462 ICSI cycles. INTERVENTION(S): Testicular sperm aspiration, microdissection testicular sperm extraction. MAIN OUTCOME MEASURE(S): The main clinical outcome parameters were cumulative clinical pregnancy rate, cumulative live birth delivery rate, and no embryo suitable for transfer cycle rate. RESULT(S): Compared with the control group, the AZFc deletion group exhibited poorer ICSI outcome, with significant differences between the 2 groups for cumulative clinical pregnancy rate (45.39% vs. 67.49%; odds ratio [OR], 2.843; 95% confidence interval [CI]), cumulative live birth delivery rate (35.15% vs. 53.44%; OR, 2.234; 95% CI), no embryo suitable for transfer cycle rate (15.07% vs. 8.23%; OR, 0.565; 95% CI), fertilization rate (46.80% vs. 53.37%; adjusted ß, -0.074; 95% CI), implantation rate (28.63% vs. 31.26%; adjusted ß, -0.075; 95% CI) separately. The poor ICSI outcome of the AZFc deletion group was related to AZFc microdeletions by linear and logistic regression analyses. CONCLUSION(S): AZFc microdeletions adversely affect ICSI outcome; patients with AZFc deletion should be informed that they have reduced opportunities to be biological fathers.

Homozygous mutations in C14orf39/SIX6OS1 cause non-obstructive azoospermia and premature ovarian insufficiency in humans.

Human infertility is a multifactorial disease that affects 8%-12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still poorly understood. Synaptonemal complex (SC) is a conserved tripartite structure that holds homologous chromosomes together and plays an indispensable role in the meiotic progression. Here, we identified three homozygous mutations in the SC coding gene C14orf39/SIX6OS1 in infertile individuals from different ethnic populations by whole-exome sequencing (WES). These mutations include a frameshift mutation (c.204_205del [p.His68Glnfs∗2]) from a consanguineous Pakistani family with two males suffering from non-obstructive azoospermia (NOA) and one female diagnosed with premature ovarian insufficiency (POI) as well as a nonsense mutation (c.958G>T [p.Glu320∗]) and a splicing mutation (c.1180-3C>G) in two unrelated Chinese men (individual P3907 and individual P6032, respectively) with meiotic arrest. Mutations in C14orf39 resulted in truncated proteins that retained SYCE1 binding but exhibited impaired polycomplex formation between C14ORF39 and SYCE1. Further cytological analyses of meiosis in germ cells revealed that the affected familial males with the C14orf39 frameshift mutation displayed complete asynapsis between homologous chromosomes, while the affected Chinese men carrying the nonsense or splicing mutation showed incomplete synapsis. The phenotypes of NOA and POI in affected individuals were well recapitulated by Six6os1 mutant mice carrying an analogous mutation. Collectively, our findings in humans and mice highlight the conserved role of C14ORF39/SIX6OS1 in SC assembly and indicate that the homozygous mutations in C14orf39/SIX6OS1 described here are responsible for infertility of these affected individuals, thus expanding our understanding of the genetic basis of human infertility.

Combined Use of Whole Exome Sequencing and CRISPR/Cas9 to Study the Etiology of Non-Obstructive Azoospermia: Demonstration of the Dispensable Role of the Testis-Specific Genes C1orf185 and CCT6B.

The genetic landscape of male infertility is highly complex. It is estimated that at least 4000 genes are involved in human spermatogenesis, but only few have so far been extensively studied. In this study, we investigated by whole exome sequencing two cases of idiopathic non-obstructive azoospermia (NOA) due to severe hypospermatogenesis. After variant filtering and prioritizing, we retained for each patient a homozygous loss-of-function (LoF) variant in a testis-specific gene, C1orf185 (c.250C>T; p.Gln84Ter) and CCT6B (c.615-2A>G), respectively. Both variants are rare according to the gnomAD database and absent from our local control cohort (n = 445). To verify the implication of these candidate genes in NOA, we used the CRISPR/Cas9 system to invalidate the mouse orthologs 4930522H14Rik and Cct6b and produced two knockout (KO) mouse lines. Sperm and testis parameters of homozygous KO adult male mice were analyzed and compared with those of wild-type animals. We showed that homozygous KO males were fertile and displayed normal sperm parameters and a functional spermatogenesis. Overall, these results demonstrate that not all genes highly and specifically expressed in the testes are essential for spermatogenesis, and in particular, we conclude that bi-allelic variants of C1orf185 and CCT6B are most likely not to be involved in NOA and male fertility.

Predictive model to estimate the chances of successful sperm retrieval by testicular sperm aspiration in patients with nonobstructive azoospermia.

OBJECTIVE: To explore predictors of successful sperm retrieval (SR) and to identify potentially suitable candidates for testicular sperm aspiration (TESA), a more straightforward, less traumatic, and less costly procedure than open surgical SR methods. DESIGN: Retrospective chart review. SETTING: Academic tertiary medical center. PATIENTS: A total of 297 patients with nonobstructive azoospermia. INTERVENTIONS: All patients underwent full clinical evaluation before undergoing a staged SR procedure, starting with TESA and proceeding to microsurgical testicular sperm extraction (microTESE). Predictors of positive SR with TESA were selected using the least absolute shrinkage and selection operator (LASSO) regression analysis using k-fold cross-validation. The obtained regression coefficients were used to create a predictive model, and a receiver operating characteristic (ROC) curve was obtained to express its predictive ability. Cut-off values for each significant predictor were also identified using ROC analysis. MAIN OUTCOME MEASURE(S): Development of a prediction model for positive SR with TESA. RESULTS: Overall, a positive SR was observed in 23.6% of patients undergoing TESA. Average testis size (P = .017) and serum follicle-stimulating hormone (FSH) level (P < .001) were the significant predictors of positive SR identified by LASSO regression analysis. The predictive model had an AUC of 0.742 with a sensitivity of 73.9% and specificity of 69.9%. Patients presenting with an average testis size >7.75 mL and serum FSH level <8.5 IU/L had a TESA-positive SR of 43%. CONCLUSIONS: TESA may be a suitable alternative to microTESE in selected nonobstructive azoospermia patients presenting with an average testis size >7.75 mL and serum FSH level <8.5 IU/L.

Testicular steroid sulfatase overexpression is associated with Leydig cell dysfunction in primary spermatogenic failure.

BACKGROUND: Decreased testosterone (T) to LH ratio and increased 17ß-estradiol (E2) serum concentrations represent a common finding among patients with severe spermatogenic failure, suggesting a concurrent Leydig cell steroidogenic dysfunction. Aromatase overexpression has been associated with increased serum and intratesticular E2 in these patients. However, it is unknown whether the sulfatase pathway contributes to the increased availability of active estrogens in patients with primary spermatogenic failure. OBJECTIVES: To assess estrogen sulfotransferase (SULT1E1) and steroid sulfatase (STS) mRNA abundance in testicular tissue of patients with Sertoli cell-only syndrome (SCOS) and normal tissues, its association with serum and intratesticular hormone levels, and to explore the mRNA and protein testicular localization of both enzymes. MATERIALS AND METHODS: Testicular tissues of 23 subjects with SCOS (cases) and 22 patients with obstructive azoospermia and normal spermatogenesis (controls) were obtained after biopsy. SULT1E1 and STS transcripts accumulation was quantified by RT-qPCR. For mRNA and protein localization, we performed RT-qPCR in Leydig cell clusters and seminiferous tubules isolated by laser-capture microdissection and immunofluorescence in testicular tissues. Serum and intratesticular hormones were measured by immunoradiometric assays. RESULTS: SULT1E1 mRNA accumulation was similar in both groups. The amount of STS mRNA was higher in cases (p = 0.007) and inversely correlated with T/LH ratio (r = -0.402; p = 0.02). Also, a near significant correlation was observed with intratesticular E2 (r = 0.329, p = 0.057), in agreement with higher intratesticular E2 in cases (p < 0.001). Strong STS immunoreaction was localized in the wall of small blood vessels but not in Leydig cells. Both SULT1E1 and STS mRNA abundance was similar in Leydig cell clusters and the tubular compartment, except for lower SUTL1E1 mRNA in the seminiferous tubules of SCOS patients (p = 0.001). CONCLUSIONS: Our results suggest that an unbalance of the STS/SULT1E1 pathway contributes to the testicular hyperestrogenic microenvironment in patients with primary spermatogenic failure and Leydig cell dysfunction.

Substance P restores spermatogenesis in busulfan-treated mice: A new strategy for male infertility therapy.

Male infertility has become an important health problem that is primarily caused by testicular dysfunction with abnormal spermatogenesis. In this study, we demonstrated that the neuropeptide, substance P (SP), is essential for spermatogonia proliferation in a seminiferous tubule culture system. In addition, SP (5 nmol/kg) treatment markedly restored spermatogenesis, improved sperm quality, and increased the number of ZBTB16+ or LIN28+ undifferentiated spermatogonia as well as STRA8+ differentiated spermatogonia in a busulfan-induced non-obstructive azoospermic mouse model. Furthermore, 100 nM SP treatment in vitro significantly stimulated the proliferation of GC-1 spg cells (a spermatogonia cell line) via activation of the Erk1/2 signaling pathway. Moreover, the sperm quality and the number of spermatogonia were significantly reduced after treatment with RP67580, a selective NK-1 receptor antagonist, suggesting that SP-NK1R signaling plays an important role in spermatogenesis. Taken together, these results suggest that SP may be a potential therapeutic agent for male infertility by accelerating the restoration of spermatogenesis.

Testicular Testosterone and Estradiol Concentrations and Aromatase Expression in Men with Nonobstructive Azoospermia.

CONTEXT: Spermatogenesis is strictly regulated by the intratesticular hormonal milieu, in which testosterone (T) and estradiol (E2) play pivotal roles. However, the optimal expression of aromatase and intratesticular T (ITT) and E2 (ITE2) levels are unknown. OBJECTIVE: To investigate ITT/ITE2 and aromatase expression in men with nonobstructive azoospermia (NOA) and to elucidate the roles of aromatase in spermatogenesis, as determined based on sperm retrieval by microdissection testicular sperm extraction (micro-TESE). DESIGN AND SETTING: A retrospective study at a reproductive center using serum, testicular specimens, and intratesticular fluid. PATIENTS: Seventy-six men with NOA, including 4 men who received 3 months of anastrozole administration prior to micro-TESE, and 18 men with obstructive azoospermia. INTERVENTIONS: Testicular aromatase expression was evaluated using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (RT-PCR). Intratesticular T and ITE2 levels were determined using liquid chromatography-tandem mass spectrometry. RESULTS: Aromatase was mainly located in Leydig cells, and the levels of its transcript and protein expression levels were increased in men with NOA. No correlation was observed between serum T/E2 and ITT/ITE2 levels, whereas significant associations were observed between decreased ITT and increased ITE2, aromatase expression, and sperm retrieval. Treatment with anastrozole increased the ITT/ITE2 ratio and decreased aromatase expression. CONCLUSIONS: A close association between the expression of aromatase in Leydig cells and ITT/ITE2 was shown. Leydig cell aromatase is a factor that is independently correlated with spermatogenesis, and aromatase inhibitors may open a therapeutic window by increasing ITT/ITE2 in selected patients.

Effects of testicular sperm aspiration upon first cycle ICSI-ET for type 2 diabetic male patients.

Male diabetes mellitus (DM) can affect erectile function and sperm quality. In severe cases, DM can lead to retrograde or no ejaculation, so testicular sperm aspiration (TESA) is combined with intracytoplasmic sperm injection (ICSI) to treat subfertility and infertility for DM couples. However, the effect of TESA upon ICSI (TESA-ICSI) for DM patients remains unclear. This research investigated the effect of TESA-ICSI on first cycle ICSI-embryo transfer (ICSI-ET) for type 2 diabetic mellitus (T2DM) patients and the potential mechanisms. The subjects consisted of 1219 male patients with azoospermia or retrograde ejaculation who were treated with TESA-ICSI from 2015.01 to 2019.11. They were classified into two groups, the T2DM group (n = 54) and non-diabetic control group (n = 1165). Sperm selection for injection was performed using motile sperm organelle morphology examination criteria. The number of available embryos and the high-quality embryo rates following a single ET as well as cleavage, fertilization, implantation, clinical pregnancy and the abortion rates were noted. Compared with the non-diabetic group, the available embryo rate (75.20 ± 26.40% vs.78.36 ± 23.25%) and high-quality embryo rate (46.49 ± 30.37% vs. 47.55 ± 28.57%) in the T2DM group were lower and the abortion rate (20.83% vs. 8.88%) was higher, but these differences were not statistically significant. There were no significant differences in clinical pregnancy, implantation, normal fertilization, and cleavage rates between the two groups. The results show that TESA for male T2DM patients does not influence the effect of ICSI. For T2DM patients with severe oligozoospermia, asthenospermia, teratozoospermia, or retrograde ejaculation that do not meet ICSI criteria, TESA-ICSI may perhaps be considered for reproductive assistance. ABBREVIATIONS: DM: diabetes mellitus; TESA: testicular sperm aspiration; ICSI: intracytoplasmic sperm injection; ICSI-ET; ICSI-embryo transfer; LH: luteinizing hormone; mL: milliliter; TES: testosterone; FSH: follicle-stimulating hormone; P: progesterone; HCG: human chorionic gonadotropin.

Novel micro-straw for freezing small quantities of human spermatozoa.

OBJECTIVE: To evaluate a novel micro-straw as an efficient, simple method for freezing a small number of human spermatozoa for intracytoplasmic sperm injection (ICSI). DESIGN: Prospective cohort study. SETTING: Sperm bank. PATIENT(S): Men with severe oligozoospermia or azoospermia undergoing a total of 143 ICSI cycles at the CITIC-Xiangya Hospital of Reproduction and Genetics from June 1, 2015, to June 31, 2019, and 20 donors at the Hunan Province Human Sperm Bank from 2001 to 2016. INTERVENTION(S): Analysis of sperm samples and clinical outcomes after sperm use. MAIN OUTCOME MEASURE(S): Clinical information, including number of motile sperm before and after freezing, freeze-thaw survival rates, two-pronuclear fertilization rates, clinical pregnancy, and early pregnancy loss rates after sperm use. RESULT(S): In the feasibility experiment using the micro-straw, we found a freeze-thaw survival rate of 73% ± 8.3% and no difference in normal sperm morphology, normal acrosome integrity, or DNA fragmentation index between the micro-straw and 1.8-mL cryotubes. The prospective cohort included 1,325 cases, and we collected sperm from testicular, epididymis, and ejaculation sources. We observed motile sperm in 1,294 (97.6%) of 1,325 frozen-thawed samples. Postthaw sperm were available for ICSI in 140 (97.9%) of 143 of cycles. The fertilization, cleavage, and high-quality embryo rates were 1,007 (81.7%) of 1,233; 995 (98.8%) of 1,007; and 537 (53.9%) of 995, respectively. Sixty-nine (49%) clinical pregnancies were achieved, and the miscarriage rate was 6 (8.6%) of 69. CONCLUSION(S): The micro-straw is suitable and clinically useful for the cryopreservation of small numbers of spermatozoa.

Intronic variation of the SOHLH2 gene confers risk to male reproductive impairment.

OBJECTIVE: To evaluate whether SOHLH2 intronic variation contributes to the genetic predisposition to male infertility traits, including severe oligospermia (SO) and different nonobstructive azoospermia (NOA) clinical phenotypes. DESIGN: Genetic association study. SETTING: Not applicable. PATIENT(S): Five hundred five cases (455 infertile patients diagnosed with NOA and 50 with SO) and 1,050 healthy controls from Spain and Portugal. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genomic DNA extraction from peripheral blood mononuclear cells, genotyping of the SOHLH2 polymorphisms rs1328626 and rs6563386 using the TaqMan allelic discrimination technology, case-control association analyses using logistic regression models, and exploration of functional annotations in publicly available databases. RESULT(S): Evidence of association was observed for both rs6563386 with SO and rs1328626 with unsuccessful sperm retrieval after testicular sperm extraction (TESE-) in the context of NOA. A dominant effect of the minor alleles was suggested in both associations, either when the subset of patients with the manifestation were compared against the control group (rs6563386/SO: P=.021, odds ratio [OR] = 0.51; rs1328626/TESE-: P=.066, OR = 1.46) or against the group of patients without the manifestation (rs6563386/SO: P=.014, OR = 0.46; rs1328626/TESE-: P=.012, OR = 2.43). The haplotype tests suggested a combined effect of both polymorphisms. In silico analyses evidenced that this effect could be due to alteration of the isoform population. CONCLUSION(S): Our data suggest that intronic variation of SOHLH2 is associated with spermatogenic failure. The genetic effect is likely caused by different haplotypes of rs6563386 and rs1328626, which may predispose to SO or TESE- depending on the specific allelic combination.

Rates of hypogonadism forms in Klinefelter patients undergoing testicular sperm extraction: A multicenter cross-sectional study.

INTRODUCTION AND OBJECTIVES: Adult patients with Klinefelter syndrome (KS) may present with testicular volume loss and a decrease in circulating testosterone (T) levels. However, the actual rate of hypogonadism in adult KS men is unknown. We aimed to (a) assess the prevalence of different forms of hypogonadism in a cohort of KS patients with non-obstructive azoospermia (NOA); and (b) investigate potential preoperative predictor of positive sperm retrieval (SR) at surgery in the same cohort of men. METHODS: Complete data from 103 KS men with NOA who underwent testicular sperm extraction (TESE) between 2008 and 2019 at five centers were analyzed. Comorbidities were scored with the Charlson Comorbidity Index (CCI). Patients were categorized into four groups of hypogonadism as follows: eugonadism [normal total T (tT) (≥3.03 ng/mL) and normal luteinizing hormone (LH) (≤9.4 mUI/mL)], secondary hypogonadism [low tT (≤3.03 ng/mL) and low/normal LH (≤9.4 mUI/mL)], primary hypogonadism [low tT (≤3.03 ng/mL) and elevated LH (≥9.4 mUI/mL)], and compensated hypogonadism [normal tT (≥3.03 ng/mL) and elevated LH (≥9.4 mUI/mL)]. Descriptive statistics tested the association between clinical characteristics and laboratory values among the four groups. RESULTS: Median (IQR) patients age was 32 (24, 37) years. Baseline follicle-stimulating hormone and tT levels were 29.5 (19.9, 40.9) mUI/mL and 3.8 (2.5, 11.0) ng/mL, respectively. Eugonadism, primary hypogonadism, and compensated hypogonadism were found in 16 (15.6%), 34 (33.0%), and 53 (51.4%) men, respectively. No patients had secondary hypogonadism. Positive SR rate at TESE was 21.4% (22 patients); of 22, 15 (68.2%) patients underwent assisted reproductive technology and five (22.7%) ended in live birth children. Patients' age, BMI, CCI, FSH levels, and positive SR rates were comparable among hypogonadism groups. No preoperative parameters were associated with positive SR at logistic regressions analysis. CONCLUSIONS: Findings from this cross-sectional study showed that 15.6% of adult KS men have normal tT values at presentation in the real-life setting. Most KS patients presented with either compensated or primary hypogonadism. Sperm retrieval rates were not associated with different forms of hypogonadism.

Testicular Microlithiasis Is Associated with Impaired Spermatogenesis in Patients with Unexplained Infertility.

INTRODUCTION: Testicular microlithiasis (TML) was shown to be associated with an increased risk of infertility. However, the association of TML with spermatogenesis in patients with unexplained infertility is still unknown. In this study, we therefore investigated the effect of TML on hormones and sperm parameters in a large cohort of infertile men without major factors for impaired fertility and azoospermic men serving for comparison. METHODS: Over a period of 10 years, we retrospectively analyzed 2,914 patients who attended our centre with the diagnosis of unexplained infertility and sperm count >1 million/ejaculate, as well as 281 patients with unexplained azoospermia. From the 2,914 patients, we identified 218 patients with TML as revealed by ultrasound imaging. Further, 26 out of 281 azoospermic patients showed TML. Subsequently, we performed a thorough analysis of reproductive parameters and their association with TML. RESULTS: The overall incidence of TML in patients with unexplained infertility and in unexplained azoospermic men was 7.5 and 9.3%, respectively. Patients with unexplained infertility and TML showed significantly smaller testicular volume, elevated FSH level, and lower sperm count and motility. Impaired spermatogenesis was not associated with the amount of microlithiasis, considered after classification into subgroups (<5 vs. ≥5 microliths/testis), and instead was associated with presence or absence of TML. TML in unexplained infertile azoospermic patients was not significantly associated neither with andrological reproductive parameters nor with sperm retrieval rate in microsurgical testicular sperm extraction. DISCUSSION/CONCLUSION: TML itself, and not the number of microliths, is associated with impaired spermatogenesis in patients with unexplained infertility. The parameter TML alone is not sufficient to predict spermatogenic impairment in azoospermic patients. This study highlights the importance of ultrasound imaging in the clinical evaluation of infertile men, taking into account that TML is a negative co-factor for male fertility.

Reproductive outcomes with donor sperm in couples with severe male-factor infertility after intracytoplasmic sperm injection failures.

PURPOSE: To evaluate reproductive outcomes of artificial insemination and IVF with donor sperm (AID or IVF-D) for male-factor couples with a history of unsuccessful ICSI attempt. METHODS: This retrospective cohort includes couples with severe male-factor infertility who failed ICSI treatment, and subsequently underwent semen donation treatment. We report the following outcomes: (1) live birth rates in AID and IVF-D treatment for couples with severe male infertility factors and prior ICSI failures; (2) paternal impact on embryo development of the same oocyte cohort; (3) prognostic factors in obtaining a live birth with donor semen. RESULTS: Of 92 women with failed ICSI cycles (26 with multiple attempts), 45 couples underwent AID treatment. Live birth rate per cycle of AID was 18.9%. Fifty-three patients underwent IVF-D including 6 couples who previously did not conceive with AID. Embryological outcomes including fertilization, viable cleavage embryos, and blastocyst formation rates were significantly lower in ICSI cycles with partner sperm compared with IVF-D (P < 0.01). Logistic regression analysis showed that female age and the severity of spermatogenetic disorder are prognostic factors in obtaining a live birth with donated sperm. CONCLUSION: Couples with severe male infertility factor (azoospermia or extreme oligoasthenospermia) and a history of unsuccessful ICSI cycles benefit from treating with donor sperm. ICSI fertilization, embryo viability, and progression of the embryo to the blastocyst stage are significantly deteriorated by semen parameters. The prognostic factors identified may help couples plan their treatment and prepare for their parenthood journey.

Prediction of microdissection testicular sperm extraction outcome in men with idiopathic nonobstruction azoospermia.

The aim of the present study is to assess whether the preoperative clinical indicators have an impact on sperm retrieval rate (SRR) in men with idiopathic nonobstructive azoospermia (NOA).We retrospectively studied 241 consecutive men with NOA who underwent microdissection testicular sperm extraction from 2016 to 2019 in the Reproductive Medicine Center, including 154 patients diagnosed with idiopathic NOA. They were grouped according to preoperative indicators, including average testicular volume, follicle-stimulating hormone (FSH), luteinizing hormone, Testosterone (T), and pathology, respectively.The overall SRR was 20.0% (31/155). Men with testicular volume of ≤5 mL had significant higher SRR than men with testes 5 to 10 and ≥10 mL (35.6% vs 12.3%, P = .002; 35.6% vs 16.2, P = .049, respectively). The SRR in men with FSH ≥ 24.8 mIU/mL was significant higher, compared with FSH level of 12.4 to 24.8 mIU/mL (32.6% vs 15.8%, P = .033). Men with Sertoli cell-only had significantly lower SRR than other pathological type (8.1%). Men with an FSH ≥ 24.8 mIU/mL in testicular volume ≤5 mL group had a significantly higher SRR than FSH level of 12.4 to 24.8 mIU/mL in testicular volume of ≤5 to 10 mL group (44.0% vs 11.4%, P = .002). Men with a luteinizing hormone level of 8.6 to 17.2 mIU/mL in testicular volume of 5 to 10 mL group had a poor prognosis, with an SRR of only 6.5%.Severely reduced testicular volume (≤5 mL) and severely increased FSH level (≥24.8 mIU/mL) had the better sperm retrieval outcome, which can be used as independent predictors in men with idiopathic NOA. And a combination of testicular volume and the hormone seemed to be useful in further increase predictive value.

Obesity with High Body Mass Index Does Not Influence Sperm Retrieval in Males with Azoospermia.

BACKGROUND The aim of this study was to evaluate the influence of male body mass index (BMI) on the retrieval of sperm from azoospermic patients who were undergoing testicular sperm extraction (TESE). MATERIAL AND METHODS The study included retrospective data of male patients suffering from non-obstructive azoospermia (NOA). Age, BMI, testicular volumes, the serum concentration of the follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and prolactin were investigated and collected. RESULTS A total of 75 azoospermic males were evaluated between 2014 and 2019, including 35 patients (46.7%) with positive sperm retrieval. The majority of patients (57.3%) had normal BMI (between 20 kg/m² and 25 kg/m²) or first degree obesity (from 25 kg/m² to 30 kg/m²). No statistically significant correlation between BMI and positive sperm retrieval or hormone levels (LH, FSH, SHBG, prolactin) were found. However, lower serum testosterone levels were observed in patients with higher BMI (P=0.035). Receiver operating characteristic curve analysis showed that none of the hormones could potentially predict the positive outcome of TESE. CONCLUSIONS The hormonal levels or patient's BMI could not predict positive sperm retrieval outcome, however a negative correlation between serum testosterone and BMI levels was calculated implicating influence on fertility.