Severe Oligo Research Articles

P-469 Micro-vapor fast freezing of human spermatozoa: development of a new method to use low number of cryopreserved spermatozoa

Abstract Study question Can Vapor Fast Freezing conducted in 10µl tips (microVFF/tips) efficiently preserve sperm viability and motility in banking samples with very low sperm concentration? Summary answer MicroVFF resulted able to efficiently preserve sperm viability and motility in patients with severe oligozoospermia or criptozoospermia What is known already Sperm cryopreservation is usually performed by conventional slow or fast freezing using carriers containing large semen volume, such as 500ul High Security Straws (500µl HSS). These carriers however are not suitable for patients with very low sperm concentration and many devices have been tested to freeze small volumes or even singularly isolated sperms (i.e.SpermVD®, Cryoloop, Cell Sleeper). Recently, our group showed that micro-VFF better preserve sperm viability and motility or DNA integrity with respect to a VFF conducted with 500µl HSS, in normozoospermic samples. Study design, size, duration The study was designed for 20 oligospermic or criptozoospermic subjects afferent to Semen Cryopreservation and Andrology Laboratory of Careggi Hospital for semen analysis, but during 24 months up to 34 patients were recruited for the trial. 13 samples were subsequently thawed and, due to the low sperm number, only motility and viability was compared between the two methods. Participants/materials, setting, methods 34 semen samples with low sperm concentration (0.06-7.7mil/ml) were cryopreserved with either microVFF/tips or the conventional method (VFF/ 500µl HSS). Thawing was conducted by keeping sample at room temparature and immediately observed by light microscopy at 20x and 40x. Total motility was scored as rapid/slow/non-progressive motility (a+b+c). Viability was evaluated by eosin test. For each method, the recovery of motility and viability was calculated as: post-thaw percentage/pre-thaw percentage Main results and the role of chance The microVFF method showed a better recovery of motility than the conventional method: 0.38 (±0.21)% vs 0.21 (±0.14)% (n = 13, p < 0.05). A similar statistically significant difference was observed for the mean recovery of viability: 0.43 (±0.20) vs 0.29 (±0.13) (n = 13, p < 0.5). for micro-freezing and conventional method, respectively. As frozen samples are used in the ART setting, microVFF/tips also shows the technical advantage to discharge directly the sample in fresh clean medium bridge in the ICSI plate, where swimming spermatozoa are washed from cryoprotectant compounds. Skipping conventional centrifugation/washing step after thawing highly protect from sperm motility/viability loss, as recently demonstrated by our group. Limitations, reasons for caution Results need to be confirmed in a larger number of patients. Although we previously showed that microVFF/tips also better preserve sperm DNA integrity than conventional method, in this study no other evaluations were possible due to the low number of spermatozoa/sample Wider implications of the findings Present results appear to be promising to enlarge the population who can be offered semen cryopreservation in our laboratory, including Klinefelter patients, severe oligo- and criptozoospermic menand patients undergoing testicular biopsy. Trial registration number not applicable

A Clinical study on the efficacy of Udvartana and Nasya on Ovulatory functions w.s.r. to Female infertility in Moderate Obese (Sthula) individuals

Childlessness and infertility are rising dramatically in cities. The increase may be due to many reasons like the way of living, coping with stress, job pressure, obesity, postponing parenthood, galloping urbanization, sexually transmitted infections and many others. Hence treating infertility is the challenging job for the doctors in present era. The failure to ovulate is the major problem in approximately 40% cases of female infertility. This can be anovulation or severe oligo ovulation. In the latter case even though the ovulation does occur, its relative infrequency decreases the womanand#39;s chance of pregnancy. Hence the infertility and especially ovarian factor induced female infertility needs an immediate attention from alternative medicines. Over last decades, fertility therapy has expanded more than any other field of medicine. Hormonal therapy, In vitro Fertilization (IVF), Embryo Transfer (ET), Gamete Intra Fallopian Transfer (GIFT) etc., all these have a minimal success rate. Additionally these procedures are associated with adverse effects andamp; are not affordable to all. Ayurveda may give a promising hand to cure this condition with the various treatment modalities mentioned in the classics. For the present clinical study, an effort was made to study the efficacy of Udvartana and Nasya followed by Shamanaushadhis in reversing the pathology of this disease and giving a “Never-Ending Joy” of parenthood to the couples through the Ayurvedic treatment.

The prevalence of midline prostatic cysts and the relationship between cyst size and semen parameters among infertile and fertile men.

When is the investigation and treatment of midline prostatic cysts (MPC) of clinical value in the work-up of males of infertile couples? With a prevalence of 10.2% in infertile men, MPC should be investigated according to a seminal algorithm detecting a MPC volume >0.117 ml, which may impair semen parameters, and could be treated to improve sperm count and achieve natural pregnancy. MPC are frequent and are considered a correctable cause of male infertility. However, they have been poorly investigated in an infertility setting. In addition, no study has investigated clinical and ultrasound (US) characteristics of men with MPC. A cross-sectional analysis was carried out of 693 consecutive subjects consulting for couple infertility from September 2012 to March 2017. As a control group, 103 age-matched healthy, fertile men were studied. Furthermore, a longitudinal evaluation of 11 infertile men undergoing trans-rectal ultrasonically-guided cyst aspiration (TRUCA), semen analyses 1 and 3 months after TRUCA and a follow-up 1 year after TRUCA to assess natural pregnancy were performed. All subjects underwent, in our outpatient clinic, clinical, hormonal, scrotal and transrectal US evaluation and semen analysis within the same day. Of 693 males of infertile couples, 648 (37.1 ± 7.9 years, mean+SD) without genetic abnormalities were studied, along with 103 fertile men (36.6 ± 5.0 years). Eleven infertile men underwent TRUCA and were followed-up as reported above. A MPC was present in 66/648 (10.2%) males of infertile couples and in 6/103 (5.8%) fertile men. MPC occurrence and volume were higher in patients with severe oligo- or azoospermia than in fertile men (all P < 0.05). Infertile men with a MPC showed a lower seminal volume and sperm count and a higher prevalence of azoospermia than the rest of the infertile sample or fertile men, and a higher frequency of US signs suggestive of ejaculatory duct obstruction. MPC volume was negatively associated with total sperm count (r = -0.452, P < 0.0001). In fertile men, the highest MPC volume was 0.117 ml, suggesting it as a biological threshold not compromising semen quality. In infertile men, using receiver operating characteristic curve analyses, a MPC volume >0.117 ml identified subjects with severe oligo- or azoospermia with an overall accuracy of ~75% (both P < 0.005). Eleven men with infertility, semen abnormalities and large MPC (>0.250 ml) underwent TRUCA, which led to sperm count improvement in all patients 1 month after surgery. Three months after TRUCA a lower sperm count and a higher MPC volume than 2 months before were observed (P < 0.005 and P < 0.05, respectively), although improved when compared to baseline. After TRUCA a natural pregnancy occurred in four couples. Finally, we propose an algorithm, based on semen parameters, useful in identifying a MPC in males of infertile couples. Although in line with the sample size of previous studies (n = 7-20), the number of infertile men with MPC evaluated longitudinally after treatment is limited (n = 11). In addition, although a MPC volume >0.117 ml can negatively affect the sperm count, only MPC > 0.250 ml have been treated in this study. First, the algorithm proposed is easy to use and useful for selecting patients who can benefit from a prostate US in the infertility work-up. Second, a MPC volume ≤0.117 ml may not impair semen quality, while a larger volume can lead to severe oligo- or azoospermia and could be treated. Third, TRUCA is effective, and simpler and less invasive than other surgical techniques for MPC treatment. Finally, since the MPC can increase in size and sperm count decrease over time after TRUCA, semen cryopreservation should be considered 1 month after TRUCA. Grants from the Ministry of University and Scientific Research (SIR project to F.L., protocol number: RBSI14LFMQ). No conflicts of interest.

Impact of antepartum diagnostic amnioinfusion on targeted ultrasound imaging of pregnancies presenting with severe oligo- and anhydramnios: An analysis of 61 cases

ObjectivesThe primary objective our study was to assess the role of diagnostic antepartum amnioinfusion on the yield from targeted ultrasounds performed in pregnancies with severe oligo- and anhydramnios. Study designThis was a retrospective and descriptive study, conducted in the fetal medicine units of two private tertiary care referral centers in south India. The details of all the cases of diagnostic amnioinfusion performed at these two centers from January 2009 to June 2016 were collected and analyzed. Inclusion criteria were pregnancies between 17 and 26 weeks of gestational age with severe oligo- or anhydramnios. Pregnancies with obvious preterm premature rupture of membranes (PPROM) were excluded. The primary outcome measure was the improvement in diagnostic information pertaining to cause of severe oligo- and anhydramnios, and the nature of such anomalies. ResultsA total of 61 cases of were identified. The median gestational age at performance of the procedure was 22 weeks [IQR, 19.5–23]. The mean volume of normal saline infused was 314±54ml. A significant increase in the single vertical pocket (SVP) was observed following the procedure (pre-procedure SVP=0.6±0.9cm, post procedure SVP=3.4±1.7; paired t test, p<0.001). In 37 cases (37/61, 60.7%), there were no pre-procedure ultrasound findings. There was significant overall detection of abnormalities post procedure (mean pre-procedure findings=0.39±0.49, mean post procedure findings=1.59±1.24; paired t test, p<0.001). The most frequent group of anomalies/abnormalities were renal (36/61, 59%), followed by PPROM (13/61, 21.3%) and finally fetal growth restriction (11/61, 18%). Conclusion(s)Antepartum amnioinfusion is a valuable ancillary technique in prenatal diagnosis as it increases the diagnostic yield from pregnancies presenting with severe oligo- and anhydramnios.

Understanding Male Infertility and Its Causes : A Review

Infertility is a distressing condition affecting 60-80 million couples all over the world. Infertility can be due to male factor, female factor or a combination of both of them. The male factor accounts for 40-50% of infertility and affects approximately 7% of all men. This paper covers the various etiologies attributing to male infertility, assessment of male factor and treatment options made available through years of research. The modifiable risk factors like obesity, smoking, alcohol intake and cell phone use should be looked for and addressed. Environmental factors like ionising radiations, pesticides and heavy metals should be avoided. The correctable causes should be identified. Antioxidants help in reducing oxidative stress and improving semen parameters. Surgical retrieval of sperms is an important technique in males with azoospermia and severe oligo asthenospermia.

Genotyping of Israeli infertile men with idiopathic oligozoospermia.

Microdeletions of the long arm of the Y chromosome involving the azoospermia factor (AZF) region are associated with severe oligo- or azoospermia. Abnormal androgen receptor (AR) structure or function has also been implicated in male infertility. To assess the contribution of these genetic defects to male infertility, 61 Israeli men with severe oligo- (n = 15) or azoospermia (n = 46), were screened for Y chromosome microdeletions, and the AR-(CAG)n repeat length. Fifty fertile Israeli men were similarly analyzed. PCR amplification of 20-54 simple tag sequences (STSs) located at Yq was used to determine the rate and extent of Y chromosome microdeletions. PCR with primers flanking the AR-(CAG)n region and subsequent size fractionation on gradient acrylamide gels were used to determine AR-(CAG)n length. Five azoospermic individuals (5/61-8.2% and 5/46-10.8% of azoospermic patients) displayed Y chromosome microdeletions. The mean CAG repeat number in infertile men was 18.6 +/- 3.0 compared with 16.6 + 2.7 in fertile men (n = 50), a statistically significant difference (p = 0.003). Y chromosome microdeletions contribute to male infertility in our azoospermic population, and the mean length of the AR-CAG is significantly longer in our infertile population than in fertile men.

Levonorgestrel Implants (Norplant II) for Male Contraception Clinical Trials: Combination with Transdermal and Injectable Testosterone

Recent studies demonstrate that combinations of androgens and progestagens are highly effective in the suppression of spermatogenesis in normal volunteers. To test whether progestagen and androgen delivery systems designed to produce steady serum levels will be as effective as other androgen plus progestagen combinations, we compared Norplant II and testosterone (T) transdermal patch to T patch alone on the suppression of spermatogenesis in normal men. Thirty-nine healthy male volunteers (age, 20–45 yr) were randomly assigned to one of two groups. Group 1 (n = 19) received two transdermal T patches daily (Testoderm TTS, each patch designed to deliver about 5 mg/d T) alone, and group 2 (n = 20) received combined Norplant II [Jadelle, four capsules delivering ∼160 μg/d levonorgestrel (LNG)] plus T patch. Neither of these regimens were very effective, with suppression of spermatogenesis to severe oligozoospermia occurring in less than 60% of subjects. We then expanded the study to include two more groups to determine whether T patch or Norplant II was the main factor causing the inadequate suppression of spermatogenesis. Another 29 subjects were randomized to one of two groups. Group 3 (n = 15) received oral LNG (125 μg/d) plus T patch, and group 4 (n = 14) received Norplant II plus T enanthate (TE) injection (100 mg/wk im). After a pretreatment phase of 4 wk, all subjects received treatment for 24 wk, followed by a recovery period of 12–24 wk. Steady-state serum LNG levels (800–1200 pmol/liter) were achieved from wk 3–24 after Norplant II insertion and decreased rapidly after the removal of the implants at wk 24. Trough serum LNG levels after oral LNG administration were at a comparable range (940–1300 pmol/liter). Azoospermia was achieved in 24%, 35%, 33%, and 93%, and severe oligozoospermia (<1 × 106/ml) developed in 24%, 60%, 42%, and 100% of the subjects in groups 1, 2, 3, and 4, respectively, during treatment phase. All subjects in the Norplant II plus TE groups had persistent sperm concentrations less than 3 × 106/ml from wk 12 until the end of treatment. Concomitant with the marked suppression of spermatogenesis in the Norplant II plus TE group, serum FSH and LH levels were most decreased in this group compared with all other groups. In the T patch-only group, serum SHBG was not suppressed, and total serum T was higher than baseline levels. In the other three groups administered progestagens, serum SHBGs were significantly suppressed, and serum total T remained similar to baseline levels. Serum free T levels were not changed in any group. Except for a suppression of serum high-density lipoprotein cholesterol, there was no significant change in weight, hematocrit, clinical chemistry, or prostate-specific antigen levels in any of the treatment groups. Although more efficacious than T patch alone, Norplant II or oral LNG plus T patch was not as effective in suppressing spermatogenesis to severe oligo- or azoospermia as in previous reports using oral LNG plus TE. This relative lesser efficacy occurred despite the achievement of serum LNG levels by Norplant II that were equivalent to those reported after administration of oral LNG. Substituting the transdermal T delivery system with TE injections resulted in very effective suppression of sperm output. The difference in spermatogenesis suppression of these combined regimens is likely due to less T delivered by the transdermal patch compared with the TE weekly injections. We conclude that Norplant II implants plus TE 100 mg/wk were very efficient in suppressing spermatogenesis to a level acceptable for contraceptive efficacy. This study demonstrates that the dose or route of administration of androgens is critical for sperm suppression in combined androgen-progestagen regimens for hormonal male contraception.

Effect of oral probenecid coadministration on the chronic toxicity and pharmacokinetics of intravenous cidofovir in cynomolgus monkeys.

In animals and humans, intravenous administration of the antiviral nucleotide analogue cidofovir results in a dose-limiting nephrotoxicity characterized by damage to the proximal tubular epithelial cells. Probenecid, a competitive inhibitor of organic anion transport in the proximal tubular epithelial cells, was evaluated for its effect on the chronic toxicity and pharmacokinetics of cidofovir. Cynomolgus monkeys (5/sex/group) received cidofovir for 52 consecutive weeks as a once weekly intravenous bolus injection at 0 (saline), 0.1, 0.5, or 2.5 mg/kg/dose alone or at 2.5 mg/kg/dose in combination with probenecid (30 mg/kg/dose via oral gavage 1 h prior to cidofovir administration). Cidofovir-associated histopathological changes were seen only in the kidneys, testes, and epididymides. Nephrotoxicity (mild to moderate cortical tubular epithelial cell karyomegaly, tubular dilation, basement membrane thickening) was present only in monkeys receiving 2.5 mg/kg/dose cidofovir without probenecid. The incidence and severity of testicular (hypo- and aspermatogenesis) and epididymal (severe oligo- and aspermia) changes were increased in monkeys administered cidofovir at 2.5 mg/kg/dose, either alone or in combination with oral probenecid. Renal drug clearance was decreased between Weeks 1 and 52 in the 2.5 mg/kg/dose groups and resulted in an increased systemic exposure to cidofovir (as measured by AUC) that was significantly greater in monkeys administered cidofovir alone (312% increase in males, 98% in females) than in those coadministered probenecid (32% increase in males, 3% in females). These results demonstrate that oral probenecid coadministration protects against the morphological evidence of nephrotoxicity and the accompanying decrease in renal clearance in monkeys receiving chronic intravenous cidofovir treatment.

Successful testicular sperm extraction (TESE) in spite of high serum follicle stimulating hormone and azoospermia: correlation between testicular morphology, TESE results, semen analysis and serum hormone values in 103 infertile men.

Spermatozoa recovered from testicular biopsies can be used through intracytoplasmic sperm injection (ICSI) to achieve a pregnancy. To assess the likelihood of successful testicular sperm extraction (TESE) in men suffering from severe oligo- or azoospermia, bilateral biopsy specimens were obtained. Following semi-thin sectioning, the morphology of testicular samples was graded according to a modified Johnsen score. TESE was performed in parallel to this histological examination. The number of isolated spermatozoa was assessed in a semiquantitative way. From 103 patients investigated, 64 (62.1%) showed azoospermia in a preceding semen analysis and 29 (28.2%) patients had sperm concentrations between 0.1 and 1 x 10(6)/ml. In 10 patients who had higher sperm counts, most spermatozoa were non-motile. Spermatozoa could be detected after TESE in the testicular tissue of 49 (77%) azoospermic men. When follicle stimulating hormone (FSH) concentration was normal, most patients had detectable spermatozoa after TESE. Nearly one-third of patients with mildly elevated FSH had no spermatozoa. Thirty-nine percent of patients in whom FSH was elevated to more than twice normal and 50% of patients with grossly elevated FSH had no detectable spermatozoa. In all, 82.8% of men with sperm concentrations between 0.1 and 1x10(6)/ml in their ejaculate showed spermatozoa in the tissue sample after TESE. Our data demonstrate that, contrary to previous recommendations, infertile men with azoospermia and high FSH values should be reconsidered for testicular biopsy, provided that tissue samples can be cryopreserved for later TESE/ICSI treatment.

Effect of Oral Probenecid Coadministration on the Chronic Toxicity and Pharmacokinetics of Intravenous Cidofovir in Cynomolgus Monkeys

In animals and humans, intravenous administration of the antiviral nucleotide analogue cidofovir results in a dose-limiting nephrotoxicity characterized by damage to the proximal tubular epithelial cells. Probenecid, a competitive inhibitor of organic anion transport in the proximal tubular epithelial cells, was evaluated for its effect on the chronic toxicity and pharmacokinetics of cidofovir. Cynomolgus monkeys (5/sex/group) received cidofovir for 52 consecutive weeks as a once weekly intravenous bolus injection at 0 (saline), 0.1, 0.5, or 2.5 mg/kg/dose alone or at 2.5 mg/kg/dose in combination with probenecid (30 mg/kg/dose via oral gavage 1 h prior to cidofovir administration). Cidofovir-associated histopathological changes were seen only in the kidneys, testes, and epididymides. Nephrotoxicity (mild to moderate cortical tubular epithelial cell karyomegaly, tubular dilatation, basement membrane thickening) was present only in monkeys receiving 2.5 mg/kg/dose cidofovir without probenecid. The incidence and severity of testicular (hypo- and aspermatogenesis) and epididymal (severe oligo- and aspenmia) changes were increased in monkeys administered cidofovir at 2.5 mg/kg/dose, either alone or in combination with oral probenecid. Renal drug clearance was decreased between Weeks 1 and 52 in the 2.5 mg/kg/dose groups and resulted in an increased systemic exposure to cidofovir (as measured by AUC) that was significantly greater in monkeys administered cidofovir alone (312% increase in males, 98% in females) than in those coadministered probenecid (32% increase in males, 3% in females). These results demonstrate that oral probenecid coadministration protects against the morphological evidence of nephrotoxicity and the accompanying decrease in renal clearance in monkeys receiving chronic intravenous cidofovir treatment.

Testicular fine needle aspiration cytology as a diagnostic tool in dog infertility.

The results of testicular aspirate cytology taken from clinical patients with a history of infertility were compared with the clinical and histological findings. Azoospermia was the most common and the most rewarding indication for the examination. Samples were also taken from cases with suspected testicular tumours, orchitis, epididymitis, severe oligo- and teratozoospermia, lack of libido and unilateral testicular atrophy. Histological and cytological findings were found to correlate well. Identification of cell types from normal germinal epithelium was relatively easy. No immediate adverse effects of aspiration were noted. Five normospermic dogs were monitored for two to six months after aspiration, and there were no marked deleterious effects on testicular consistency, testicular histology or semen characteristics. Testicular cytology obtained by fine needle aspiration may, at least to some extent, be used to assist clinical diagnosis, especially in azoospermic dogs and dogs with palpable changes of testicular tissue.

Failure of high-dose sustained release luteinizing hormone releasing hormone agonist (buserelin) plus oral testosterone to suppress male fertility.

Previously we have demonstrated that sperm counts of normal young men decreased during constant subcutaneous infusion of the LHRH agonist buserelin (118 or 230 micrograms/d). In order to test whether azoospermia can be achieved with higher doses, seven young men received 450 micrograms buserelin subcutaneously daily for 12 weeks via extracorporeal osmotic minipumps. To avoid symptoms of androgen deficiency, oral supplementation with 80 mg/d testosterone undecanoate (TU) was initiated in week 5 and was increased to 120 mg/d by week 8. Follow-up after treatment lasted for another 12 weeks. In order to evaluate possible psychotropic effects of treatment-related endocrine changes, continuous psychometric testing was performed focusing on personality, emotions and sexuality. After an initial rise, both serum LH and FSH returned to normal. FSH was below normal during the 3rd-5th week following treatment. LHRH stimulation tests performed at the end of treatment showed pituitary desensitization. Serum T (always measured between 0800 and 1300 h at least 12 h after last TU) tended to decrease by week 7 and remained slightly depressed until the end of treatment while libido, potency and emotional well-being remained unchanged. While testicular volumes showed a reduction from week 7 of treatment to week 10 post-treatment, sperm counts decreased only insignificantly from 65 +/- 10 to 44 +/- 14 million per ml in week 12 post-treatment. Severe oligo- or azoospermia was not observed in any of the seven men. It is concluded that full androgen substitution by TU can drastically delay if not abolish the antifertility effect of LHRH-induced pituitary desensitization.