Testicular Sperm Motility Research Articles

Quality of testicular spermatozoa improves with changes in composition of culture medium

BackgroundSpermatozoa retrieved from the testis and epididymis are deprived of the beneficial effects of seminal fluid. Thus applying an artificial medium with normal seminal fluid characteristics, known as artificial seminal fluid (ASF), may provide an appropriate condition for improving some sperm parameters in azoospermia. The objective was to investigate the impact of in vitro exposure of testicular and epididymal spermatozoa to ASF on sperm quality. The study was conducted on testicular (n = 20) and epididymal (n = 20) sperm specimens obtained from azoospermic men. Each sample was divided into two equal parts: Part I) for processing and incubation with Ham’s F10 medium; Part II) for processing and incubation with ASF.ResultsAfter 2 h incubation, testicular sperm motility was significantly higher in ASF than in Ham’s F10 medium. In comparison to 0 h, mitochondrial membrane potential levels of testicular spermatozoa were significantly higher after 2 h and 24 h in ASF and after 24 h in Ham’s F10 medium. Furthermore, the data indicated significantly lower rates of epididymal spermatozoa with high MMP in both media after 24 h. There were no significant differences in the DNA fragmentation index of testicular and epididymal spermatozoa between ASF and Ham’s F10 medium at different time points.ConclusionThe results demonstrated that in vitro incubation of testicular spermatozoa improved their motility more effectively than Ham’s F10 medium in the short term (2 h), but had no effect on epididymal spermatozoa. Since the physiology of testicular spermatozoa is different from that of ejaculated spermatozoa, it seems that a special environment should be designed and used for each of them.

A cryoprotectant supplemented with pentoxifylline can improve the effect of freezing on the motility of human testicular sperm.

This study examined the effect of a cryoprotectant with and without pentoxifylline supplementation on the motility and viability of human testicular sperm, both before and after freezing. Testicular samples were obtained from 68 patients with azoospermia who came to the Andrology Service of West China Second University Hospital, Sichuan University, for testicular biopsies from December 2019 to April 2020. All patients were assigned randomly to two groups: experimental, whose testicular sperm were added to the cryoprotectant with pentoxifylline, and the control, whose testicular sperm were added to the cryoprotectant without pentoxifylline. Both groups used the same freezing and thawing methods. Testicular sperm motility in the experimental group was significantly higher than that of the control group, both before and after cryopreservation. The recovery rate of sperm motility in the experimental group was significantly higher than that of the control group. The percentage of samples with motile testicular sperm in the experimental group was significantly higher than that of the control group after thawing. Sperm viability was unchanged between the experimental and control groups, both before and after freezing. Overall, a pentoxifylline-supplemented cryoprotectant can significantly improve the motility of testicular sperm before and after cryopreservation.

The impact of fresh and frozen testicular tissue quality on embryological and clinical outcomes.

Our ability to predict the potential of testicular spermatozoa to support embryonic development is still limited. Although motility of testicular spermatozoa is associated with embryo development, the impact of morphology and the presence of spermatozoa in the testicular sample has not been previously researched. Moreover, while the majority of data indicate no effect of cryopreservation, there are studies reporting impaired clinical outcomes due to testicular cryopreservation. In a retrospective study, 118 ICSI-TESE cycles were analysed to study the impact of (a) total quality of testicular tissue, (b) testicular tissue cryopreservation and (c) presence/motility/morphology of testicular spermatozoa in fertilisation rate, embryonic development, clinical pregnancy (CPR), ongoing pregnancy (OPR) and live birth rate (LBR). Results showed that fertilisation rate was significantly affected by both total quality of testicular tissue (p<.05) and rare presence of spermatozoa (p<.01). Moreover, total tissue quality (p<.01), cryopreservation of low-quality samples (p<.01), absence of motile testicular spermatozoa (p<.01) and poor spermatozoa morphology (p<.05) had a negative impact on the number of good quality day 3 embryos. CPR, OPR or LBR was not affected by any parameters examined. Our data suggest that the quality of testicular tissue influences both fertilisation rate and embryo development. Moreover, cryopreservation of low-quality testicular samples has a negative impact on the number of available embryos for transfer.

Effect of cryoprotectant-free vitrification versus conventional freezing on human testicular sperm motility: a prospective comparative study

This study aimed to demonstrate the effect of conventional freezing versus cryoprotectant-free vitrification on the recovery of testicular sperm motility. Testicular samples were obtained from 50 patients with azoospermia for testicular biopsy ± potential sperm storage. We retrieved 100 spermatozoa from each patient divided equally into two straws. They were frozen using conventional freezing as a control group and cryoprotectant-free vitrification in micro-capillary system using open–pulled straws. Seven days later, cryopreserved straws were thawed and assessed in duplicate. The mean sperm motility between the original spermatozoa sample and the post warming sample was reduced after conventional freezing compared to cryoprotectant-free vitrification (4.48 ± 2.09% versus 3.25 ± 1.92%, p < 0.001; 4.48 ± 2.09% vs 3.68 ± 1.93%, p < 0.001, respectively). There was a significant difference between the two methods regarding the mean sperm motility after warming (3.38 ± 1.86% versus 3.76 ± 1.88%, p = 0.015). The mean recovery percent of testicular sperm motility from the original sperm sample was lower (p = 0.02) after conventional freezing compared to cryoprotectant-free vitrification (78.4 ± 28.17% versus 85.37 ± 23.63%). Overall, the rate of post-thaw recovery of human testicular sperm motility improved using cryoprotectant-free vitrification compared to conventional freezing.

Role of Ozone Therapy in Preventing Testicular Damage in an Experimental Cryptorchid Rat Model.

BackgroundCryptorchidism is the most common developmental abnormality of the male reproductive system. If left untreated, it results with infertility and testicular cancer. According to current evidence, surgery is the mainstay of treatment, and hormonal therapy approaches are still under investigation. For the protection of testicular functions, antioxidants have emerged as novel options. This study aimed to evaluate the protective properties of ozone, a strong antioxidant, on testicular tissue.Material/MethodsThirty-five male Wistar-albino rats, 1-month-old, were used for the study. Groups were formed as follows: 1) control, 2) sham surgery (cryptorchidism), 3) cryptorchidism plus ozone, 4) cryptorchidism plus human chorionic gonadotropin (hCG), and 5) ozone plus hCG. Surgical procedures were performed on all rats except the control group. All rats except the control group were used to create an experimental cryptorchidism model, and left testes of animals were surgically placed into the abdomen. After 1 month of surgery, groups 3, 4, and 5 were given corresponding treatments intraperitoneally for 4 weeks. At the end of the study period, testicular atrophy index (TAI) and testicular sperm motility (TSM) were assessed and biochemical, histopathological, and immunohistochemical tests were performed.ResultsTAI and TSM were higher in the ozone, hCG, and ozone plus hCG groups than in the sham surgery group (p=0.001). TSM in the ozone group was significantly higher than in the hCG and ozone plus hCG groups. In biochemical analyses, the parameters of oxidative stress (GPx1, MDA, CAT, GSH, SOD) indicated increased oxidative activity in cryptorchidism, which was resolved by applying ozone and hCG (p=0.001). In addition, apoptotic markers, Caspase 3 and bcl-2 were significantly decreased by applying ozone and hCG (p=0.001).ConclusionsResults of this study suggest that ozone therapy, either as a single agent or in combination with hCG, is a promising approach for protection of testicular functions.

Outcome of ICSI with motile testicular spermatozoa obtained through microscopically assisted testicular sperm extraction in relation to the ovarian response

IntroductionTo determine the relationship between AFC, basal FSH level, woman's age, the number of oocytes retrieved and the outcome of ICSI with testicular spermatozoa obtained with microscopically assisted testicular sperm extraction. Material and methodsIn this retrospective cohort study, a total of 340 couples who underwent ICSI treatment with testicular sperm were enrolled. Women aged≤40years and the first cycles of couples were included. ICSI was performed with motile testicular spermatozoa obtained from 89 men with obstructive azoospermia and 251 men with nonobstructive azoospermia. GnRH-antagonist protocol was used for ovulation induction. Simple linear regression was carried out to analyze relationship between the AFC, basal FSH, woman's age, the number of oocytes, and the live birth rate (LBR). Receiver operator characteristic curves (ROC) were formed to detect cut-off values below which LBR was significantly decreased. ROC curve analysis demonstrated that the cut-off level of the number of oocytes retrieved to predict the LBR was 7. According to this cut-off level, all patients were divided into two groups. Women with retrieved<7 oocytes were included in Group 1 and women with retrieved≥7 oocytes were included in Group 2. ResultsThe mean age of men was 35.1±4.9years. The mean age, mean FSH level and mean AFC of women were 32.1±4.9years, 6.9±2.7 IU/L, 7.6±3.4, respectively. Significant correlations were found between AFC, the number of oocytes retrieved, and the LBR per ICSI cycle with testicular spermatozoa. The LBR was significantly lower in women with AFC<8 than those with AFC≥8. Independently, the LBR was significantly lower in cycles with<7 oocytes retrieved compared to those with ≥7. Embryo transfer was not achieved in 37 cycles with<7 oocytes (37/167, 22.1%) and 18 cycles with≥7 (18/173, 10.4%) because of the absence of transfer-quality embryos (P=0.005). The LBRs were the lowest in cycles with one or two oocytes available (8.3 and 8.3%, respectively), but these rates were not statistically different than those in cycles with 3, 4, 5 and 6 oocytes (14.2, 17.2, 18.5, 17.6%, respectively, P=0.810). ConclusionsAFC and the number of oocytes retrieved are important prognostic factors in an ICSI cycle with testicular sperm in women ≤40years, yielding significantly diminished LBRs with<8 antral follicles and/or<7 oocytes retrieved.

Effect of artificial oocyte activation in intracytoplasmic sperm injection using testicular spermatozoa on sibling oocytes

Artificial oocyte activation (AOA) has been proposed as a suitable means to overcome the problem of failed or impaired fertilization after intracytoplasmic sperm injection (ICSI). To analyze with calcium ionophore after ICSI using testicular spermatozoa improves fertilization, embryonic development and pregnancy outcome in patients with obstructive azoospermia (OA) or non-obstructive azoospermia (NOA). Prospective clinical analysis on sibling oocytes. This prospective study was performed between October 2013 and April 2015. All patients involved gave written consent, and institutional review board approval was granted. This study includes 22 OA and 47 NOA couples. We excluded the couples using only immotile spermatozoa for ICSI. Retrieved oocytes were incubated in culture medium (Universal IVF Medium: UIM) for 2 hours at 37C and 6% CO₂ and were underwent ICSI with motile testicular spermatozoa. When eight or more metaphase M (MII) oocytes were available, AOA was performed on half of the sibling MII oocytes. After ICSI, oocytes were incubated in UIM for 30 minutes, and exposed to 10μM of calcium ionophore A23187 for 15 minutes. The oocytes were then washed and placed in UIM. Two pronuclei (2PN) oocytes, blastocysts development, good-quality blastocysts, biochemical pregnancies, and clinical pregnancies rates were compared between two groups. In terms of OA couples, there were no significant difference in 2PN oocytes, blastocysts development, and good-quality blastocysts rates (73.0%, 56.1%, and 47.8% with AOA and 66.0%, 41.2%, and 45.7% without AOA, respectively). For NOA couples, 2PN oocytes with AOA (74.0%) was significantly higher than those without AOA (61.2%). Blastocysts development, and good-quality blastocysts rates for NOA couples were 53.8% and 38.4% with AOA and 56.7% and 40.5% without AOA, respectively (no significant differences). AOA with calcium ionophore showed favorable effect on fertilization rate in patients with NOA but OA. The sperm source was strongly affect the fertility potential or clinical outcomes. Severe male factor infertility, especially NOA, could be an indication for application of AOA.

In vitro Study of the Spermatozoa Motility in the Lizard Eutropis carinata

Reptilian epididymis is considered as an important excurrent duct system required for the sperm maturation. Reptilian epididymis synthesizes and secretes proteins which vary in different regions of the epididymis. Hence, to investigate the effect of the secretions of different regions of the epididymis on spermatozoa motility, an in vitro study was undertaken to observe the changes in the patterns of motility of the testicular spermatozoa incubated with luminal contents of different regions of the epididymis in the lizard Eutropis carinata for the first time. The non motile testicular spermatozoa from the testis exhibited different patterns of motility, when incubated with the luminal contents of different regions of the epididymis. The spermatozoa from the testis,different regions of the epididymis exhibited 8 different patterns of motility (a-h). Testicular spermatozoa incubated with the anterior and middle epididymal luminal contents showed the motility patterns almost similar to that of the spermatozoa of the anterior and middle epididymis respectively. In contrast to the spermatozoa of the posterior epididymis, none of the testicular spermatozoa showed any movement when incubated with the posterior epididymal luminal contents. This study throws light on the importance of each region of epididymis in the physiological maturation of spermatozoa.

Motility and fertilization ability of sterlet Acipenser ruthenus testicular sperm after cryopreservation

Sturgeon spermatozoa are immotile in the testis and acquire the potential for motility after contact with urine in Wolffian duct. The present study tested if in vitro incubation of testicular sperm in seminal fluid from Wolffian duct sperm leads to the acquisition of sperm fertilization ability. Sterlet sperm was taken from the testes, matured in vitro and cryopreserved. The fertility and motility of cryopreserved semen were tested. Matured testicular sperm showed freeze–thaw survival rates similar to Wolffian duct sperm, which is commonly used in sturgeon artificial propagation. Matured testicular sperm and Wolffian duct sperm post-thaw motility rate and curvilinear velocity were not significantly different, while duration of matured testicular sperm motility was significantly shorter than that of Wolffian duct sperm. Development rates of embryos obtained with post-thaw matured testicular sperm and Wolffian duct sperm were not significantly different. In vitro maturation of sterlet testicular sperm can potentially be useful in sperm cryobanking.

Outcome of intracytoplasmic sperm injection cycles with fresh testicular spermatozoa obtained on the day of or the day before oocyte collection and with cryopreserved testicular sperm in patients with azoospermia

To compare the outcome of intracytoplasmic sperm injection (ICSI)-ET cycles with fresh testicular spermatozoa obtained on the same day or the day before oocyte retrieval with frozen-thawed spermatozoa. Retrospective cohort study. Fertility center. The first ICSI-ET cycle of 337 couples with motile testicular spermatozoa of azoospermic patients. Microdissection testicular sperm extraction (TESE), sperm cryopreservation, ICSI-ET. Fertilization, implantation, clinical pregnancy rates (PRs) and delivery rates. Testicular sperm retrieval was performed on the day of oocyte retrieval in 166 cycles (group A), the day before oocyte retrieval in 42 cycles (group B), and the frozen-thawed testicular spermatozoa were used in 129 cycles (group C). The groups were comparable in terms of the ages of male and female patients, ovarian response to stimulation, as well as the number of oocytes injected. The number of cycles with nonobstructive azoospermia and obstructive azoospermia was evenly distributed in each group. Fertilization rates were 70.7%, 68.7%, and 67.3%, clinical PRs 31.3%, 30.9%, and 25.5%, and delivery rates 28.9%, 28.5%, and 23.2% for groups A, B, and C, respectively. The outcomes of patients with nonobstructive azoospermia did not differ from those of patients with obstructive azoospermia within and among the groups. Neither the timing of TESE (on the day of or the day before oocyte retrieval) nor the use of frozen-thawed testicular sperm affects the outcome of ICSI-ET cycle when motile spermatozoa are obtained in azoospermic men. In addition, etiology of azoospermia does not have any influence on the outcome with different timing of microdissection TESE procedure for ICSI.

Pharmacological stimulation of sperm motility in frozen and thawed testicular sperm using the dimethylxanthine theophylline

To evaluate whether the use of theophylline improves sperm motility and treatment outcome in frozen-thawed testicular sperm extraction (TESE). Artificial sperm activation was offered to azoospermic patients between January and October 2010 in two different centers (identical lab conditions). IVF units of public hospitals. Sixty-five patients participated and gave informed consent. Sibling oocytes were split into a study (intracytoplasmic sperm injection [ICSI] with thawed testicular sperm treated with theophylline) and a control group (ICSI with thawed untreated sperm). Sperm motility, time for sperm selection, rates of fertilization, implantation, clinical pregnancy, and live birth. All patients but one (98.5%) showed a significant improvement in testicular sperm motility when theophylline was used. In addition, sperm selection took significantly less time in the study as compared with in the untreated control group. Corresponding rates of fertilization (79.9% vs. 63.3%) and blastulation (63.9% vs. 46.8%) were significantly increased. Significantly more patients achieved clinical pregnancy if embryos/blastocysts derived from oocytes that had been injected with pharmacologically stimulated testicular spermatozoa were transferred (53.9% vs. 23.8%). This also holds true for the implantation rate. Theophylline turned out to be a reliable tool in stimulating testicular spermatozoa after thawing. Its immediate effect allows for faster and more accurate selection of viable sperm, which in turn improved fertilization and pregnancy outcome in this prospective study.

271 THE EFFECT OF TEMPERATURE ON HUMAN TESTICULAR TISSUE TO OPTIMIZE THE IN VITRO MATURATION OF PRE-FREEZE MOTILITY

The development of intracytoplasmic sperm injection has made the use of testicular sperm a viable option for infertile men with obstructive and nonobstructive azoospermia. Over the past decade, testicular biopsies have been handled and processed using a variety of different methods. Whole biopsy pieces can be effectively cryopreserved in a 10% glycerol diluent (Schiewe et al. 1997 67, S115 abst); however, the ability to find viable, motile sperm post-thaw is improved when prefreeze motility exists. The purpose of this study was to comparatively document in vitro sperm motility enhancement over time at different temperatures, and to prove that an intermediate temperature (28 to 30°C) would optimize sperm longevity for up to 1 week. In this study, 10 men with obstructive azoospermia underwent a surgical, open testicular biopsy procedure. Each biopsy was placed in HEPES buffered-human tubal fluid (mHTF) medium supplemented with 5% human serum albumin (HSA; Irvine Sci., Santa Ana, CA, USA) and transported to the laboratory at room temperature. Each testis biopsy (TBx) was dissected into 8 equal pieces (approximately 2 × 2 × 1 mm). Five intact pieces of TBx were cryopreserved in separate cryovials for future use. The remaining TBx tissue was subdivided into 1 of 3 temperature treatment groups (24, 30, or 37°C) for extended IVM. The 30°C incubation condition was achieved by placing the dish(es) in a Styrofoam box placed on a 37°C warming plate. Each TBx was placed into a separate 100 × 35 mm Falcon dish in 150-μL droplets of mHTF under oil and shredded by needle dissection to disperse the contents of the seminiferous tubules. Reminant tissue was placed into another droplet for additional dissection, as needed. Sperm were analyzed for motility (graded as Type I = twitching, II = undulating, III = slow progression, and IV = rapid progression) at 0, 24, 96, and 144 h without replacing the IVM medium. The percentage increase in motility, compared with 0 h, was statistically contrasted across temperature treatments by chi-squared analysis. Testicular sperm motility ranged from 5 to 25% at 0 h and increased in all groups at 24 h. There was no difference in total motility at 24 h, but progressive motility was higher (P &lt; 0.05) at 37°C compared with 24°C. Significant differences (P &lt; 0.05) were observed in total percentage of motility/percentage of progressive motility at 96 h with treatment differences (*) being 30°C (66%*/44%*) &gt; 24°C (42%*/14%) &gt; 37°C (18%*/10%). This statistical trend continued at 144 h with 30°C (42%*/23%*) &gt; 24°C (24%*/8%) &gt; 37°C (9%*/5%). During this study, a viable pregnancy was achieved using a 30°C sample 8 days post biopsy, exceeding 2 previous healthy triplet pregnancies, which were successful using 4-day-old TBx specimens in 1996 and 2004. This study confirms that an intermediate IVM temperature of 30°C is optimal to enhance TBx cryopreservation, which in our laboratory is generally performed at 24 to 72 h of IVM. Our routine pregnancy success in applying ICSI with IVM, thawed TBx sperm discounts the concern some individuals express regarding DNA fragmentation of sperm over time.

Effects of l-carnitine and l-acetyl carnitine on in vitro sperm maturation

Received: 13 November 2010 Revised: 16 May 2011 Accepted: 15 June 2011 Abstract Background: Sperm cells extracted from testes (TESE) have poor chromatin quality and motility. Various substances are used in the laboratory to increase sperm motility and improve the ART outcomes; however, there are few research which considered improving both sperm motility and chromatin quality. Objective: The aim of this investigation was to evaluate the improvement of the testicular sperm motility and chromatin quality exposed to L-carnitine (LC) and Lacetyl-carnitine (LAC), which are normally concentrated in testis and epididymis, compared with Pentoxifylline (PF), which used for sperm motility enhancement in IVF procedures. Materials and Methods: TESE samples from 30 male mice divided into four parts. The sperm samples were added to Ham' F10 (control) or the media contained 1.76mM of LC, LAC or PF), then, the samples were kept in the room temperature for 30, 90 and 180 min. At each time step, sperm motility and chromatin quality were assessed. Chromatin quality was evaluated by chromomycin A3 and aniline blue. Statistical analysis was performed using one way analysis of variance (ANOVA). A p-value less than 0.05 were accepted as a statistically significant difference. Results: The results showed LC, LAC and PF significantly increased the sperm motility. However, sperm chromatin quality only improved significantly by administration of LC and LAC. Conclusion: Administration of LC and LAC to the testicular sperm samples can lead to improve both sperm motility and chromatin quality. It may be because they can mimic in vivo sperm condition during late spermatogenesis.

Sperm motility and cryopreservation of spermatozoa in freshwater gobies

Testicular sperm motility and methods for the cryopreservation of spermatozoa in the freshwater goby Rhinogobius sp. CB (Cross Band type) were examined. Spermatozoa were almost immotile upon dilution with 300 mOsm kg−1 of NaCl, KCl and mannitol solutions but began to swim in solutions with concentrations &lt;200 mOsm kg−1. The highest percentage and longest duration of motility was obtained in the 0 and 100–200 mOsm kg−1 solutions, respectively. The highest post‐thaw motility, c. 50% of motility before cryopreservation, was obtained when spermatozoa were diluted with an extender of 10% methanol and 90% artificial seminal plasma, cooled at −10·0 ± 1·1° C min−1 (mean ±s.e.) to −50° C and plunged into liquid nitrogen. Spermatozoa were cryopreserved in a 50 μl acrylic haematocrit tube to store the small amount of milt. As the cryopreservation method described above was applicable to the endangered Rhinogobius sp. BI (Bonin Island type), it is probable that this method can be used for other species of freshwater gobies.

Testicular tissue processing: maximizing viability for cryopreservation and clinical use

Background and Significance: Schiewe et al. has previously shown that testicular tissue could be effectively cryopreserved as whole biopsy pieces. Interestingly, abnormal sperm were twice as likely to survive freeze-thawing than normal sperm (32% vs 15% viable, respectively). However, in the absence of post-thaw motility, structurally normal late spermatids (i.e., cell wall encasing the spermatozoal head) had the highest post-thaw viability (>60%). Although non-motile, thawed late spermatids have been used to create viable pregnancies, the goal of testicular tissue processing should be to promote and maintain sperm motility pre- and post-cryopreservation, respectively.Objective: The purpose of this study was to determine to what degree in vitro culturing of testicular sperm enhanced the motility of fresh and frozen-thawed specimens.Materials and Methods: Fresh and frozen-thawed testis biopies (n=10 each) were obtained from NOA and OA patients whose spouse was undergoing COH for an ICSI cycle. Testicular sperm extraction was attained by an open biopsy procedure. Specimens were placed into 2ml of H-HTF medium + 10% SS in a 35 × 10mm Falcon dish prior to dissection into 1–2mm3 pieces. Four pieces of each biopsy were placed into each of four 150ul droplets of H-HTF/SS under oil and minced by needle every other day. The dishes were maintained under ambient conditions (22–27°C) and evaluated daily for motility and progression (I= twitching, II=undulating, III= forward, linear movement to IV= rapid motility). Differences in percent motility between intervals was assessed by chi-square analysis.Results: Initial sperm motility varied in fresh and frozen-thawed biopies (10 to 60% and 2 to 25%, respectively), with a majority of sperm having a progession index of I-III for fresh tissue and I-II for frozen biopies. In fresh biopies, motility and progression steadily increased over 96hr (1.5 to 5-fold, P>0.01) in all specimens. Whereas, sperm motility peaked by 24 hr in the frozen-thawed specimens.Conclusion: Good testicular sperm motility post-thaw: 1) decreases processing and ICSI intervals; 2) enhances ICSI outcomes; and 3) increases the probability of achieving a successful pregnancy. In this study we determined that testicular sperm motility in vitro increases over time for fresh tissue, suggesting that cryopreserving a biopsy four days after retrieval may optimize frozen-thawed motility outcomes. Furthermore, thawing samples 1 day prior to egg retrieval will increase the efficacy of using actively motile sperm for ICSI. Background and Significance: Schiewe et al. has previously shown that testicular tissue could be effectively cryopreserved as whole biopsy pieces. Interestingly, abnormal sperm were twice as likely to survive freeze-thawing than normal sperm (32% vs 15% viable, respectively). However, in the absence of post-thaw motility, structurally normal late spermatids (i.e., cell wall encasing the spermatozoal head) had the highest post-thaw viability (>60%). Although non-motile, thawed late spermatids have been used to create viable pregnancies, the goal of testicular tissue processing should be to promote and maintain sperm motility pre- and post-cryopreservation, respectively. Objective: The purpose of this study was to determine to what degree in vitro culturing of testicular sperm enhanced the motility of fresh and frozen-thawed specimens. Materials and Methods: Fresh and frozen-thawed testis biopies (n=10 each) were obtained from NOA and OA patients whose spouse was undergoing COH for an ICSI cycle. Testicular sperm extraction was attained by an open biopsy procedure. Specimens were placed into 2ml of H-HTF medium + 10% SS in a 35 × 10mm Falcon dish prior to dissection into 1–2mm3 pieces. Four pieces of each biopsy were placed into each of four 150ul droplets of H-HTF/SS under oil and minced by needle every other day. The dishes were maintained under ambient conditions (22–27°C) and evaluated daily for motility and progression (I= twitching, II=undulating, III= forward, linear movement to IV= rapid motility). Differences in percent motility between intervals was assessed by chi-square analysis. Results: Initial sperm motility varied in fresh and frozen-thawed biopies (10 to 60% and 2 to 25%, respectively), with a majority of sperm having a progession index of I-III for fresh tissue and I-II for frozen biopies. In fresh biopies, motility and progression steadily increased over 96hr (1.5 to 5-fold, P>0.01) in all specimens. Whereas, sperm motility peaked by 24 hr in the frozen-thawed specimens. Conclusion: Good testicular sperm motility post-thaw: 1) decreases processing and ICSI intervals; 2) enhances ICSI outcomes; and 3) increases the probability of achieving a successful pregnancy. In this study we determined that testicular sperm motility in vitro increases over time for fresh tissue, suggesting that cryopreserving a biopsy four days after retrieval may optimize frozen-thawed motility outcomes. Furthermore, thawing samples 1 day prior to egg retrieval will increase the efficacy of using actively motile sperm for ICSI.

Treatment of freeze-thawed testicular sperms with Human Follicular Fluid (HFF) improves their motility and is useful to select viable sperms for ICSI procedure

Objective: The motiltiy of sperms obtained from testicular biopsy is generally very low.Since in some cases, it is nessesary to freeze the biopsied tissue in order to prevent of repeated testis biopsy, and for future use of testicular sperms in ICSI,on the other hand, many of these cells may lose their viability during cryopreservation, it is reasonable to find a way which may help us to select viable sperms after thawing, for injecting them into oocytes. According to previous studies on the positive effects of hFF on sperm motility and its capacitation,we tried to take advantage of this property and to evaluate the motility of freeze-thawed testicular sperms, following culture in different combinations of hFF and Ham’s F-10. Design: Comparison of testicular sperm motility immediately after thawing,and following culture in different concentrations of hFF. Materials and Methods: The biopsied testicular tissue (n=8) was subjected to mechanical mincing by two glass slides and recovered testicular sperms were freezed with conventional sperm freeze solution (glycerol 15%). After thawing, samples were allocated to four groups as follow: Group 1(Control);Ham’s F-10/without culture,Group 2; Ham’s F-10+0% hFF/with 24 hrs culture, Group 3; Ham’s F-10 +25% hFF/with 24hrs culture, Group 3; Ham’s F-10 +50% hFF/with 24hrs culture.Culture was done under 5% CO2 and 37° C .In group 1 (Control), motility was evaluated immediately after thawing .In Group 2,evaluation was done to determine if culture alone has a positive effect on motility and in remainig groups(3 and 4), the effect of different concentrations of hFF on testicular sperm motility was determined. At least 100 sperms were counted for motiltiy scoring in each group. Statistical analysis was performed by paired t-test between control and experimental groups,and a comparison of Mean+SD for motility between groups 2,3 and 4 was done to define the best concentration of hFF for testicular sperm culture after thawing. P<0.05 considered significant. Results: Sperm motility immediately after thawing was 3.00+ 4.40%(Group 1).After 24 hrs of culture, motility was 21.25+12.61% (Ggroup2),30.75+16.23% (Group 3) and 43.0+17.00% (Group 4) respectively.The differences between control and other groups(cultured in FF) was statistically significant. Also the motility was significantly higher in group 4 compared with other groups(2,3). Conclusion: These results suggest that 24 hrs culture of freeze-thawed testicular sperms in media containing 50% hFF may improve its motility,and, in addition of helping us to select viable sperms for ICSI, may be beneficial for these cells in aspect of optimizing their quality and ability to fertilize oocytes.

Comparison of the motility of testicular sperm cultured at different culture conditions: Human follicular fluid (HFF) temperatures

Objective: The motility of sperm obtained from testicular tissue is generally very low. In our previous studies, we have achieved better results by culturing testicular sperm in vitro for 24 to 48 hrs compared with simple culture or no culture. Despite the low initial sperm quality, a high percentage of the prepared testicular sperm showed increased motility after 24 or 48 hrs of culture at 37°C. However, the previous reports suggested that the optimum culture condition was 32°C. Therefore, we tried to evaluate the testicular sperm motility following different culture conditions.

Outcome of ICSI Using Fresh and Cryopreserved-Thawed Testicular Spermatozoa in Patients With Non-Mosaic Klinefelter’s Syndrome

Several deliveries have recently been achieved by intracytoplasmic sperm injection (ICSI) of testicular sperm taken surgically from patients with nonmosaic Klinefelter's syndrome. This study, in 12 azoospermic patients with a cytogenetic diagnosis of nonmosaic KS, was intended to compare the outcome of ICSI using fresh and cryopreserved-thawed testicular spermatozoa. Fresh ejaculates showed mature but nonmotile sperm in 2 patients and no spermatozoa in 10. ICSI using nonmotile sperm failed to achieve fertilization, so all patients underwent testicular sperm extraction (TESE). ICSI was done using both fresh and cryopreserved-thawed spermatozoa. Ovulation induction and oocyte retrieval were carried out by midluteal pituitary suppression with a gonadotropin-releasing hormone agonist followed by human menopausal gonadotropins. After oocyte retrieval aided by vaginal ultrasound, three or (in repeated attempts) four embryos were transferred. Progestational luteal support was routinely provided. TESE yielded motile testicular spermatozoa in 5 of 12 cases (42%); excess testicular tissue was cryopreserved. All testicular biopsy specimens demonstrated hyalinized tubules with no spermatozoa. ICSI using fresh testicular sperm led to a fertilization rate of 66%. Transfer of three embryos in each instance resulted in three pregnancies, and all 15 embryos had a good morphologic grade. There were two singleton pregnancies, one delivered and one ongoing, and a triplet pregnancy resulting in twin delivery (when a 47,XXY embryo was eliminated). ICSI using cryopreserved-thawed testicular spermatozoa yielded a fertilization rate of 58%. Sixteen of the 18 embryos transferred had a good, and 2, an average morphologic grade. Two pregnancies resulted in one twin delivery and one early spontaneous abortion. Both types of spermatozoa were associated with a good cleavage rate; there were no significant differences in fertilization, embryo cleavage, or implantation rates. ICSI is a practical measure in patients with nonmosaic Klinefelter's syndrome. If only nonmotile sperm are found, it remains possible to obtain motile testicular spermatozoa after TESE, and these may lead to pregnancy and delivery. Comparable results were achieved in the present study using fresh and cryopreserved-thawed testicular spermatozoa.

Intracytoplasmic sperm injection using non-motile testicular spermatozoa selected by modified hypo-osmotic swelling test.

Objective: The aim of this work was to explore the fertilizing capacity of totally non-motile testicular spermatozoa selected by a modified hypo-osmotic swelling test (consisting of 50% culture medium and 50% milli-Q water), compared to motile testicular spermatozoa in patients with infertility due to non-obstructive azoospermia.Design: A comparison of fertilization, clinical pregnancy and on-going pregnancy rates in azoospermic patients with non-motile testicular spermatozoa selected by a modified hypo-osmotic swelling test versus azoospermic patients with motile testicular spermatozoa.Materials/Methods: Fifty five cycles in infertile couples due to non-obstructive azoospermia treated with intracytoplasmic sperm injection (ICSI), consisting of 16 cycles where ICSI was performed using non-motile spermatozoa and 39 cycles where ICSI was performed with motile testicular spermatozoa. In the 16 cycles with total absence of motility, the spermatozoa used for injection were selected by a modified hypo-osmotic swelling test (a solution consisting of 50% culture medium and 50% milli-Q water), while in the 39 cycles with motile spermatozoa, these were randomly selected.Results: The fertilization rate was 32.4% in the non-motile sperm group compared to 51.6% in the motile sperm group (P < 0.005) and embryos were available for replacement in the motile sperm group in 76.9% of the cycles compared to 50% of the cycles in the non-motile sperm group (P < 0.05). The clinical pregnancy rate was 12.5% in the non-motile group compared to 12.8% in the motile group (P = 0.9742) and the delivery/ongoing pregnancy rate was 6.25% in the non-motile sperm group compared to 7.69% in the motile sperm group (P = 0.8516).Conclusions: ICSI performed with totally non-motile testicular spermatozoa selected by the modified hypo-osmotic swelling test results in comparable pregnancy rates ICSI using motile testicular spermatozoa, in cases of non-obstructive azoospermia despite the fact that the fertilization and cleavage rates are lower in the non-motile sperm group.Supported by: Alexandria Fertility Centre. Objective: The aim of this work was to explore the fertilizing capacity of totally non-motile testicular spermatozoa selected by a modified hypo-osmotic swelling test (consisting of 50% culture medium and 50% milli-Q water), compared to motile testicular spermatozoa in patients with infertility due to non-obstructive azoospermia. Design: A comparison of fertilization, clinical pregnancy and on-going pregnancy rates in azoospermic patients with non-motile testicular spermatozoa selected by a modified hypo-osmotic swelling test versus azoospermic patients with motile testicular spermatozoa. Materials/Methods: Fifty five cycles in infertile couples due to non-obstructive azoospermia treated with intracytoplasmic sperm injection (ICSI), consisting of 16 cycles where ICSI was performed using non-motile spermatozoa and 39 cycles where ICSI was performed with motile testicular spermatozoa. In the 16 cycles with total absence of motility, the spermatozoa used for injection were selected by a modified hypo-osmotic swelling test (a solution consisting of 50% culture medium and 50% milli-Q water), while in the 39 cycles with motile spermatozoa, these were randomly selected. Results: The fertilization rate was 32.4% in the non-motile sperm group compared to 51.6% in the motile sperm group (P < 0.005) and embryos were available for replacement in the motile sperm group in 76.9% of the cycles compared to 50% of the cycles in the non-motile sperm group (P < 0.05). The clinical pregnancy rate was 12.5% in the non-motile group compared to 12.8% in the motile group (P = 0.9742) and the delivery/ongoing pregnancy rate was 6.25% in the non-motile sperm group compared to 7.69% in the motile sperm group (P = 0.8516). Conclusions: ICSI performed with totally non-motile testicular spermatozoa selected by the modified hypo-osmotic swelling test results in comparable pregnancy rates ICSI using motile testicular spermatozoa, in cases of non-obstructive azoospermia despite the fact that the fertilization and cleavage rates are lower in the non-motile sperm group. Supported by: Alexandria Fertility Centre.

The use of a modified hypo-osmotic swelling test to select non-motile but viable testicular spermatozoa for intracytoplasmic sperm injection.

Objective: The aim of this work was to explore the fertilizing capacity of totally non-motile testicular spermatozoa selected by a modified hypo-osmotic swelling test (consisting of 50% culture medium and 50% milli-Q water), compared to motile testicular spermatozoa in patients with infertility due to non-obstructive azoospermia.